A POTPOURRI OF NEMATOLOGICAL

                   METHODS AND TECHNIQUES

 

                                                    

 

                                    Armen Charles Tarjan, Professor Emeritus

                         Department of Entomology & Nematology

                         University of Florida, Gainesville 32611

                                                  (dated 15-Jan-05)

                                    

 

 

 

Foreword, Acknowledgements & Introduction .... Section A

 

Abstracts ................................................................Section B

 

Subject/Acronym ................................................... Section C

 

References & Periodicals........................................ Section D

 

Author/Reference Citations .................................... Section E

 

 

 

SECTION A --  FOREWORD

 

     It continually remains a tragedy that many unique methods which were devised for specific purposes or problems and described in  published research-related literature are overlooked, disregarded or otherwise ignored.  One might somewhat rightfully conclude that the majority of such methods are nothing more than an unknowing duplication or modification of older known methods.  Yet, the solution to any problem usually is dependant upon the investigative method selected. Thus it becomes of cardinal importance that applicable, cogent techniques are utilized and, preferably, remembered.

 

   Methodology long had been a favorite area of interest for me and one in which I and former co-workers had previously collaborated in compiling information. Unfortunately, that project was necessarily left unfinished in 1986. Hence, the inevitability of my pursuing a completion to that effort in year 2001 became pertinent

 

    Attempting to accumulate a potpourri of diverse methods proposed over a period of 60-years has become a Herculean task. Even my original plan of eventually releasing the undertaking in four sections has been abandoned. Instead, the work will be released in alphabetically sequential segments, the first being for methods beginning with A to D, the second from E to H, etc.

 

 

ACKNOWLEDGEMENTS

 

    My thanks are extended to the people who, in the past, had contributed to this project, namely : Drs. B. A. Adams, R.P. Esser, J. H. O'Bannon and G. C. Smart, Jr. Dr. Zafar Handoo has been very helpful in submitting a considerable amount of information obtained from the U. S. Department of Agriculture Library.  Dr. N. B. Khuong's input has been invaluable in transposing this work into HTML and S. E. Lasley, Senior Systems Programmer has also extended his expertise.  I offer special thanks to Dr. J. L. Capinera, Chairman of the Dept. of Entomology & Nematology who has allowed me use of an office and laboratory, disregarding my "emeritus" status, and has encouraged the development of the present compilation.

 

        There is nothing original about the present work other than it's mainly a compilation of various innovative techniques which other workers discovered or developed. Two sources have been very largely responsible for the information and the syntax, as presented herein. I can not praise too highly the thorough, meticulous work by  E. J Cairns, 1960,. Methods in Nematology: a Review. Chapter 5, pgs.33-84  in J. N. Sasser :& W. R. Jenkins (eds.). Nematology fundamentals and recent advances with

emphasis on plant parasites and soil forms. Univ. North Carolina Press, Chapel Hill.  His work most closely approximates what I am doing, some four decades later.   Accordingly I have used many of his descriptions of older work which is unavailable to me. Although my syntax, at times, may differ from his, it is his work that essentially is reproduced herein. The second publication on which I have relied most heavily is: the outstanding illustrated work edited by J. F. Southey, 1986. Laboratory methods for work with plant and soil nematodes. Reference Book 402, Ministry of Agriculture, Fisheries and Food,  202pp. This also is a storehouse of essential nematological methodology.

 

 

 INTRODUCTION

 

     The inherent inadequacy of the present undertaking lies in the obscurity or relative unavailability of desired information on the subject of  "methods' . More often, when a method applicable to the task at hand isn't found, the investigator's ingenuity and resourcefulness must be called into play and a new method or technique is born.  All too often, however, such novel techniques are either concealed or surrounded by a fortress of verbiage thus neatly obfuscating the reader.  It is the purpose of the present work , in part,  to resuscitate such methods from their relative obscurity. As additional information is located pertaining to alphabetical sections of this work already on the internet, the new material will be added as soon as possible

 

        Only a part of the intended compilation has been completed. It now contains 2000 published methods pertaining to 124 subject headings as of Jan 1, 2005.  The complete reference citation ,and usually a 2 to 3 sentence description of each novel method, is presented. In addition to the abstracts which constitute the bulk of the file, there is a Subject/Acronym section which coordinates with the Author/Reference section.  Finally, there is a lengthy Publications File identifying the publications and scientific journals in which the methods have appeared.  With the placement of the material on the internet, work primarily continues searching for applicable new methods for inclusion.  

 

IMPORTANT:  There will be errors in this work despite the 'careful' formulation and reading of each entry. The author would appreciate being informed about such errors. Inevitably, some pertinent published novel methods have been overlooked. In such cases, if the complete citation AND accompanying abstract is sent to me, these will be included into this methods collection.

                                                    Armen Charles Tarjan --- (tarjan@mail.ifas.ufl.edu)

 

 

 

 SECTION B --  ABSTRACTS ALPHABETICALLY ARRANGED

                           ACCORDING TO SUBJECT

 

  The acronym or 3-letter "identifier" that follows each underlined subject category relates to the Author-Reference section.  At the end of each listed reference, the reader gains immediate knowledge of the subject about which each author wrote by virtue of the acronym.

                                                                        

          ----- A -----

 

AERATION -- AER            

                                                                                   

 Flegg, 1967 -- Annals of Applied Biology  60: 429-437.

Using two Xiphinema  species, the Baermann funnel method was compared with a  dish method designed to provide enhanced oxygenation. The latter method produced more active nematodes than the funnel method.

 

Nicholas & Jantunen, 1963 -- Nematologica 9: 332-336.

Caenorhabditis briggsae was cultured axenically in a biotin-free medium supplemented with chick embryo extract. The addition of avidin, which is known to combine with biotin, is an inhibiting factor. The effect of avidin is nullified by excess biotin. It was concluded that biotin is a vitamin required by the nematode.

 

Nicholas & Jantunen, 1964 -- Nematologica 10: 409-418.

The rhabditid nematode, Caenorhabditis briggsae, was studied under anaerobic conditions for effects on eggs, larval stages and adults.  Such conditions were produced by exposing the eelworms, in drops of media, to a stream of carefully purified  moist nitrogen or hydrogen. The nematodes were maintained under axenic conditions while anaerobic. It is concluded that swimming ablity is impaired by lack of oxygen, leading to complete paralysis.

  

Oostenbrink, 1960 -- in Sasser & Jenkins, Nematology Fundamentals and recent advances with emphasis on plant parasites and soil forms,  Univ. North Carolina Press, Chapel Hill, pgs. 85-102.

Nematodes are extracted from fine debris obtained by washing soil and afterwards placed on cottonwool filters. To facilitate an even distribution of nematodes in the suspension for counting,  the nematode-water mixture is agitated using a small electro-magnetic air pump.

 

Tarjan, 1960 -- Plant. Disease Reporter   44: 574-577

A test was designed to compare the utility of  one-mil thick polyethylene bags versus glass jars as incubation chambers for extracting nematodes from citrus roots. The plastic bags proved to yield more nematodes from the roots. It was theorized that this may be due  to the ability of the plastic to delay dissapation of soil gases and yet admit oxygen.

 

Wallace, 1956 -- Annals of Applied Biology 44: 57-66.

The rate of emergence of larvae from cysts of Heterodera schachtii increases with aeration. Studies on soil structure, rather than just an mechanical analysis of the soil should be emphasized.

 

 

AGAR, CALLUS TISSUE -- ACT

 

Corbett, 1970 --Nematologica 16: 156.

Towards the goal of maintaining nematode cultures under mineral oil, Pratylenchus fallax and Aphelenchoides ritzemabosi were established on alfalfa callus in test tubes on agar. Tubes were inspected at the end of six and nineteen months with living nematodes being in evidence.

 

Krusberg, 1961 -- Nematologica 6: 181-200.

Krusberg used the medium proposed by White, 1943 (see below) for growing alfalfa callus on which Ditylenchus dipsaci, Aphelenchoides ritzemabosi, pratylenchu penetrans and P. zeae were cultured  Details of nematode feeding and symptoms of injury are described.  Sugars, organic acids and amino acids were subsequently examined in the alfalfa tissues.

 

Krusberg & Blickenstaff, 1964 -- Nematologica 10: 145-150.

Alfalfa callus tissue grown on agar media containing 2,4-D supported maximum numbers of  Ditylenchus dipsaci, Pratylenchus penetrans, and P. zeae. Kinetin increased the reproductive rate of  D. dipsaci, but reduced the rate of reproduction of P. penetrans  and  P. zeae.

 

Reidel, Foster & Mai, 1973 --- Journal of Nematology 5: 71-72.

A simplified sucrose-yeast extract-2,4 D agar medium was beneficial in allowing reproduction of Ditylenchus dipsaci and Pratylenchus penetrans on onion and alfalfa callus cultured in 25x150mm tubes respectively. The average population of  D. dipsaci was10.500/tube. For P. penetrans,  populations averaged at 20,460 nematodes /tube.

 

Schroeder & Jenkins, 1964 -- Nematologica 9: 327-331.

Found that callus tissue is often a better host for nematodes than the differentiated tissue from which it is derived. They used  agar slants  of  various media to produce callus from eleven crop plants and found that pea, alfalfa and cucumber callus were best for the reproduction of Pratylenchus penetrans.

 

Tamura & Mamiya, 1976 -- Nematologica 21: 449-454.

The authors discovered that Bursaphelenchus lignicolus successfully reproduced on alfalfa callus tissues.  The simplified agar culture medium containing yeast extract, sucrose and 2,4-D reported by Reidel , Foster & Mai in 1973 was found to be  the best.

 

Webster & Lowe, 1966 -- Parasitology 56: 313-322.

Six races of Ditylenchus dipsaci were cultured on alfalfa and red clover callus tissues on nutrient agar Red clover callus supported large populations of Aphelenchoides ritzemabosi while red clover seedlings did not.. Heterodera rostochiensis did not reproduce in callus tissue.

 

White, 1943 -- A handbook of  Plant Tissue Culture, Ronald Press Co., New York.

An agar medium  which contains 20 different substances is described. It is suggested that the medium could serve as the basis for studies culturing callus tissue which, in turn, could support nematodes.

 

AGAR, CULTURES -- AGA           

                                                                                       

Bingefors & Eriksson, 1963 -- LantbrHoegsk. Annlr. 29: 107-118.

Sterilized clover seed by soaking in concentrated sulfuric acid for 30 min, then washing with sterile water, then with a solution of streptomycin sulphate. The seeds were germinated on water agar. 

 

Cohn, 1970.-- Journal of  Nematology 2: 167-173.

Cohn studied the the feeding habits of Longidorus  and Xiphinema spp. on grape seedlings, Urtica urens and  Bidens tripartita. Both short-term and long-term  symptoms were observed. All Xiphinema spp.caused a distinct thinning and darkening of root systems. Longidorus spp. caused stubby and swollen root tips.

 

.Dallimore,1966 -- Phytopathology  56: 874-875.

Aseptic plugs of potato were embedded in potato-dextrose, corn meal or water agar and then inoculated with a piece of infested tissue from a young lesion of infested  potato. Potato plugs prepared in this manner proved an excellent sourse of inoculum when placed in the soil near growing potato plants.

 

Dropkin & Webb, 1967 -- Phytopathology 57: 584-587.

Axenic tomato seedlings were grown on agar slants of a modified 'White's medium' for studing resistance of the seedlings to species of Meloidogyne. It was found that plants  resistant to M. hapla lacked necrotic responses and that larvae generally induced gall formation but the number of juveniles inresistant seedlings was much less than in susceptible plants.

 

Feder & Feldmesser, 1957 -- Phytopathology 47: 11.

The burrowing nematode, Radopholus similis, was cleaned by soaking nematodes in mercuric chloride 1/1,000 for 5 minutes followed by 3 serial washings and centrifugations in sterile distilled water. The cleaned animals were fully infective when placed on citrus roots and retained their motility when placed on various types of sterilized agar media such as potato-dextrose, nutrient, potato-dextrose-peptone, and yeast extract.

 

Goodrich, Hechler & Taylor,1968 -- Nematologica 14: 25-36.

 Two predacious species of  Mononchoides were cultured on water agar using Aphelenchus avenae as the food source. The article deals primarily with the morphology and systematics of Mononchoides changi n. sp. and M. bollingeri  n. sp.

 

 Hechler, 1963-- Proceedings  Helminthological Society of Washington 30; 182-185.

The biological development of the predacious nematode Seinura tenuicaudata was studied on potato dextrose  agar cultures using Aphelenchus avenae as the food source.

 

Khera & Zuckerman, 1963 -- Nematologica 9: 1-6.

Studied feeding habits of seven different nematode species on roots of different plants growing in 1% water agar. Concentrated extracts of the nematodes from soil or hand-picked, sterilized specimens were placed close to the roots of  the 3 to 7-day-old seedlings. Hemicycliophora similis. Trichodorus christiei and  Tylenchorhynchus claytoni fed on most of the thirteen plants tested.

 

Russell & Perry, 1966 -- Phytopathology 56: 357-358.

The behavior of Trichodorus christiei (=Paratrichodorus minor) on wheat roots growing in agar. was studied. An agar block containing nematode eggs, and a section of root  was placed on a cover slip and inverted into the concavity of a microculture slide. The cover slip was ringed with petroleum jelly. This allowed for close oservation of nematode activity.

 

Wyss,  1970 -- Nematologica 16: 55-62.

0bserved the feeding of Pratylenchus penetrans, Rotylenchus robustus, Tylenchorhynchus dubius and Longidorus elongatus on strawberry seedlings growing in water agar. L. elongatus was found to be highly pathogenic in contrast to damage caused by the other three species. Plants attacked by L. elongatus show characteristic root deformations and suppressions of growth.

 

AGAR,  SPECIAL METHODS -- ASM

 

Culbreath, Rodrigues-Kabana & Morgan-Jones, 1984 -- Nematropica 14: 145-154.

An agar-disc method was developed for estimating the level of fungal colonization  of nematode eggs in soil and for isolation of involved fungi. A field study demonstrated a significant level of egg colonization by fungi in a soil with five different fertilizer treatments.  Soils deficient in nitrogen had a higher level of egg colonization than those with adequate nitrogen fertilization.

 

den Ouden, 1958 --Tijdschrift voor Plantenziekten 64: 269-272.

A method for growing plants in thin layers of agar is described. Enough agar is introduced into polyethylene bags under sterile conditions to form a thin, even layer containing numerous air bubbles. Sterile germinated seeds  are inserted through a cut in the bag which is then resealed . Cuts are subsequently made for the shoots to emerge. Sterile nematodes are introduced as is water, from time to time. The method  can be used for the observation of nematodes and other root paprasites while attacking growing roots.

 

den Ouden, 1960 -- Nematologica 5: 255-259.

An improved agar medium for making thin agar layers in polyethylene bags was presented. The medium is prepared by mixing one part of a 1% solution of methylcellulose in water (prepared 12 hours before mixing) with one part of 5% agar dissolved in a double concentration of a suitable nutrient solution. Seedlings are introduced ito the bags and subsequently inoculated with nematodes.

 

Feldmesser, 1967 -- Nematologica 13: 141-142.

Plants were grown on an agar medium contained in Petri dishes the lids of which

contained holes through which the stem grew. Meloidogyne incognita inoculum consisted of 100 to 200 surface-sterilized nematodes. This technique was devised to test the effect of stem- and foliage-applied nematocides on the nematodes in or near the roots .

 

Grewal, 1990 -- Revue de Nematologie 13: 121-122.

The use of agar as a cover-glass support for quick preparation of mounts of nematodes is described. The described agar method removes the need for coverglass supports when the area occupied by the mountant exceeds a quarter of the cover glass area.. The method is simple, quick and inexpensive.

 

Mountain, 1955 -- Proceedings Helminthological Society of Washington  22: 49-52.

A technique of rearing plant parasitic nematodes under aseptic conditions on root tissiue cultures is described. White's culture medium (White, 1973) is used without modification except that the solution is made up into a 0.75% agar medium on which seeds of corn, tobacco and red clver were planted. Once germinated, the root tips were severed and transferred to nutrient agar and inoculated with nematodes.

 

O'Bannon & Taylor, 1968 -- Phytopathology 58: 385.

2-4mm carrot discs  from carrots previously cut, washed, dipped in 95% ethanol then flamed.  were placed on 1% water agar.   Pratylenchus brachyurus or Radopholus similis were pippetted onto the agar beside the discs or first added to the agar then the discs were placed on top.. This proved to be a rapid method of obtaining large  nematode  populations for greenhouse experiments.

 

Tiner, 1961 -- Experimental Parasitology 11: 231-240.

Pratylenchus  penetrans from corn roots are treated 1 hr. with 0.2 mg/ml aqueous mercurochrome and suspended in sterile water. Roots  from agar-flask cultures are placed in special "trap units". Quantitative studies included infection buildup in excised roots, abandonmenmt of damaged roots by the parasites, and toxicologic experiments. 

 

AGAR, SPECIAL PREPARATIONS  -- ASP

 

Chen, Kilpatrick & Rich, 1961 -- Phytopathology 51: 799-800.

Axeniic cultures of Pratylenchus penetrans were established on roots of white-clover seedlings which were growing under flourescent light in tubes on agar containing modified Hoagland's and  Knop's  nutrients. The method provided a pure culture technique for studying damage to the plants caused  by the nematodes alone.

 

Grootaert & Jaques, 1979 -- Nematologica 25: 203-214.

Butlerius degrissei n. sp., a predatory diplogasterid, was maintained in xenic culture on 0.8% bactoagar  using Panagrellus redivivus as its food source. Details on its culture, reproduction, feeding habits, and life-cycle are presented.

 

Grootaert & Maertens, 1976 -- Nematologica 22: 173-181.

  Mononchus aquaticus was cultivated on dishes of 1% bacto-agar using Panagrellus redivivus as its. food source. Optimum temperature for mass cultivation was found to be 22C. The role of the excretory gland during ecdysis was discussed, and information was presented on feeding behaviour, embryology and moulting.

 

 Nigon, 1949 -- Annales Scientifique Naturelle 11: 1-132.

The author used an agar medium containing magnesium sulphate, potassium phosphate sodium chloride, potassium nitrate, peptone, lethicin and water. His studies determined that the agar medium could be used with success for agnotobiotic cultures of  families of non-parasitic soil nematodes.

 

Pillai & Taylor, 1968 --  Nematologica 14: 285-294.

The authors cultured the predacious  Demaniella basili n. sp. on a buffered sucrose-tryptose-agar medium in mixed culture with an intermediate coliform bacterium and the amoeba, Naegleria gruberi. The paper essentially is a population study.

 

Thornton, 1922 -- Annals of Applied Biology 9: 241-274.

The author recommended the use of an asparagine-mannitol agar for culturing the bacterial-feeding nematodes occurring in environments poor in putrifying materials.

 

AGING -- AGE

 

Bollinger & Willett, 1978 -- Nematologica 24: 398-403.

A method for age synchrony of  Panagrellus redivivus in xenic culture is described. Synchronous cultures  are begun with 1-2 day old larvae and resynchronized at 7 days of age and every 3 days thereafter. Synchrony is obtained by separating parents from their progeny, and females from males, on sucrose gradients.

 

Bollinger & Willett, 1981 -- Nematologica 26: 491-493.

In a second paper suggesting a method for synchrony of adult Caenorhabditis elegans, the authors propose a method that uses settling and filtration to obtain synchronized populations of adults in sufficient quantity for life history studies. The synchrony method suggested allows for an adequate number of individuals in each of the adult phases to be obtained for comparison.

                                                                                                          

Giovanola, 1936 -- Journal of  Parasitology  22: 207-218.       

To distinguish glycogen from other polysaccharides, control slides were placed in a solution of filtered saliva in distilled water for an hour @37C. This destroyed all of the glycogen present by the action of glycolitic enzymes.

 

Rogers, 1939 -- Journal of  Helminthology 17: 195-202.

These tests were designed to investigate at regular intervals the activity, infectivity, and fat content of ageing infective juveniles of  Ancylostoma caninum. Two lots of juveniles were stored in jars containing water.  One jar was stored at 37C and one at 7C Each week nematodes from each lot were examined for the presence of glycogen and fat, infectivity and activity.

 

Rogers, 1940 -- Journal of  Helminthology 18: 183-192.

The infectivity, fat content and activity of  Haemonchus contortus juveniles was examined. Forms in which the line of  fat along the intestine was well marked, broad, and in which activity was over 70 moves per minute are regarded comparatively as highly infective. Juveniles in which fat globules were smaller, separated, and round and the nematodes had an activity of 40 moves per minute had low infectivity.

 

ANAESTHETIZING -- ANS 

                                                                                   

Ellenby & Smith, 1964 -- Nematologica 10: 342-343.

Panagrellus redvivus and Globodera rostochiensis juveniles were immobilized within 1/2 hr when placed  in the lower strengths of a 0.5 to 1.0% soln. of  propylene phenoxetol in tap water.

 

Goodey, 1957 -- Technical Bulletin 2, Ministry of Agriculture, Fisheries and Food, 47pp.

Add 50ml of water into a small stoppered bottle and add 2 drops of dichlorethyl-ether. Shake well and allow the contents to clear. Nematodes placed in the solution will become immobilized but will recover quickly when placed in fresh water.

 

Nelson, Albert & Riddle, 1983 -- Journal of  Ultrastructural Research 82: 156-171.

Nematodes quickly became anaesthetized after being immersed in 0.01M concentration of  sodium azide. They were reactivated after rising with fresh water.

 

Noel & Maggenti, 1976 -- Journal of Nematology 8: 271-272.

Nematodes were immobilized by carbon dioxide perfusion.  The specimens were placed in a drop of water on a slide next to the "dry ice" (frozen CO2) beneath an inverted beaker.

 

Peters, 1955 --  in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pg. 417.

Active nematodes can be immobilized by immersion into a solution of bis-chloroethyl ether (25 dps/ 100ml) and killed by a solution of 0.1% iodine solution.

 

Townshend, 1984 -- Journal of Nematology 29: 357-360.

The effects on three nematode species of four concentrations of propylene phenoxetol, as well as a weak solution of dichlorodiethyl ether,  were observed. The immobilized nematodes recovered when placed in fresh water.

 

ANALYSIS -- ANA    

 

Bird & Rogers, 1956 -- Experimental Parasitology 5: 449-457.

The chemical composition of the cuticle of third stage nematode larvae was investigated.

Juveniles of Haemonchus contortus, Trichostrongilus spp, and Nippostrongylius muris

were found to be soluble in boiling water, 0.2 N NaOH, and 10% sodium hypochlorite.

An extensive further analysis was done.

                                                                                  

Deubert & Gray, 1975 -- Nemtologica 20: 365-366.

A new technique which enables workers to quantify chemical components in individual nematodes was reported. Analyses were conducted with a Varian-Techtron AA5R atomic absorption spectrophotometer equipped with a Model 61 carbon rod atomizer with Mini-Massmann type rods. The technique requires two attendents. One analysis could be carried out in 6-8 minutes.

 

Fenwick, 1952 -- Annals of Applied Biology 39: 457-467.

The effect of diluting potato root diffusate was investigated. It was found that hatching ability was inversely proportional to the logarithim of its dilution. There is a "threshold value" of dilution for each sample beyond which it becomes inactive. The slopes of the dilution curves for the diffusates were all parallel. These relationships can be used for determining sample strength.

 

Kaul, 1962 -- Nematologica 8: 288-292.

Studies were conducted on a phenolic complex obtained from the cysts of Heterodera rostochiensis. The material studied, a pigment, was obtained from the cyst walls of the nematode. It was extracted with ethanol and later with carbon tetrachloride to free it from fat and other liupids. It was concluded that the cyst wall contained no tannin group substance and that the cysts contained a hydrostable condensed catechin substance.

 

Marlatt, Morton & McKittrick, 1959 -- Plant Disease Reporter 43: 1073-1077.

The authors sought to determine if a relationship existed between plant parasitic nematodes and cantaloupe (muskmellon) vines suffering from "crown blight", a disease of unknown origin. Cantaloupe vines were planted in greenhouse pots in soil from both diseased and healthy areas. No relationship was found between nematodes which might be parasitic and crown blight

 

Morgan & McAllan, 1962 -- Nematologica 8: 209-215.

A simple viscometer is depicted for analysis of enzymes in nematodes. The viscometer is a capillary tube of 1mm bore with a central reservoir of 1.5ml capacity. The time for the reservoir to empty between two scratch marks is recorded by a stopwatch calibrated in tenths of seconds. The water bath has temperature fluctuation of +/- 0.01C and pumps 10L/min thru a water jacket so as to maintain a constant temperature in the viscometer.

 

Noel & Maggenti, 1976 -- Journal of Nematology 8: 271-272.

The authors proposed a method using vaporphase perfusion of carbon dioxide for the anesthetization of nematodes. The method allows for the starting of greenhouse cultures with large, pure populations of the desired species.

 

Tastet, Bossis Gauthier, Renault & Mugniery, 1999 -- Nematology 1: 301-314.

Studies were conducted determining the variation of total soluble protein extracts from females of Meloidogyne chitwoodi and  M. fallax using two-dimensional gel electrophoresis. The results from a clustering analysis allowed differentiation between both species and showed intraspecific variation among the M. chitwoodi isolates to be equal to that among the M. fallax isolates.

 

Ye & Robinson, 2004 -- Journal of Nematology 36: 207-218.

For a new approach in species identification, a cluster analysis of Longidorus species is suggested.  Cluster analysis dendograms visually illustrate the grouping and morphometric relationships of the species and populations. It provides a computerized statistical approach to assist by helping to identify and distinguish species, by indicating morphometric relationships, and by assisting with new species diagnosis.

 

AXENIZATION -- AXE      

 

Bolla & Jordan, 1982 -- Journal of Nematology 14: 377- 381.

The pine wilt nematode, Bursaphelenchus xylophilus, was cultured axenically in vitro on

soy peptone/yeast extract or modified Caenorhabditis medium supplemented with cholesterol and hemoglobin. This medium was best for nematode growth and reproduction.

.

Buechner & Hansen, 1971 -- Journal of Nematology 3: 199-200.

Mass culture of axenic nematodes using continuous aeration was reported. The nematode species Caenorhabditis elegans, Turbatrix aceti, Panagrellus redivivus, Neoaplectana glaseri, and N. carpocapsae were tested for growth under continuous aeration. Each species went through one or two generations. Counts were up to more than 10 times greater than in control testtube cultures. and increased up to 600-fold over the inoculum.

 

Buecher, Hansen & Myers, 1970 -- Journal of Nematology 2:189-190.

Continuous axenic culture of an Aphelenchoides species is reported. It is suggested that development of large populations of the nematode in a medium composed of commercially available ingredients, and establishment of continuous axenic cultures on glass wool columns will facilitate study of nutritional requirements of the nematode. The culture medium "CbMM".is described.   A table of ingredients is presented also showing population numbers at 3 weeks and at tempersture of 20C aqnd 23C.

                                                                                

Cryan, 1963 -- Journal of Parasitology 49: 351-352.

A method for axenizing large numbers of nematodes involves a procedure in which both larval and adult stages free themselves of debris by migrating through paper and glass beads while the axenizing effect of concentrated antibiotics followed by prolonged holding in dilute antibiotics is achieved automatically without manipulation of the worms.

 

Hansen & Cryan, 1966 -- Nematologica 12: 138-139.

A new method for continuous thin film axenic culture for nematodes involving a minimum of handling results in larger populations and higher proportions of adults than is attained in test tube culture with identical media.The increase in population may be attributable to improved physiological conditions which enhance gas exchange.

 

Hansen & Myers, 1970 -- Journal of Nematology 2: 189-190.

Continuous axenic culture of an Aphelenchoides species is reported. It is suggested that development of large populations of the nematode in a medium composed of commercially available ingredients, and establishment of continuous axenic cultures on glass wool columns will facilitate study of the nutritional requirements of the nematode.

 

Myers, Buechner & Hansen, 1971 -- Journal of Nematology 3: 197-198.

An oligidic medium for axenic culture of  Aphelenchoides  sp is described as containing 3% Bacto Soytone (Difco Laboratories), 2% yeast extract (Nutritional Biochemicals Corp.), and  10% chick embryo extract.(Grand Island Biological Company).

 

Nicholas & McEntegart, 1957 -- Journal of Helminthology 31: 135-144.

A technique for obtaining axenic cultures of rhabditid nematodes is described as two techniques which depend on the killing and superficial sterilization of gravid females with a chemical sterilising agent and their transfer to a medium containing antibiotics.

The young worms hatched from the eggs are collected aseptically. In one method merthiolate is used as a sterilizing agent, in the other hydrogen peroxide is used.

 

Pertel, 1964 -- Nematologica 10: 343.

A crude liver medium capable of supporting the axenic cultivation of Caenorhabditis briggsae and C. elegans was proposed. It is 'Bacto-Liver', a product of Difco Laboratories. The medium will support the growth and reproduction of the two species and is prepared by filtering a 10% suspension of the powdered liver.

 

Riedel, Foster & Mai, 1973 -- Journal of Nematology 5: 71-77.

A simplified medium for monoxenic culture of Pratylenchus penetrans and Ditylenchus dipsaci involves maintaining the nematodes on callus tissue produced with simplified nutrient agar. Sterile onion and alfalfa seedlings were cultured on a described nutrient medium then inoculated with the nematodes and maintained in the dark for 8 to 10weeks.

 

Sanwal, 1959 -- Canadian Journal of Zoology 37: 707-711.

A simple method for rearing pure populations of the foliar nematode, Aphelenchoides ritzemabosi, in the laboratory involes a direct infestation method. A mature female is placed in a droplet of water on the ventral surface of the leaf. Once the nematode enters the leaf tissue reproduction takes place. The resulting progeny are transfered to new leaves and the procedure is continued.

 

Viglierchio, Maggenti & Johnson, 1969 -- Journal of Nematology 1, 76-83.

Axenic ovarial explants from the marine nematode Deontostoma californicum on culture media were prepared. These new techniques were necessary since there were no publications of the application of tissue culture techniques to explant organs or nematodes in vitro. The results indicated that those conditions suitable for adults on culture medium are not necessarily  suitable for eggs, larvae or tissue explants.

 

 

           ----- B ----- 

 

BIOASSAY -- BIO        

                                                                                   

Bird, 1967 --  Nematologica  12: 471-482.

Using sterile juveniles of Meloidogyne javanica bioaassay was conducted for kinin  and gibberellin. Tests were conducted for the presence of amylase, cellulase, and pepsin.

 

Cairns, 1958 -- Proc. S-19 workshop in Phytonematology, 1957. Univ. Tennessee, July 1 - 6, 1957, 196pp, mimeo.

The use of tomato plants as indicators was found to be superior to elutriation of soils by a modification of the Seinhorst technique for the detection of low infestations of root-knot juveniles in winter collection of soils.

 

Carroll, Heyns, Johnson & Todd, 1958 -- Nematologica  3: 154-167.

A series of procedures are described  to further concentrate and purify the potato eelworm (golden nematode) hatching factor. Areas covered in the article are "Bioassay of the hatching factor", "Elimination of salts in the concentrates", "Silica gel chromatography", and "Ion exchange chromatography".

 

d'Herde, Kips & van den Brande, 1956 -- Nematologica 1: 14-19.

The authors propose new techniques (in French) regarding experimentation with potatoes and the potato cyst nematode. Conditions of the soil, the nematocide, plant resistance, and cyst collection are discussed.

 

Dropkin, 1952 -- Journal of Parasitolology 38: 18.

 Dropkin describes the method of using a single juvenile at each generation of root-knot nematode to maintain a pure line of the organism  The unfertilized female produces an egg mass which hatches and individual juveniles are laced singly on marked roots.

 

Dropkin. 1954 -- Phytopathology 44: 43-49.

Infectivity of Meloidogyne larvae to seedling roots is measured by placing a 4mm diam. drop of water containing the larvae on a circular cover glass. A drop of 0.5% agar is added to prevent rapid drying of the larvae.  The coverslip is placed in a Petri drish with the drop uppermost and the tip of the seedling in the drop.  The preparation is incubated for 24 hours.

 

Dropkin, 1959 --  Phytopathology 49: 18-23.

The host-parasite interaction between 19 varieties of soybeans and four Meloidogyne species are presented as a basis for distinguishing  races of root-knot nematodes.

 

Dropkin, Martin and Johnson, 1958 -- Nematologica 3: 115-126.

Having observed that juveniles of Meloidogyne javanica failed to emerge from eggs kept in dilute fertilizer solutions, the authors made a detailed study in  sodium chloride and other salts on concentrations necessary to prevent hatching. The study showed that the response of certain nematode eggs to an environmental factor and  moisture stress

enabled the nematodes to survive long periods of drought in the soil.

 

Duggan, 1958 -- Economic Proceedings of the Royal Dublin Society, Ireland 4: 83-89.

A test for beet root eelworm was devised by growing beet seedlings in glass tubes containing infested soil. Infection caused by one cyst  per 200cc soil can be detected by the formation of new root cysts.

 

Fenwick, 1952 -- Annals of Applied Biology 39: 457-467.

The author proposed, for research involving Heterodera rostochiensis,  that hatching ability is inversely proportional to the logarithim of its dilution. The slopes of dilution curves for different diffusates are all parallel. Use is made of such relationships in estimating the strength of any diffusate sample.

 

Franklin, 1940 -- Journal of Helminthology 18: 63-84.

Three methods were used in an attempt to find a quick means of identifying strains of Heterodera schachtii.  In infection experiments using possible host plants, a strain of the nematode occurring on wild clovers appeared to be different from the pea, oat and beet strains.  A strain parasitic on Myosotis appeared to differ from the clover strain. Juveniles from the potato strain appeared to be stimulated by root secretions from several solanaceous plants. The oat strain forms and those of  Heterodera  punctata were considerably longer than the other strains investigated.

 

Godfrey, 1934 -- Soil Science 38:  3-27.

A method of estimating the degree of infestation is described. Counts of galls on roots of indicator plants are made for cases of  heavy infestations, or the percentage of plants infested is determined when infestations are light.

 

Godfrey & Nolf, 1956 -- Journal of  Parasitology 42: 16.

Specific enzyme systems were studied by the authors using the technique of spectroscopic observation of packed specimens of Trichinella spiralis.

 

Goffart & Heiling, 1962 -- Nematologica 7: 173-176.

Observations on the enzyme content of the saliva of plant parasitic nematodes were investigated. It was found that the nematodes produce amylase, invertase and pectin in the secretion of their salivary glands. It was found that these not only are liberated in living plant cells but also in cell-free substrrates such as oatmeal agar.

 

Hague, 1958 --  Nematologica 3: 149-153.

 A method for the concentration of potato root diffusate  is described. The concentrating procedures are conducted at either 5C or 30C.

 

Jones & Gander, 1962 -- Nematologica 8: 39-50.

In order to obtain cyst batches of equal size, a pippette is fitted with a rubber bulb and plastic measure. Heterodera cysts are placed in a beaker with enough water to form a thick suspension. A vibrator is introduced for proper mixing. The desired amount of suspension is  drawn off and placed on a watch glass together with water.

 

Lloyd, 1946 -- Annual Report for 1946, Agriculture and Horticulture Research Station, University of Bristol, pgs 153-156.

A method was devised for estimating soil populations of  Ditylenchus dipsaci which were infecting red clover plants. Soil samples from the infested areas were placed in boxes and planted to red clover. Estimations on the suitability of the field for growing clover were made on the basis of the symptoms observed on the plants in the boxes.

 

Loewenberg, Sullivan & Schuster, 1960 --  Phytopathology 50: 215-217.

Studies were made determinng the effect of pH and environmental mineral composition on hatching and survival of Meloidogyne incognita incognita juveniles. It was found that Heller's solution at a pH of 6.5 greatly stimulated the emergence and survival of juveniles. 

 

Marlett, Morton & McKittrick, 1959 -- Plant Disease Reporter 43:1073-1077.

A test involving species of  Acobeles, Aphelenchus, Cephalobus, Dorylaimus, Eucephalobus, Pratylenchus, Prismatolaimus, Rhabditis, Trichodorus and Tylenchorhynchus was conducted on cantaloupes in desert soil. The test  included collection from and comparison of  nematode counts from soil and root samples. It was found that both collections generaly gave the same results.  It was  suggested that that  "it might not have been necessary"  to collect nematodes from both.

 

Peters, 1928 -- Journal of Helminthology 6: 1-38.

It was found that the vinegar eelworm, Tubatrix aceti has little need for oxygen. It exhibits a negative geotaxis and it is non-pathogenic to humans. It is sensative to iodine, ammonia and chloroform and it can tolerate temperatures up to 40C.

 

Rau, 1944 -- Planter's Chronicle 39: 347.

The author used indicator plants for determining the presence of root-knot nematodes

in tea nurseries. Tephrosia vogelii, which is highly susceptible to nematode attack proved satisfactory  Soil was brought back from suspect areas in the field to experimetal facilities, placed in "tins" with 12 to 15  Tephrosia seed sown in each tin. This approach has been able to detect very light infestations which the usual soil extraction method failed to reveal.

 

Sasser, 1954 -- Bulletin of the Maryland Agricultural Experiment Station., A-77, 31pp.

The author did an exhaustive study on the species of root-knot nematodes which were  proposed by Chitwood five years earlier. The bulletin lists several approaches to the study of this important group of parasites.

 

Sayre & Mountain, 1959 -- Phytopathology 49:  549.

A method of bioassay is described using symptom expression by onion plants to Ditylenchus dipsaci. Equal numbers of seedlings are grown in a gradient series in muck soil infested with the nematode. After 7 weeks, seedling mortality, bloating and reduced green weight is evaluated.  The most accurate manner of detecting low population levels was by means of the number of seedlings bloated and the numbers of recoverable nematodes.

 

Scheetz, 1966 -- Phytopathology 56: 586.

To demonstrate contrast between resistant and nonresistant roots, 30-day-old infected roots are quick frozen, sectioned in a cryostat, stained with Sudan IV, and photographed. Peripheral lining of lysigenomata is dark orange red in susceptible, and light orange red in resistant roots.

 

Seinhorst, 1957 -- Nematologica 2: 351-361.

The author conducted a series of tests attempting to differentiate between the different races of the stem nematode, Ditylenchus dipsaci. He demonstrated distinct differences

in the reaction of 11 biological races of the nematode to nine plant species. He further investigated the rate of increase of Ditylenchus on different crops growing in a clay soil and a sandly soil.

 

Smith & Ellenby, 1967 -- Nematologica 13: 395-405.

Biochemical changes in the cyst contents of  Heterodera rostochiensis during maturation were investigated by the authors who found considerable amino acid present. It was found that changes in the proportions of the different acids and a decrease in quantity were correlated with the activity of transaminase enzymes. The behavior of yellow and brown pigments, and of related colorless compounds in chromatography, electrophoresis, and other procedures indicates their affinity with tyrosine derivatives.

 

Tracey, 1958 -- Nematologica 3: 179-183.

To determine cellulase activity, suspensions of nematodes are washed then centrifuged resulting in a thick paste of organisms which is ground and made into a thick slurry with water.. This is centrifuged then ground again. Investigation was conducted for Chitinase and Cellulase activity. is then determined. There was evidence for the presence of polygalacturonase.

 

 

BIOCHEMISTRY -- BCM   

 

Bird, 1958 -- Nematologica 3: 205-212.

The chemical composition and structure of the adult female cuticle and egg sac of the genus Meloidogyne was studied. It was found that the egg sac is a tanned glycoprotein and that the adult female cuticle consists of a thin tanned lypoprotein layer. The cuticle of the genus Meloidogyne differs markedly from that of the genus Heterodera in that frozen sections do not show two obvious parts, the exo- and the endo-cuticle.

                                                                                                                 

Giovanola, 1936 -- Journal of  Parasitology  22: 207-218.       

To distinguish glycogen from other polysaccharides, control slides were placed in a solution of filtered saliva in distilled water for an hour @37C. This destroyed all of the glycogen present by the action of glycolitic enzymes.

 

Krusberg, 1960 -- Phytopathology 50:  9-22. 

Concentrated suspensions of nematodes are pelleted by centrifugation and the supernatant is removed by pippette.Most bacteria and soluble substances are removed by resuspension and centrifugation a few seconds at 680u, 8x using distilled water and twice with buffer or sucrose solutions.

 

Krusberg, 1961 -- Nematologica 6: 181-200.

In addition to studying details on the culture and histopathology  of Ditylenchus dipsaci and  Aphelenchoides ritzemabosi, Krusberg conducted extensive biochemical investigations on plant tissue that had been parasitized by nematodes.

 

Lapp & Triantaphyllou, 1969 -- Journal of  Nematology 1: 296.

NA determinations of selected ventral-chord nuclei of some Heteroderidae were made according to the two-wavelength method with a Leitz MPV microscope photometer equipped with a Xenon arc lamp, an interference-graded line filter, and a Photovolt 520-M photometer.

 

Lees, 1951 -- Journal of Helminthology 25: 97-104.

The author conducted a series of tests on Panagrellus silusiae determining the effects of digestive enzymes, temperature and lack of oxygen on the nematode.When fed to mice, it was found to remain alive a short time in the stomach but was killed when passed to the intestine. It also was shown to invade the vagina where it remained for three days causing some irritation to the host.

 

Littrell, 1966 -- Phytopathology 56 : 540-544.

Several methods are presented whereby the composition of giant cells caused by Meloidogyne incognita acrita could be analysed .  

 

Morgan and McAllan, 1962 -- Nematologica  8: 209-215.

Cellulase activity is measured in a viscometer using  0.75% methyl cellulose.  The reaction mixture consists of 4ml of 0.75% methyl cellulose buffered to pH 4.8 with acetate buffer and 0.02ml of the enzyme solution.. Special procedures are described for determining enzymatic activity, concentration of cellulose in nematodes. and pectinase activity.

 

Myers & Krusberg 1965 --  Phytopathology  55: 429-437.

Methods are presented whereby organic substance discharged by nematodes

can be analysed. Microchemical tests on solutions in which Ditylenchus triformis was incubated were positive for amino acids, amines ammonia, proteins, 1,2-diboxylic acids and aldehaydes but negative for primary alphatic amines, formic acid, methanol, alcohols, and a number of other substances.

 

Nicholas, Hansen & Dougherty, 1962 -- Nematologica 8: 129-135.

So as to determine which of the B vitamins are required by the free-living nematode, Caenorhabditis briggsae, it was cultured axenically in a known medium together with chick embryo extract. The concentration of each vitamin added to the medium was varied, and each was omitted in turn.  It was found that the omission of thiamine, riboflavin, folic acid, calcium pantoythenate, niacinimide and pyroxidine was deleterious.

 

Nicholas & Jantunen, 1963 -- Nematologica 9:  332-336.

Caenorhabditis briggsae was cultured axenically in a biotin-free medium supplemented with chick embryo extract. The addition of avidin, which is known to combine with biotin, is an inhibiting factor. The effect of avidin is nullified by excess biotin.It was concluded that biotin is a vitamin required by the nematode.

 

Peters, 1928 -- Journal of  Helminthology 6: 1-38.

In this lengthy article, Peters conducted a thorough investigation of the vinegar eelworm, Turbatrix aceti.  The work on this subject of other authors was also reviewed and new procedures were devised for obtaining additional information. Some of the conclusions arrived at by the author were that the worm has very little need for oxygen and displays a negative geotaxis which is independent of dissolved oxygen as a stimulus. The nematode also is very susceptile to drying, and very sensative to iodine, ammonia, and chloroform, but extraordinarily resistant to most chemical substances. Re the vinegar industry, it was demonstrated to be harmful only when it occurs in the acetifiers, from which it can be eliminated by a suitably high temperature of 40C.

 

Popham & Webster, 1979 -- Nematologica 25: 67-75.

The use of osmium tetroxide-salt mixtures in localizing ions in Caenorhabditis elegans was employed so as to precipitate ions in situ and to further clarify the problem of ion regulation. The results suggested that chloride ion regulation may occur in the hypodermal and intestinal cells rather than in the excretory tubules of the nematode.

 

Robinson, 1986 -- Revue de Nematologie 9: 307.

A versatile method for generating linear gradients of dissolved gases in  semi-solid gels during nematode behavior experiments is proposed. Continuous observations of nematodes and rapid changes in gaseous conditions can be made without physically disturbing nematode suspensions. The technique consisted of suspending nematodes within a 1-mm thick film of agar or agarose within a 2 x 3 x 50mm inside a transparent acrylic chamber. 

.

Rogers, 1940 -- Journal of  Helminthology 18: 183-192.

The infectivity, fat content and activity of  Haemonchus contortus juveniles was examined. Forms in which the line of  fat along the intestine was well marked, broad, and in which activity was over 70 moves per minute are regarded comparatively as highly infective. Juveniles in which fat globules were smaller, separated, and round and the nematodes had an activity of 40 moves per minute had low infectivity.

 

Rubenstine & Owens, 1964 -- Contributions Boyce Thompson Institute 22: 491-502.

The incorporation of tritium-labeled thymidine and uridine into tomato root galls caused by Meloidogyne incognita acrita was studied using microautoradiographic techniques. It was shown that the developing syncytium is a region of intense ribo- and desoxyribonucleic acid biosynthesis. DNA synthesis within a syncytium was found to be dependent upon the close association of a feeding nematode, whereas RNA synthesis once initiated is apparently independent of the nematode.

 

Smith & Ellenby, 1967 -- Nematologica 13: 395-405.

Biochemical changes in the cyst contents of  Heterodera rostochiensis during maturation were investigated by the authors who found considerable amino acid present. It was found that changes in the proportions of the different acids and a decrease in quantity were correlated with the activity of transaminase enzymes. The behavior of yellow and brown pigments, and of related colorless compounds in chromatography, electrophoresis, and other procedures indicates their affinity with tyrosine derivatives.

 

Tracey, 1958 -- Nematologica 3: 179-183.

To determine cellulase activity, suspensions of nematodes are washed then centrifuged resulting in a thick paste of organisms which is ground and made into a thick slurry with water. This is centrifuged then ground again. Investigation was conducted for chitinase and cellulase activity.  There was evidence for the presence of polygalacturonase.

 

 Veech & Endo, 1969 -- Journal of Nematology  1: 265-276.

The sites of alkaline phosphotase, acid phosphotase, esterase, peroxidase, adenosine triphosphatase, and cytochrome oxidase were demonstrated histochemically in fresh sections of  'Lee" soybeans  infected with Meloidogyne incognita acrita by various analytical techniques described in the literature.

 

Wallace, 1956 -- Annals of  Applied  Biology 44: 57-64.

Experiments show that the rate of larval emergence increases with aeration and that emergence did decrease as the oxygen consumption of the soil increased. This implicitly suggests that an increased oxygen supply to cysts will stimulate increased emergence.

 

Webster & Lowe, 1966 -- Parasitology 56: 313-322.

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) was investigated using nematodes in monoxenic culture. It was found that some callous tissue was induced by 2,4-D.  Some plants normally resistant to plant parasitic nematodes lost that resistance. Studies were conducted on Aphelenchoides ritzema-bosi,  Ditylenchus dipsaci, and Heterodera rostochiensis  raised on callous tissue in agar culture. Nematode colonies multiplied the most in callous tissue that grew the quickest.

 

 

          ----- C -----

 

CHROMATOGRAPHIC TECHNIQUES --CHR  

                                                     

 Benton & Myers, 1967 -- Nematologica 12: 495-500.

Separation of proteins from nematode homogenates is made by disc electrophoresis in an acrylamide gel.  A lengthy account is given on reagents and procedures used.

 

Bird & Rogers, 1956 --  Experimental Parasitology 5: 449-457.

The chemical composition of the cuticle of the third stage nematode larvae of  Haemonchus contortus, Trichostrongylus  spp. and Nippostrongylus muris was studied using chromatographic as well as other techniques. The cuticles were found to contain proline, hydroxyproline, aspartic acid, cysteic acid, glutamic acid, alanine, leucine, glycine, and valine.

 

Fife,1954 -- Proceedings of the American Society of Sugar Beet Technologists, General Meeting 3: 207-211.                                                             

By using paper chromatography, striking differences were found in the relative concentration of certain amino acids in the juice expressed from healthy and diseased beet leaves and in he phloem exudate collected from healthy and diseased sugar beet roots. Distinct differences were found between the papergram patterns made from the phloem exudate collected from resistant and susceptible varieties of sugar beet roots and from mangels.

 

Wallace, 1961 -- Nematologica 6: 7-16.

A technique for identifying polyphenols in leaf tissue is presented. About 2 grams of leaf tissue is macerated in methyl alcohol, filtered and the liquid concentrated in a rotary evaporator under vacuum and then stored at O degrees C. The leaf extracts are spotted on chromatograph paper and two-way chromatograms are conducted. After drying, the papers are examined under ultra-violet light.

 

Wong & Mai, 1972 -- Journal of Nematology 4: 237.

An elaborate technique for studying the influence of oxygen on the parasitizing of soil-grown lettuce by Meloidogyne hapla is described. A Buchner funnel with a fine porous plate connected to a water manometer is used.

 

CLASSIFICATION -- CLA

 

Adamson, 1987 -- Canadian Journal of Zoology 65: 1478-1482

In this study, the author examines characters upon which  various versions of the classification are based to identify plesiomorphic and derived character states. These are used to construct a phylogenetic tree using the Wagner algorithm.

 

Thorne, 1949 -- Proceedings of the Helminthological Society of Washington 16: 37-73.

A system of classification of the Tylenchida, new order (Nematoda, Phasmidia) was proposed.  This monumental work presents numerous subclassifications which belong within the new order, as well as descriptions and drawings of nematode belonging to the underlying families and siubfamilies.

 

COLLECTION, BAERMAN FUNNEL -- COF

 

Andersson, 1970 -- Nematologica 16: 222-226.

A method for the separation of Heterodera cysts from organic debris is based on the methods presented by Buhr, 1954 and Seinhorst, 1964.  The cylindrical container and the filter strips used in those methods are replaced by the use of a funnel  and by round filter paper

 

Dikmann, 1936 -- Proc. Helminthological Society Washington 3: 64.

A method for obtaining adults of  Stephanofilaria stilesi involved placing ground pieces of skin showing characteristic lesions in a Baermann funnel containing water or physiologic saline  at a temperature of 48-50C. The liquid is drawn off and centrifuged. The residue contained enbryos, larvae, and adult males of the species.        

 

Hirling, W, 1971 -- Zeitschrift fuer Pflanzenkrankheiten und Pflanzenschutz 78:335-348.

A simple method of isolating nematodes from plant roots is to fill the Baermann funnel in which the roots are immersed with diluted hydrogen peroxide instead of just water.  This technique is regarded to be very efficient and time-saving as compared to conventional methods.

 

Hohmaier & Meyer, 1937 -- Science 86: 568.

The authors suggested for the collection of nematodes from material in water within a Baermann funnel that the funnel stem could be connected directly to collection vials or to centrifuge tubes into which the nematodes settle.

 

Meyer & Nelson, 1971 -- Plant Disease Reporter 55: 899-900.

An improved method for concentrating nematodes from the Baermann funnel is a modification of an earlier method reported by Zuckerman, 1960. The method of concentrating the nematodes also may be used to collect living nematodes prior to gathering specific species from a random sample.

 

Shorb, 1936 -- Proceedings of the Helminthological Society of Washington 4: 52.

A method of separating infective larvae of  Haemonchus contortus (Trichostrongyllidae) from free-living nematodes is described.  Mixed suspensions of the nematode and other free-living nematodes recovered from soil by the Baermann funnel method were treated by adding concentrated hydrochloric acid to the suspension of larvae so that the final dilutions varied from 1 : 2 to 1 : 300.

 

Taylor, 1968 -- Nematologica 14: 596.

A  multiple Baermann funnel rack which accomodates 36 funnels and is constructed from standard fittings is described and illustrated. The frame of the rack is made from two 'Gondola Units' and two 90 cm spacer bars. The unit has three 18cm shelves, with support brackets, and three support bars are attached at suitable intevals to each side of the frame

 

Trudgill, 1967 -- Nematologica 13: 263-272.

In a paper dealing with the effect of environment on sex determination in Heterodera  rostochiensis, the author uses an apparatus for collecting males that is a 7-inch Baermann funnel with rubber tubing closed off with a pinch clamp. The funnel is nestled in a plant pot and is filled with nutrient solution. A plastic cover lays over the top of the pot and a potato plant with roots in the nutrient solution is held in place by the plastic cover A tube acting as an aerator is also inserted through the cover into the nutrient solution

 

van Volkenberg, 1936 -- Proc. Helminthological Society Washington 3: 65.

A method for recovering the strongyle larvae of the horse is presented.  The fresh manure in the form of balls is collected. Within 4 days, ensheathed, infective larvae are present. The balls are broken up and placed in a Baermann funnel. By changing the manure in the funnel 4 times at 3-hour intervals,  approximately 9000 larvae have been recovered from the manure of a heavily infested animal.

 

COLLECTION, MISCELLANEOUS -- COL 

 

Alam,  1975 -- Nematologica 21: 264-266.

A simple device is reported for the rapid selection of nematodes from a mixed population in aqueous suspension. The accompanying illustration shows a filter funnel connected to a separating funnel, both of which are connected to rubber tubing with a rubber bulb. The tubing terminates in a glass jet equipped with a bulb. This apparatus is supported by a retort stand.

 

Buecher, Yarwood & Hansen, 1974 -- Nematologica 19: 565-566.

A screen to separate young larvae of  Aphelenchus avenae from adults and eggs was developed to aid in the development of a simple method for providing a continual supply of newly-hatched larvae.

                                   

.Christie, 1937 -- Journal of Agricultural Research 55: 353-364.

In working with Mermis subnigrescens, a nematode parasite of grasshoppers, a simple and practical method of obtaining the parasite in large numbers was to collect infected grasshoppers and confine them in cages until the parasites emerge.To facilitate the collection process, wheat was planted and allowed to grow a few inches high before being placed in the cage and the collected grasshoppers put into the cage.

 

Cobb, 1918 -- Estimating the nema population of soil.Agricult. Tech.Circular 1, pp 1-48.

A soil-sampling tube for soil-census work is described. It is made of tin or galvanized iron, 2.84 inches internal diameter, with the rim of one end reinforced and the other end sharpened. The area of the internal cross-section is one-millionth of an acre. The tube is forced into the soil using one's foot.

 

Cobb, 1929 -- Contributions to a Science of Nematology XXIX:, pp 411-412.

An initial stratigraphic survey of nemas in the upper 20mm of marine beach sand, near the low tide mark was conducted. Illustrations accompany the aricle showing two types of novel apparatus constructed for conducting such a survey.

 

Cooper, 1955 -- in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pp 269-280.

A mechanical device for concentrating  Heterodera cysts is described. An electrically operated vibrator separates the cysts from soil debris obtained by floatation in water. The device is well illustrated and described. They state that the device has already reduced time and drudgery in several operations and claim that by eliminating the personal factor it may add to the accuracy of laboratory soil examination.

 

Cuany & Rodolphe, 1980 -- Revue de Nematologie 3: 37-50.

A procedure for collecting and interpreting data from field experiments with nematocides is described. The first discussed is a procedure to record, qualitative variables (plant vigor, gall index). The second part of the paper is a statistical approach. The techniques involved are the non-parametric and newly developed multivariate statistical methods such as factor analysis, discriminant analysis, Friedman's variance analysis and rank correlations.

 

Fenwick & Mohammed, 1967 -- Nematologica 13: 467-468.

A refinement to the Peters Screening technique (cf  Peter's, 1952) is proposed which makes use of the nematode's negative geotaxis for cleaning.  The equipment proposed allows living worms  migrating upwards and congregating upwards to be pippetted off for experimental use. 

 

Green & Hornsey, 1983 -- Nematologica 29: 362-365.

Apparatus for collecting and surface sterilizing nematodes and similar small aquatic organisms is described,  illustrated, and the opening statement suggests that the equipment described can be bought or made with a drill and glass-working blow-lamp. Three drawings illustrate the necessary components of the process.involving collection of the nematodes.

 

Hussey, 1971 -- Journal of Nematology 3: 99-100.

A macerating technique for obtaining quantities of living Meloidogyne females is reported.which sufficient quantities of cleans adult female nematodes for biochemical investigations. Cleaned infected roots were cut into 2.5cm sections and macerated in a 50% solution of Pectinol 59L and shaken at 180 ocillations/min for approximately ten hours at 25C. Collected root debris and females were suspended in 20% sucrose solution and centrifuged 2 1000g for 10 minutes.

 

Lapage, 1933 -- Nature 3310: 583-584.

In an article titled "Cultivation of Nematodes", the author outlines a method for obtaining large numbers of nematode larvae parasitic in sheep in media for which the composition can be accurately controlled. Ten "steps" are presented for conducting the method but the author finally honestly alludes to "innumerable problems which have arisen during conduction of the proposed method .

 

Pableo, 1981 -- Nematologica 27:  242-243.

A method which results in the rapid separation of female Meloidogyne from root debris suspension involved macerating infested roots with enzymes and  then placing them in commercial sucrose solution. Collected females again were centrifuged on a 10-50% sucrose gradient at 5,000 rpm for 15 min in an ultracentrifuge. with a swinging bucket rotor at 4 degrees.

 

Raski, 1953 -- Phytopathology 43:259- 263. 

Methods of detecting and investigating plant parasitic nematodes are presented .The procedure of making mass collections of unmounted nematodes is also referred to.

 

Scognamiglio, 1963 -- Att.delle giornate fitopatologiche for 1963, pp 201-209.

A new technique for collecting randomized soil samples in nematological research suggests that more precise results would result by obtaining 3 to 4 samplings before treatment, and as many samples after treatment. The data obtained would provide a much more reliable index

than data obtained from a single sampling.

 

Stekhoven, Berends & Soelistio, 1954. -- Mitteilungen aus der Biologischen Zentralanst                                                                                            fhr Land- und Forstwirtschaft.  Berlin-Dahlem  83: 128-129.

A new method is offered which allows living nematodes to be easily separated from dead nematodes. In addition, the authors suggest that they can catch a large number of nematodes in a small dish within a very short period of time. Details of the apparatus used are presented as well as the information that they were able to accumulate about 200 specimens of Ditylenchus dipsaci divided into 8 dishes within 10 minutes.

 

Tiner, 1960 -- Experimental Parasitology 11: 231-240.

A trap for the collection of axenic nematodes is described as being constructed of ground-joint glassware, sterilizable plastics  and hypodermic needles plus their tubing adaptors. The author used the plant-parasitic nematode, Pratylenchus penetrans, obtained from infection-damaged corn roots as test organisms. The nematodes were treated one hour with 0.2 mg/ml aqueous mercurochrome, and suspended in sterile water.

 

Vrain, 1977 -- Journal of Nematology 9: 249-251.

A technique for the collection of larvae and eggs of Meloidogyne spp. was described using heavily galled roots of tomato. Mist chambers and treatment with 0.53% sodium hypochlorite and use of four nested metallic sieves were employed to obtain the necessary hatch data.

 

COLLECTION -- SIEVING, SUCTION, SIPHON -- COM

 

Caveness, 1975 -- Nematropica 5: 30-32.

A simple siphon method for separating nematodes from excess water is to concentrate nematodes. The collected nematode-water mixture is allowed to settle in a wash bottle for 5 or more hours. Rubber tubing forms a siphon and excess water is emptied leaving a nematode concentrate of  10 to 45 ml.

 

Ford, 1957 -- Plant Disease Reporter 41: 89-90.

A source of controlled vacuum for pipetting nematodes is described. Drawings of an apparatus which applies a source of constant controllable suction are presented  Suction within a closed system is obtained by a descending column of water. The suctioning effect is transmitted thru rubber tubing to which a finely drawn pippette is attached. Nematodes are thus sucked into the orifice of the pippette and subsequently released into a container.

 

Guentzel, 1981 -- Nematologica 27: 246-248.

A rapid and efficient method for concentrating  nematodes from a suspension is described and illustrated.   It uses the combination of a nylon mesh with a membrane filter. The drawing depicts a supported funnel, over nylon mesh, over a membrane filter, then a steel mesh, a safety collar of plastic fixed to a brass tube and finally a rubber bung to fit on a suction flask.

 

Hafkensheid, 1971 -- Nematropica 17: 535-541.

During virus transmission tests with Trichodorus pachydermus, many specimens showed little or no motility, therefore the extraction method was modified. A method was developed preventing the nematodes from coming in contact with copper ions which were found to be the toxic principle. A sieve made of plastic was placed in 75% Hoaglands solution, a combination that yielded the most nematodes and the highest percentage of active nematodes after an extraction period of 2.5 days.

 

Hussey & Barker, 1973 -- Plant Disease Reporter 57: 1025-1028.

Methods of collecting inocula of  Meloidogne spp.were reported upon, including a n ew technique where sodium hypochlorite  at 0.53% or 1.05% was used. Nematode-infected tomato roots were shaken in the solution for four minutes and the freed eggs were collected by sieving. Then the chemical was used as a  2.65% solution, , it had a detrimental effect on larval infectivity when assessed  for reproduction.

 

Juhl, 1973 -- Nematologica 19,  399-400.

A new suction trap with hand aspirator for collecting cysts and vermiform nematodes is described and figured. The device is made of perspex and consistes of a suction trap and a hand aspirator. Up to 97% of Heterodera avenae larvae that were caught could be recoverered on the nylon net of a perspex ring.

 

Jutras & Tarjan, 1964 -- Proceedings Soil & Crop Science Soc.of  Florida 24:154-158.                                                                 

A vibrating, translocatable sieve mounted on a vehicle and positioned directly below a soil auger was developed for the collection of citrus root samples  Operating power is supplied by a hydraulic pump on the vehicle. The auger is lowered into the soil while rotating, then raised. The vibrating sieve is positioned directly below and catches the soil and roots shaken off the auger. The soil passes through while roots are retained on the sieve.

 

Madamba, 1960 -- Phillipine Agriculturalist 54: 146-148.

Techniques for collecting soil nematodes are modified so as to gain their advantages and eliminate some of their drawbacks in processing Phillipine soils. Sieving and collection of nematodes by use of one sieve which will trap adults of the nematode parasites is described. The method eliminates the possibility of large scale nematode escape.

 

Minteer, 1965 -- Nematologica 11: 644.

A device for rapid selection of nematodes from aqueous suspension is described and illustrated. Collecting rates of  100 nematodes in four minutes are common from suspensions> 25/ml of the desired species. Operation of the device depends upon a pressure drop produced by cooling a gently heated copper tube which induces a flow of water (and nematodes) into the tip of an attached pippette and attached hollow stainless

steel  needle.

 

Shukla, Nath & Swarup, 1971 -- Indian Journal of Nematology 1: 87-88.

A modification of  Minteer's device for rapid selection of large numbers of nematodes and  eggs from aqueous suspensions is proposed.  The modification proposed primarily consists

of rubber tubing and a rubber bulb and it is illustrated.

 

Staniland, 1954 -- Journal of Helminthology 28: 115-117.

A length of glass tubing drawn out to a fine point and flame sealed was inserted into rubber tubing  at the end of a funnel stem. Nematodes collected at the tip could be drawn off in a small amount of water by breaking the glass tip which then could be resealed.

 

Thorne, 1940 --  Proceedings Helminthological Society Washington 7: 53-54.

Three methods for clearing screen residue in separating nematodes from soil are presented.

Residues from finer screens used for separating nematodes from the soil contain suspended material that impedes clear vision while using the microscope.: The use of three substances which eliminate this difficulty are the sap of cactus, Opuntia sp., saliva, and fresh or condensed milk.

 

Varma, 1979 -- Indian Journal of Nematology 7: 94-95.

A device for the rapid collection of nematodes from aqueous suspensions is described and depicted. The device employs a used "Dextrose Normal Saline Intrafix set" which is a hospital waste mateial. The device operates by a suction created from the user's mouth which is regulated by movement of a knob. A nylon bristle at the end of the apparatus in the dish flicks up the desired nematode which is then sucked into the collecting apparatus.

 

 

CONTROL CHEMICAL, USING  GASES -- CONG

 

Byars, 1919 -- Phytopathology 9: 93- 106.  

Hydrocyanic-acid gas as liberated in loam soil by the application of sodium cyanide and ammonium sulphate failed to eradicate the root-knot nematode from the soil, even when applied at the rate of 3000 and 5400 lbs/acre respectively. No infection was found, however, at the end of the first season on dasheen plants grown in the treated plots.

 

Fenwick, 1942 --- Journal of Helminthology 20: 41-50.  

"In vitro" experiments showed that sulphur dioxide is very lethal to the cysts of the potato root eelworm, especially when cysts are moist instead of dry. It was found that fumigation of seed potatoes for 24 hrs resulted in complete destruction of the adhering cysts.  Treatment had no visible effect on the potato tubers.

 

Vassallo, 1967 -- Nematologica 13: 155.

The nematocidal power of ammonia is derived from its capacity to generate high osmotic pressures, even in low concentrations. The technique was reputed to make it possible to calculate mathematically the exact point at which the physical effects of osmotic pressure became lethal to the nematodes.

 

CONTROL CHEMICAL, USING  LIQUIDS -- CONL

 

Bijloo, 1966 -- Nematologica 11: 643-644.  

Pea seedlings were inoculated with (1) untreated nematodes, (2) nematodes in a 1000ppm solution of physostigminesulphate,  and (3) nematodes treated with tap-water after having been immersed in a 1000ppm solution for 24 hrs. at about 20C. The seedlings were inoculated with 50 Ditylenchus dipsaci between the cotyledons and the growing point. Plants grown from pea seeds which had been soaked in the chemical solution for 24 hrs and were ioculated with the nematodes did not develop symptoms of attack.

 

Brashears & Orr, 1975 -- Journal of Nematology 7: 315-316.  

An improved 4-row liquid nematocide applicator for research plots was designed and constructed.  Its advantages are: (1) the operator isn't exposed to chemicals being applied, (2) changes of chemicals and rates are quickly accomplished, and (3) the metering device provides a more accurate application rate.

 

Chitwood & Buhrer, 1945 -- Proceedings Helminthological Soc. Washington 12:39-41.  LA series of tests using various nematocides to control Heterodera rostochiensis were conducted on Long Island, New York.  It was concluded that a heavy dose of D-D at the point of application , combined with wide spacing, caused injury to some plants the following Spring. A lighter dose at the point of application, combined with closer spacing, gave no injury and better nematode kill.

 

d'Herde & van den Brande, 1959 --Mededelingen van de Landbouwhogeschool en de Opzoekingsstations van de Staat te Gent,  Belgium.24:637-644. (in Flemish)

A new applicator for soil fumigants allows a correct distribution of very low to normal doses of fumigants at the desired depth of soil. The mechanism by which rate of flow is controlled is of simple construction. Flow rate is not altered due to speed modification  Two prototypes are illustrated

 

Goberdhan, 1964 -- Nematologica 9; 353-360.  

 To protect coconut roots against infection by Rhadinaphelenchus cocophilus, a 1/1000 dilution of "Nemafos" 0,0-diethyl 0-2 pyrazinyl phosphorothioate was injected into coconut roots Exposure to treatment for six days appeared to be the minimum time required for treatment. six trees after one treatment and one subsequent exposure to infection did not contract the disease, while six other trees similarly infected without treatment showed typical symptoms of  red ring disease within  9 to 20 weeks.

 

.Gurner, Dube & Fisher, 1980 -- Nematologica 26: 448-454.  

Chemical control of the cereal cyst nematode, Heterodera avenae, on wheat was accomplished by using a new low-volume applicator. The applicator uses microtubes which allows the accurate placement in soil of very small volumes of nematocides or other liquids. When injecting ethylene dibromide to wheat for cereal cyst nematode control, the initial cost of the  applicator and the chemical was recovered from the increased yield from two hectares.

 

Hague & Omidvar, 1962 -- Nematologica 7: 219-230.  

Tests with Heterodera rostochiensis cysts were based on hatch in root diffusate, larval invasion of roots and final cyst population and were used to determine the efficacy of fumigants against the nematode.  The final cyst population gave more reliable results than either the hatcing test or larval invasion.

 

Nicholls, 1984 -- Nematropica 30: 244-247.   

A hand-operated sprayer for applying nematicides around trees is descried and partially illustrated.  Whereas two or more applications of material usually are necessary during a single treatment, this novel applicator with semicircular spray head allows for treatment of a tree with a single insertion under the canopy. Labor is saved  because the spray-head is inserted only once under each tree canopy and there is no formation of basins or furrows.

 

O'Bannon, 1958 -- Plant Disease Reporter 42: 857-860.    

Application of emulsifiable dibromochloropropane in irrigation water as a preplanting soil treatment on nematode-infested cotton production soils. Distribution and penetration were evaluated by use of the onion test for detecting bromine and found to be reasonably uniform over the rows to a depth of 12 inches.

 

O'Bannon & Bistline, 1969 -- Plant Disease Reporter 53:  799-802.  

A simple device for injecting methyl bromide into a replant site was developed and tested. A hollow probe was injected into the soil to depths of 1 to 4 feet and methyl bromide in 1-pound cans  was released into the site. Citrus nematodes, Tylenchulus

semipenetrans were controlled effectively at each of the depths.

 

Oteifa, Shafiee & Eissa, 1965 -- Plant Disease Reporter 49: 598-599. 

The efficacy od DBCP (dibromochloropropane) flood irrigation in established citrus groves in Egypt was reported  The soils type was a light sand containing 78% sand, 15% gravel and 7% clay, with a moisture-holding capacity of  about 18%.Treated trees showed a remarkable improvement and yield was increased almost two-fold.

 

Ruehm, 1962 --Zeitschrift fuer Planzenkrankheiten und Pflanzenschutz 69: 278-283.   

Haltox (98% methylbromide and 2% trichlornitromethane) was successfully used against parasitic nematodes in forestry nurseries.  It was pointed out that animals at a depth of 25-30cm might not be affected by the chemical.

 

Storey, 1982 --Annals of Applied Biology 101: 93-98. 

The author reported on the ATP method for rapid assessment of the efficacy of a single application of a fumigant against  Globodera spp. in field soils. The method involved population estimates by  both visual egg counts and measurement of the adenosine triphosphate (ATP) content of cysts from 26 fields  There was some disagreement in results from the two methods but they did agree for seven non-fumigated fields and that three fields were safe for the cropping of potates.

 

 Tarjan, 1954 -- Phytopathology 44: 431-432.  

Aqueous emulsions of 3-p-chlorophenyl-5-methyl rhodanine (2g/sq.ft.) were effective in controlling Meloidogye incognita infecting roots of  potted tomato plants. Plants failed to become infected when transplanted into nematode-infested soil when the powdered chemical had been mixed in in rates as low as 0.5g per sq. ft.of surface area.

 

Taylor & O'Bannon, 1968 -- Plant Disease Reporter 52: 218-222.  

Experiments with an apparatus for subsurface application of nematocides to nursery plants in containers were reported. The equipment used was a 50-gallon  drum which served as a tank, a pump connected to a 1/3 horsepower motor, and probes made from 1/8 or 1/4 inch iron pipe. Excellent control of nematodes was obtained  when nematocides were applied with the described equipment..

 

CONTROL CHEMICAL,. MISCELANEOUS -- CONM

 

Chitwood, 1939 -- Proceedings Helminthological Society Washington 6: 66-70.  

A rapid method for determining the "k" values of nematocides suggests the I-test (horizontal) is of value in selecting chemicals  and determining dosage rates, but not for the determination of row and hole spacing in plot or field.

 

Chitwood, 1939 -- Proceedings Helminthological Society Washington 6: 70-72

Under the topic of  "Frames for spacing injections of soil nematocides", three types of frames applicable to greenhouse beds of various sizes are described. The description of each of the three is accompanied by drawings and dimensions.

 

Grainger, 1958 -- West of Scotland Agric. College Research Bull. 25, 46pp

A new production prototype soil disease control unit was constructed which was found to give good contro in the field of five soil-borne diseases, among which were those caused by the potato root eelworm and club root of turnips.  The paper is abundantly supplied with photographs and drawings.

 

Grainger, 1962 -- Horticultural Research 2: 57-58

It became necessary to develop a machine which would adequately mix non-volatile chemicals with the soil and which could be used to apply inorganic compounds of mercury to control soil-borne diseases. A machine was ultimately developed (see Grainger, 1958) from which a minimum volume of 6 cu. ft. of dust per acre, containing 2-1/2 lbs. of inorganic merciry, was delivered evenly in two layers for mixing.

 

Jensen & Page, 1954 -- Plant Disease Reporter 38: 401.  

An experimental blade-type soil fumigant applicator is illustrated and described.The applicator was designed to apply the chemical in a contnuous sheet at the 6- or 7-inch injection depth.

 

Johnson & Lear, 1962 -- Plant Disease Reporter 46: 742-743.

A method for assaying nematocidal activity of experimental chemicals is to mix nematode infested soil and experimental chemicals in polyethylene bags. This has proved to be a safe and efficient method  of dispersing candidate materials throughout a soil mass..

 

O'Bannon & Good, 1971 -- Journal of Nematology 3: 93-94.   

Applications of microwave energy were tested for controlling the root-knot nematode Meloidogyne incognita in potting soil. The treatment substantially reduced root-knot nematodes at an exposure of 15 seconds.  Based on gall formation, the nematode was eliminated at all the longer exposure periods. A 300-second exposure resulted in complete drying of the soil.

 

Overman, 1978 -- Nematropica 8: 19-20.   

Foliar sprays of  oxamyl at insecticidal rates was shown to control nematodes. Nematode control and yield response of celery, as well as to bell pepper anMd tomato, to various treatments involving oxamyl were reported  Population assays taken at harvest indicated that numbers of phytoparasitic nematodes were reduced by all treatments on all crops, except for the application of oxamyl in transplant water to pepper.

 

Tarjan, 1959 -- Plant Disease Reporter 43: 451-458.  

Pressure injection of chemicals for possible systemic action against burrowing nematodes infecting citrus was proposed using  air pressure to force aqueous nematocidal chemicals into citrus tree trunks for controlling the nematodes. Some of the materilas tested showed promise as nematocides but the duration of control appears to vary with fluctuations in nematode population during the sampling period.

 

Tarjan & Cheo, 1956 -- Misc. Publ. 47, Univ. Rhode Island Agr Exp Sta., pp. 3-26.  

A series of six tests were proposed for evaluating the nematocidal efficacy of candidate nematocides. These were a contact test, an ovicide test, a therapy test involving the foliar spray method, a therapy test involving the soil amendment method, a therapy test involving the stem injection method, and a nematode repellency test. Extensive data is presented on the performance of the large number of chemical compounds investigated.

 

Thorne, 1951 -- Proceedings of the Helminthological Society of Washington 18: 18-24. M

Diffusian patterns of soil fumigants were studied by Thorne so as to determine such patterns when applied with chisel type commercial machines or as single injections with hand applicators.

 

 

CONTROL CHEMICAL, USING SOLIDS -- CONS

 

Grainger, 1956 -- Nematologica 1: 31-46.  

Mercury compounds when used in pot experiments at rates as low as 5.4 lbs mercury equivalent per acre have resulted in over 80% control of infestation  by Heterodera rostochiensis which is the case when potatoes are grown on untreated infested ground  The performance of dusts appeared to be inversely related to the particle size of the active agent

 

McLeod, 1973 -- Nematologica 19; 236-247.   S

Thiabendazole at 2.2 to 20 ppm and benomyl at 5-20 ppm effectively inhibited the multiplication of Aphelenchoides composticola and Ditylenchus myceliophagus  on  Agaricus bisporus. for over three weeks and prevented degeneration of the mycelium.

 

Miles, 1930 -- Journal of Helminthology 8: 103-122.   S

In experimentation involving Heteradera schachtii on potatoes,, the author found that, for the year of 1929,  there were no appreciable effects from the use of crude or f lake naphthalene, and bleaching powder. The growth of potato plants was stimulated by calcium cyanide.

 

Tarjan, 1972 -- Plant Disease Reporter 56: 626-627.

The nematocide 'Nemacur' (ethyl-4-methylthio-m-tolyl-isopropylphosphoramidate),also known as BAY 68138, was prepared in a paste formulation and applied to stems of

Citrus jambhiri seedlings infected with Pratylenchus coffeae.  Reduction of root nematode populations in treated plants proved the movement of the chemical, or its breakdown products from the stems to the roots.

 

CONTROL, NON-CHEMICAL -- CNC    

 

Byars & Gilbert, 1919 -- Phytopathology 9: 49.

The possibility of using hot water as a means of disinfecting  small quantities of soil infested with the root-knot nematode and with fungi causing "damping-off" was investigated.  It was shown that immersion of 4-inch pots in boiling water for 5 min. killed all the organisms concerned. Similar results were obtained treating largers pots and infested soil as well.

 

Chantanao & Jensen, 1969 -- Journal of Nematology 1: 277-278.

The authors, working with Pristionchus lheritieri used a combination of techniques involving surface sterilization and control and exchange of bacterial flora. A test for bacterial contamination was conducted after treatment and the authors felt that such contamination will remain a serious obstacle in interelationship studies involving nematodes and other organisms..

 

Crow, Yu & Chiba, 2004 -- Journal of Nematology 36: 313.

Mustard Bran is proposed as a biochemical nematocide for turfgrasses. The bran derived from oriental mustard, Brassica juncea releases the nematocide allyl-isothiocyanate upon contact with water.Numerous experiments conducted for 4 years evaluated the effectiveness of mustard bran for management of Hoplolaimus galeatus and Belonolaimus longicaudatus

on turfgrasses.

                                                          

Crump, 1987 -- Nematologica 33: 232-243.

A method is presented for assessing the natural control of beet cyst nematode populations and for isolating the fungal parasites involved. Triangular-shaped observation chambers with removable sides are used for the making of continual assessments of the numbers of females and developing new cysts on sugar beet roots on two sides of the chamber.

 

Gommers, 1973 -- Mededelingen Landbouwhogeschool, Wageningen 73-17, 71pp.

A wide range of  Compositae were tested for their nematocidal properties using Pratylenchus penetrans as the test organism.  Suppressing effects on the populations of the nematode were found within 14 of the plant genera investigated. Further investigations were carried out on extracts from Tagetes patula.

 

Paracer, Tarjan & Hodgson, 1987 -- Journal of Nematology 19: 194-200.

When used as soil amendments, extracts from the marine algae Spatoglossum schroederi, Botryocladia occidentalis & Bryothamnion triquestrum at 0.5 to 1.0% concentrations significantly reduced root galling of tomato caused by Meloidogyne arenaria, M. incognita, and M. javanica.

 

Pena, Schroeder & Osborne, 1990 -- Nematropica 20: 51-55.

 Entomogenous nematodes of the families Heterorhabditidae and Steinernematidae were used to control the banana moth, Opogona sachari.  The entomogenous nematodes Steinernema feltiae, Heterorhabditis bacteriophora, and H. heliothidis were applied to the larvae of the banana moth infesting potato and bamboo palms. The nematodes were successfully established with a subsequent 58-100% of larval numbers of the moth.

 

Renn & Wright, 2000 -- Nematology 2 :217-222.

The authors developed a way of using a rhabditid, Steinernema feltiae, in a bait system to control the common house fly, Muscsa domestica.

 

Rodriguez-Kabana, Hoveland & Haaland, 1977 -- Journal of  Nematology 9: 323-326.

Evaluation of a seed-treatment method with acetone for delivering systemic nematocides with wheat and rye reports results on the efficacy of oxamyl, carbofuran, and phenamiphos in acetone solutions as seed treatment for control of certain plant parasitic nematodes. The nematode species involved were Hoplolaimus galeatus and Tylenchorhynchus claytoni

 

Rodriguez-Kabana, Morgan-Jones, Godoy & Gintis, 1984 -- Nematologica 14: 155-170.

The potential of several species and isolates of  Gliocladium, Paecilomyces and Verticilium  grown on autoclaved oat kernels, for use as biochemical agents against Meloidogyne arenaria was evaluated in two greenhouse experiments.The results of the tests indicated that the effectiveness of fungi for control of M. arenaria depends upon the species and individual isolates within a species.

 

Scotto la Massese, Vassy & Zaouchi -- Nematologica Meditteranea 1: 15-20.

Elimination of Tylenchulus semipenetrans by nitrogen fertilization in a Clementine orchard was attempted in an Algerian citrus orchard. It is estimated that the nematode infected about 95% of the orchards and caused losses that can reduce the value of the crop by 50%.  Nitrogen fertilization of 300g/N/tree each year for each year of age resulted in the elimination of the parasite affecting 10-year-old trees. The publication is written in French.

 

Tarjan, 1977 --  Nematropica 7: 53-56.

Application of municipal solid waste compost to nematode-infected citrus was an attempt to put to good use solid waste compost, otherwise known as "garbage". The material applied  was "supersoil", a product made of thoroughly digested and pulverized municipal waste. Results indicated a slight beneficial effect as measured by growth response, plsnt appearance and fruit yield

 

Tarjan, 1977 -- Journal of Nematology 9: 287.

Kelp derivatives for nematode-infected citrus trees were a kelp meal applied alone or with 1,2 dibromo-3-dichloropropane to the soil and a  water-soluble kelp extract applied as a foliar spray. It was concluded that the materials were beneficial and even mildly nematocidal.

 

COUNTING  EQUIPMENT -- CNS 

                                                                                   

Anderson & Yanagihara, 1955 -- Phytopathology 45: 238-239.

The lower side of a microscope slide was scribed to give squares of a size so that a single square could be seen in the field of the dissecting microscope. The top 3/8 inch of a liquor class was then cut off and cemented immediately above the scribed area. This formed a counting dish of good optical characteristics and provided a means of estimating rapidly the number of nematodes.

 

Baxter, 1968 -- Nematologica 14: 599-600.

An intermittent or continuous electromagnetic counter for use in nematology which enables rapid counting is described and diagrammed. Single intermittent counts may be recorded by depressing a button briefly for each count. A convenienient setting for the counter was suggested to be about five counts per second.

 

Cobb, 1918 -- Estimating the nema population of soil. Agricul. Tech. Circular 1, pp 1-48.

(1) An ordinary Syracuse watch glass is dipped in hot wax, set to drain and cool so that the thinnest layer of wax will be on the inside bottom of the glass. Concentric circles and radiating lines  are scratched on the bottom, the exposed glass lines are etched with hydrofluoric acid and the resulting grooves filled with white lead.

(2)A counting eyepiece with a diaphram having a square aperture for use in a compound microscope is proposed.  The square aperture should be divided into 9 or even 25 smaller squares for aiding observation. To aid observation and counting, the mechanical stage should be movable in two directions.

 

De Grisse, 1963 -- Nematologica 9: 162.

Since counting dishes usually have a vertical wall with the disadvantage that nematodes lying close to the wall are difficult to recognize, the author proposed use of a dish with a sloping wall..Dimensions of two types of such dishes are presented.

 

Doncaster, 1962 -- Nematologica  7: 334-336.

A counting dish is described and illustrated which is open, circular and has ten flat-bottomed concentric channels, each of which is separated by narrow, rounded ridges. The distribution of the nematodes falling between the ridges confirms to Poisson's Law, the accuracy of the count being dependent on the number counted.

 

Doncaster, Edwards & Shepherd, 1966 -- Nematolgica 12: 644.

A labor-saving method for making Fenwick multi-chamber counting slides for nematode counts is presented. Specific instructions and measurements are meticulously presented. The written account is accompanied by two illustrations, one showing the two alternative presses for bonding the slides simultaneously and the other a diagram showing the contruction of the counting slide.

 

Evans & Forder, 1976 -- Nematologica 22: 475-476.

An automatic counter was designed which could be operated by either a hand or a foot switch for either intermittent or automatic counting. The short article is accompanied by two circuit diagrams of the power supply and the circuit, and a diagram of the simple  intermittent or continuous counter and foot switch.

 

Fenwick, 1951-- Journal of Helminthology  25:173-176.

A new modification of the McMaster slide for use in potato-root eelworm investigations is described as affording complete security to its contained fluid. Five or more chambers can be accomodated in a single unit, thus considerably expediting the counting of a large number of samples. Extensive experimentation with the new slide revealed that counts from different samples from a suspension of  Heterodera eggs and larvae have been found to conform to a Poisson distribution.

 

Fenwick, 1967 -- Nematologica 13: 467-468.

A counting slide was constructed consisting of three sheets of 1.5mm thick perplex which are cemented together. The lower sheet is marked with lines 3mm apart, the middle sheet is a spacer, while the upper sheet is unmarked.

 

Fenwick & Mohammed, 1967 -- Journal of Helminthology 18: 155-172.

In a paper dealing with the recovery and counting of cysts of Heterodera schachtii from soils containing less than 115 moisture,  the author described a new type of cyst counting slide. A modification of the usual method for counting cysts was proposed The optimum weight of a soil sample for these procedures was found to be 1/2 lb.

 

Ferris, Mai & Lyon, 1956 -- Plant Disease Reporter 40: 182-183.

A new method for counting golden nematode cysts was developed by use of small rectangular plastic boats which are made  in the laboratory. The size and design of the boat make it possible to count cysts quickly and accurately. The boats are partly filled with water, the cyst material is placed within them, and all clumps are broken up.

 

Jimenez, 1972 -- Nematropica 2: 6.

A rapid method for building nematode counting chambers promises highly satisfactory results because of convenience in cleaning and operation. Plexiglass 3x1mm thick is  used to construct chambers which are a small reticulum engraved on a flat bottom, retangular in shape of 8.0 x 5.0 x 0.1 cm to which a frame of the same dimensions  is fixed.

 

Johnson, 1957 -- Plant Pathology 6: 75.

In a paper only two paragraphs in length, details for an improved cyst counting tray are based on a modification of an earlier described counting tray (Fenwick, J. Helminth 18:155-172. 1940).

 

Peters, 1952 -- Journal of Helminthology 26: 97-110.

For use in tests with the vinegar eelworm, Turbatrix aceti, a novel counting slide was deveoped. A detailed description of the slide and also of the counting method which gave replicate counts in agreement with Poisson expectations.

 

Singh & Menon, 1968 -- Indian Phytopathology 21: 451-453.

An easy-to-make counting dish for nematological work is reported using 2mm thick perspex.The dish has sides sloping at an angle of 45 degrees to permit examination and manipulation of nematodes lying in the peripheri. The inside bottom surface is divided into sections by raised ridges 1mm high. The underside of the dish has 3 small lugs which prevent the scoring of the base when the dish is slid over the microscope stage.

 

Tarjan, 1950 -- Phytopathology 40: 1111-1124.

 Syracuse watch glasses was specially divided so as to facilitate  relatively accurate determinations of all the nematodes in the dish, based on the careful counting of the nematodes in specified sectors.

 

van Bezooijen, 1970 -- Nematologica 16: 457-458.

A modification of Cobb's syracuse watch glass is suggested whereby the counting dish which holds 10ml is a modification of Cobb's engraved watch glass and is  made of polymethylacrylate plate. It has engraved concentric rings which are separated into 12 lined sections.

 

Williams & Winslow, 1955 --  in Soil Zoology, Proceedings University Nottingham Second Easter School in Agric., Science,  Butterworth,  pp  375-384.

Under the heading "Estimation of Cyst Contents" a counting slide is decribed and figured  After the cysts are squashed from pressure exerted by a microscope slide, the material is washed into a small contained with tap water. The suspension is then homogenized by blowing using a 10 ml pipette for 10-20 sec, quickly drawing up 10ml, and  injecting the suspension into the first chamber of a perplex counting  slide. Counting methods are discussed.

 

 COUNTING METHODS --CNM

 

Barker & Sasser, 1959 -- Phytopathology 49: 664-670.

In order to assay Ditylenchus dipsaci in roots, young seedlings were boiled for 4-5 min in a cotton blue-lactophenol solution. The seedlings are then crushed between 2 slides and the stained nematodes are counted.

 

Baxter, 1968 -- Nematologica 14: 599-600.

An intermittent or continuous counter is described and figured for use in nematolgical investigations.  An illustration of the counter and a detailed diagram of the contained circuitry are provided.

 

Beaver, 1950 -- Journal of Parasitology 36, 451-456.

Eggs of Ascaris lumbricoides or Trichuris trichiura in fecal smears were counted. Fecalsmears of varying density were evaluated using just a drop of a barium sulphate solution for each of the smears.

 

Byerly, Cassada & Russell, 1975 -- Review of Scientific Instruments 46: 517-522.

A counter is used to detect changes in the electrical current produced when a suspended particle (such as a nematode) passes between the two electrodes of a small transducer. Microprocessors compile the results for a direct reading.

 

Cobb, 1918 -- Estimating the nema population of the soil, Agricultural Technology Circular 1,   pp 1-48.

 (1) So as to obtain an even distribution of nematodes within a suspension to aid subsequent counting, it is suggested that air be bubbled through the suspension. A small propeller driven by an electric motor also can be employed for agitating the liquid.

  (2) The counting of nematodes using a microscope is facilitated by use of a two-dimensional mechanical stage and by using 2/3 and 1/6 inch objectives.

  (3) The use of a spider web for making cross-hair eyepieces is described. It is claimed that when such eyepieces are properly constructed using webs with threads of sufficiently large diameter, the diaphrams can last for years.

 

Dijkstra, 1956 -- Euphytica 5: 298-307.

Methods used for working with Ditylenchus dipsaci infecting red clover are given. Varieties of red clover which are susceptible or resistant to the nematode in a given origin within the Netherlands were susceptible or resistant in about the same degree to nematodes from a different part of the country. Intercrossing local Dutch varieties and selections and the progenies resulted in fairly resistant families.

 

Dimock & Lear, 1950 -- Phytopathology 40: 460-463.

Root galls caused by Meloidogyne spp.can be counted by floating the washed roots in one inch of water in a moist chamber which then is placed on a black background which adequately reveals the contained galls.

 

Doncaster, 1962 -- Nematologica 7: 334-336.

Because active nematodes often move from on division to another of a counting dish, a dish in which the divisions are made by ridges instead of engraved troughs is described. The dish has ten flat-bottomed concentric channels separated by narrow rounded ridges.Nematodes settling on the dividing rided soon fall off into the channels.

 

Doncaster, Edwards & Shepherd, 1966 -- Nematologica 12: 644.

A labor-saving method for making Fenwick multi-chamber counting slides for nematode counts is presented. Specific instructions and measurements are meticulously presented. The written account is accompanied by two illustrations, one showing the two alternative presses for bonding the slides simultaneously and the other a diagram showing the construction of the counting slide.

 

Fenwick, 1940 -- Journal of Helminthlogy 18: 155-172.

Methods for recovering and counting cysts of  Heterodera schachtii from soil are described. One such method recovers cysts of the nematode from soils of not more than 10% water content. Another recovers cysts by soaking the float and then subjecting it to differential floatation. The optimum size of a sample containing cysts was found to be 1/2 lb. and a new type of counting slide was described.

 

Ferris, Mai & Lyon, 1956 -- Plant Disease Reporter 40: 182-183.

A new method for counting golden nematode cysts involves the use of small rectangular plastic boats the construction of which is described. The boat is drawn slowly past the observer's visionwho is using  a stereoscopic microscope. The size and the design of the boat make it possible to count the cysts quickly and accurately.

 

Jones, 1945 -- Annals of Applied Biology 32: 351-380.

A standardized sampling technique is proposed for estimating the cyst, viable cyst, and egg content of soil. The first part of the paper deals with sampling technique and the various steps of which it is composed. Results were obtained by the application of the technique to 24 fields between the years  1937-1942. Correlation was made between the general level of eelworm population and the frequency of both when susceptible crops were grown and the amount of nematode damage to the crop.

 

Korsten, Sieben & Voskuyl, 1953 -- Euphytica 2: 135-138.

A quick and reliable method is described for making nematode suspensions of  known concentration required for an attempt at artificial infestation.  Using a colorimeter, the percent absorption of a quantity of nematodes suspended in an aqueous solution of carboxymethylcellulose is determined.

 

Lownsberry, 1951 -- Phytopathology 41: 889-896. 

In order to facilitate the counting of nematodes in water, parallel lines measuring 1/4 inch apart are scratched into the bottom of a Syracuse watch glass. A method for assaying larval emigration from cysts of the golden nematode of potatoes, Heterodera rostochiensis is described which uses large numbers of cysts and eliminates selection and counting of cysts.

 

Marlatt, Morton & McKittrick, 1959 -- Plant Disease Reporter 43: 1073-1077.

A test involving species of  Acobeles, Aphelenchus, Cephalobus, Dorylaimus, Eucephalobus, Pratylenchus, Prismatolaimus, Rhabditis, Trichodorus, and

Tylencorhynchius ws conducted on cantaloupes on desert soil.  The test included collection and comparison of nematode counts from soil and root samples.  It was found that both collections gave generally the same results.  It was suggested that it might not have been necessary to collect nematodes from both.

 

Peters, 1952 -- Journal of Helminthology 26: 97-110.

In a series of tests involving the vinegar eelworm, the author described culturing and counting methods which were used in his work with the nematode.

 

Rodriguez-Kabana, Morgan-Jones, Godoy & Gintis, 1984 -- Nematropica 14:155-170.

The effectiveness of species of Gliocladium, Paecilomyces, and Verticillium were investigated for control of Meloidogyne arenaria in field soil. The fungi were colonized on autoclaved oat kernels. Reductions in number of galls were observed in soil amended with 2 isolates of Gliocladium roseum, one isolate of G. catenulatum, one isolate of P. lilacinus and one isolate each of  Verticillium lamellicola and V. chlamydosporium.

 

Sadun, Allain & Heimlich, 1957 -- Experimental Parasitology 6: 271-279

The quantitative determination of Ascaris eggs in clear suspensions by photonephelometry was achieved and the results were compared with dilution egg counts. A linear relationship between the concentration factors and the nephelometric  readings were observed. The nephelometric determinations yielded substantially smaller variabilities and required considerably less time to perform than the dilution egg counts.

 

Stoeckli, 1943 -- Bericht der Schweizerischen  Botanischen Gesellschaft 54A: 160 .

A direct, but time consuming, examination of the sample is described. Although the method insures a high degree of accuracy it can be lengthy and tiresome.

 

Stoll, 1930 -- Parasitology 22: 116- 136.

The author suggests several methods for counting nematode ova in sheep dung. He first points out that the presence of helminth eggs in the dung of infected hosts has been the accepted diagnostic method for half a century. He reports on two techniques which had been developed to demonstrate the presence of Haemonchus contortus ova in sheep dung. The first is "Dilution egg Counting" while the second is based on "Centrifical Floatation Counts". It is suggested that extension of the presented methods, with or without modifications, would permit studies on helminthic infestations of the gastro-intestinal tract and its diverticula in domestic animals.

 

Thomas, 1965 -- Nematologica 11: 395-408.

Nematodes in agar culture are counted by placing on the agar a white cardboard disc of the same diameter and having ten 1-square cm windows in sequence. Mean total numbers of adults, juveniles, and eggs are thus recorded per square cm of agar.

 

Van der Laan, 1956 -- Nematologica 1: 112-125.

Roots infected with Heterodera rostochiensis are weighed, added to 250 cc of water, and  processed for 1 min in a Waring Blender.  The cut roots are then caught on a 100-mesh sieve, immersed in F.A. fixative, and stored in glass tubes until counts of the nematodes are made.

 

Willams & Winslow, 1955 -- Soil Zoology, Proceedings of the University of Nottingham

                                             Second Easter School in Agricultural Science, 1955.

Nematode cysts are gently squashed between a metal and a glass slide then washed into a graduate cylinder and made up to required volume with water. The suspension is homogenized with blown air and 10 ml is placed into a Perplex counting slide. Counting methods are discussed.  An additional counting method is covered using a Peters 1-ml eelworm counting slide.

 

CULTURE -- CUL     

                                                                                                           

Anderson & Coleman, 1977 -- Journal of Nematology 9: 319-322.

A system using glass microbeads in ecological experiments with bacteriophagic nematodes is described. Culture with glass microbeads was found to simulate soil  microorganisms more closely than did agar culture in terms of carbon dioxide production as well as number and size of nematodes produced.

 

Brooks, 1967 -- Nematologica 13: 472.

Polystyrene beakers were suggested for the culture of nematodes on potato plants and have been used in large scale tests involving pathotype determinations of the potato cyst eelworm.

 

Bryant, Nicholas & Jantunen, 1967 -- Nematologica 13: 197-209.

In a study dealing with aspects of the respiratory system of Caenorhabditis briggsae,

a method is described for the production of large numbers of the nematode free from culture medium. Studies were conducted on the effects of oxygen, cyanide, carbon monoxide, and sonic disruption on the worms.

 

Cairns, 1953 -- Phytopathology 43; 105-106.

A culture-reared, plant-parasitic nematode suitable for teaching and research is reported to be a species of Ditylenchus closely related to Ditylenchus destructor.  The nematode can exist for extended periods of time feeding on mushroom hyphae. It can be raised in large quantity in spawn culture bottles.

 

Chen, Hirumi & Marmarosch, 1968 -- Phytopathology 58: 1046-1047.

Aseptically reared Pratylenchus penetrans was cut into small pieces, dissociated into separate cells in 0.25% trypsin solution, and transferred to a culture medium designed to maintain the proliferation of nematode cells in vitro. Cells were observed to adhere readily to the glass surface of the culture vessels. Aggregation and proliferation of these cells became

apparent after 6 days.

 

Chen & Rich, 1962 -- Phytopathology 52: 922-923.

For determining the pathogenicity of Pratylenchus penetrans on strawberry and Ladino w

\                      

hite clover, a test-tube technique was devised which insured the absence of all other organisms.

 

Corbett, 1970 -- Nematologica 16: 156.

In a test toward maintaining nematode cultures under mineral oil, Pratylenchus fallax and Aphelenchoides ritzemabosi were established on lucerne callus growing on slopes of an agar medium with added 2,4D. After a week, each slope was covered with autoclaved paraffin oil and stored at 25C. At the end of 19 months, it was found that nematodes from all of the cultures successfully established themselves.

 

Dallimore, 1966 -- Phytopathology 56: 874-875.

The culture of  Ditylenchus dipsaci on aseptic potato plugs was accomplished by partially embedding the plugs in a few millimeters of potato-dextrose, cornmeal or water agar. After 5-6 weeks of incubation at room temperature, external symptoms of infection developed on the potato plug similar to those in advanced stages of tuber infection.

 

Darling, Faulkner & Wallendal, 1957 -- Phytopathology 47: 7.

Large pure cultures of the potato rot nematode Ditylenchus destructor have been obtained by using a modified White's medium to culture undifferentiated tissue from potato, clover, carrot and tobacco.  Uncontaminated nematodes were later introduced to the tissues. Using pure-culture techniques, it was also possible to develop large populations of nematodes in cultures of each of 37 species of 15 genera of fungi.

 

Dougherty & Calhoun, 1948 -- Proc. Helminthological Society Washington 15: 55-68.                                                                               

Rhabditis pellio and related soil nematodes were cultured after a method was devised using penicillin, streptomycin, and merthiolate for eliminating mixed bacterial flora and for establishing a single bacterial species which can, in turn, be eliminated with streptomycin. Rhabditis pellio and R. elegans were grown for several generations for the first time under axenic conditions in a complex medium.

 

Dougherty, Hansen, Nicholas, Mollett & Yarwood, 1959 -- Annals of the New York Academy of Sciences 77: 176-217.

Axenic cultivation of  Caenorhabditis briggsae with unsupplemented and supplemented chemically defined media was described. The best growth of the nematode obtained was considered sub-optimal as compared with growth of the nematode in the presence of bacteria. The authors concluded that the definition of nutritional requirements for the nematode still presents many challenges.

 

Dropkin, 1966 -- Nematologica 12: 89.

A  bacteria-free culture unit for analyzing host-parasite relationships of root-knot nematodes is described. It consists of an excised tomato root growing in a modified White's medium with 1.5% agar. The unit is adaptable for studies on host resistance and larval behavior, and for the production of synchronous populations of nematodes.

 

Dropkin & Boone, 1966 -- Nematologica 12: 225-236.

A bacteria-free culture unit to be used in large numbers for detailed analysis of host-parasite

relationships of Meloidogyne sp. is described. The unit consistes of an excised tomato root growing in agar medium in a test tube to which one nematode larva has been added.

 

Ducharme & Hanks, 1961 -- Plant Disease Reporter 45: 142-144.

A procedure was devised to grow citrus seedlings in a gnotobiotic environment, which is an environment free of all other organisms except those selected.  With this method it was possible to study penetration of burrowing nematodes, Radopholus similis, and subsequent development of  lesions in citrus rootlets and to observe the effects of the nematode on the whole plant under gnotobiotic conditions. Axenic citrus seedlings have been grown for 14 mos. in culture tubes.

 

Evans, 1970 -- Journal of Nematology 2: 99-100.

Mass culture of mycophagous nematodes was accomplished by developing a method for economically obtaining greater quantities of nematode tissue than had been produced by conventional methods. The average size of most of the species cultured  on Agaricus bisporus by this method has been greater than those grown on the same fungus in agar cultures

 

Hollis, 1957 -- Phytopathology 47:  468-473.

The nematode  Dorylaimus ettersbergensis was culktured in Petri dishes with water agar on two green algae, a protozoan, and a fungus.  The nematode inserted its stylet and fed on cells of the algae, on cysts of the protozoan, and on conidia of the fungus.

 

Hooper & Cowland, 1987 -- Nematologica 33:  488-490.

In a short contribution entitled "Courgette marrows for the mass culture of some nematodes" , the authors state that large numbers of the oat and giant races of the stem nematodes reproduce in freshly harvested fruits (courgettes) of courgette mar(squash) Cucurbita pepo. The technique of culturing the nematodes is described.

     

Lapage,1933 -- Nature 3310: 583-584.

The cultivation of parasitic nematodes from sheep is proposed using a method whereby large numbers of larvae can be obtained by use of media the composition of which can be much more easily controlled than any previosly used.

 

Lauritis, Rebois & Graney, 1982 -- Journal of Nematology 14: 422-424.

A technique for the gnotobiotic cultivation of  Heterodera glycines Ichinohe on Glycine max is described and illustrated.  The efficacy of the procedures in establishing the soybean cyst nematode on Kent sowbean was 75% effective.

 

Lower & Buecher, 1970 -- Nematologica 16: 563-566.

An aqueous extract from ruptured baker's yeast added as a required supplement to a soy-peptone Bacto yeast solution constitutes an easily prepared , inexpensive and stable medium for the axenic culture of some nematodes. Medium containing the supplement supported growth of three species of free-living nematodes and two species of  Neoaplectana, an insect parasite.

 

McKenna, Willis & Winslow, 1964 -- Record of Agricultural Research, Northern Ireland 13: 29-32.

A glass tube culture method is described for determining resistance of potatoes to the potato cyst nematode.  An example of its use is descibed and illustrated. The authors point out that in certain circumstances, it would be an economic alternative to the usual method of assaying in plant pots.

 

Moody, Lownsbery & Ahmed, 1973 -- Journal of Nematology 5: 225-226.

Culture of the root-lesion nematode Pratylenchus vulnus on carrot discs was reported by first allowing the nematodes to crawl through water agar containing a  suitable disinfestant, e.g. Aretan, a formulation containing 3% organic mercury. The axenization method  described  involves the use of dihydrostreptomycin. A detailed method is given with the finl conclusion that after seven years of culture away from the usual hosts, there is no evidence that the nematode's ability to reproduce has been altered.

 

Mountain, 1955 -- Proceedings of the Helminthological Society of Washington 22: 49-52.

A method of culturing plant parasitic nematodes under sterile conditions which stresses the growing of the eelworms in sterile media under sterile conditions in laboratory glassware is deMountain, 1955scribed with suggestions for application of the method .

 

Murphy & Doncaster, 1957 -- Nematologica 2: 202-214.

A new technique is described which has proved very suitable for studies requiring close observation of the culture subject. The culture chamber consists of a sintered-glass micro-immersion filter culture cell with syringe.

 

Nicholas, 1956 -- Nematologica 1: 337-340.

Turbatrix aceti was successfully cultured with continued growth and reproduction for more than a year under sterile conditions by serial sub-culture. Two culture media were used that contained an autoclaved liver homogenate. One of these was supplemented with an unheated liver extract while the other with an unheated chick embryo extract.

 

O'Bannon & Taylor, 1968 -- Phytopathology 58: 385.

Large numbers of the burrowing nematode, Radopholus similis, and the root-lesion nematode,  Pratylenchus brachyurus, were grown on carrot discs under sterile conditions.

The nematodes were easily extracted from the carrot discs by cutting or mincing the tissue and sieving the slurry to remove the nematodes.

 

Peters, 1952 -- Journal of Helminthology 26: 97-110.

For use in tests with the vinegar eelworm, Turbatrix aceti, various modifications of the culture medium were investigated, including temperature modifications. It was found that a medium of vinegar, 4% sugar,4% ethyl alcohol or combinations of these can result in a two-fold increase of worms each week

 

Reynolds, 1947 -- Proceedings of the Indiana Academy of Sciences 56: 84-85.

A new technique in the artificial culture of nematodes deals with the preparation and use of a clear medium in the cultural studies conducted. A 1% water agar is half-filled into test tubes.

After sterilization, surface-sterilized lettuce seeds are placed on moist filter paper for germination in petri dishes . When sufficiently developed, the seedlings are trasferred to small test tubes for further growth, and afterwards inoculated with nematodes.

 

Reidel, Foster & Mai, 1973 -- Journal of Nematology 5: 71-72.

A simplified medium for monoxenic culture of nematodes is described. Week-old sterile onion and alfalfa seedlings were cultured for 2 weeks on nutrient medium.  Thereafter, the onion and alfalfa callus tissues were inoculated with Ditylenchus dipsaci and Pratylenchus penetrans.

 

Reversat, Boyer, Sannier & Pando-Bahuon, 1999 -- Nematology  1: 209-212.

A mixture of pure silica sand and water-absorbent synthetic polymer as substrate was used for the successful culturing of some tropical nematodes.

 

Roessner, 1971 -- Nematologica 17: 320-321.

A simple method for sterile culture of nematodes was proposed.  A glass cylinder of sufficient volume is filled with moist sandy soil which is pressed down and covered with a layer of light soil. The top of the cylinder is greased with silicon paste and a glass cover with a bore hole is affixed.

 

Shepperson & Jordan, 1968 -- Proc. Helminthological Soc.Washington 35: 106-108.

Pure cultures of Meloidogyne incognita were established in aerial organs of Begonia and tomato plants. Stems of the plants were inoculated with glass capillary tubes containing sterile eggs or larvae thus producing several generations of the nematodes in a single host plant.  It was pointed out that nematodes produced in aerial organs are not exposed to soil-borne contaminants.  Resulting nematode egg sacs protruded through the epidemis, thus providing convenient accessibility.

 

Stephenson, 1942 -- Parasitology 34: 246- 252.

On the culturing of  Rhabditis terrestris n. sp., the method employed is composed of 2 parts of 0.6% agar solution and 1 part of a broth made of earthworms. 1.5cc of a culture containing large numbers of 2-day-old larvae were introduced into 15cc of the broth medium in a 10cm. Petri dish.

 

Tamura, 1978 -- Japanese Journal of Nematology 8: 24-27.

Bursaphelenchus lignicolus reared on the fungus Botrytis cinerea monoxenically in test tube culture was maintained at high population levels at either room temperature or a lower temperature of 10-14C for a long period of time by pouring sterile liquid paraffin into the tube.   The nematodes did not lose reproductivity of pathogenicity to pine trees after storage for one year. The nematode reproduced on the fungus under liquid paraffin at a temperature of 25C.

 

Tarjan & Cheo, 1955 -- Plant Disease Reporter 39: 405-406.

The use of anti-fermentative chemicals for maintaining cultures of Panagrellus redivivus

for nematocide screening implicated fermentation as a problem in the culture work. Among the anti-fermentative materials investigated, Amberlite IR-120, a cation-exchange resin, performed very well. Nematodes from cultures treated with this material were indistinguishable from nematodes obtained from non-treated cultures

 

Todd & Atkins, 1958 -- Phytopathology 48: 632-637.

"White Tip" of rice, caused by the ectoparasitic seed-borne nematode, Aphelenchoides besseyi Christie, 1942, was grown in artificial culture by introducing nematode-infested rice seed into flasks containing a substrate of steamed, unhulled rice. Fungi from the partially disinfested seed growing on the moist substrate within the flasks served as a source of food for growth and multiplication of the nematodes.

 

Viglierchio, Siddiqui and Croll, 1973 -- Hilgardia 42: 177-213

The authors conducted a series of  tests  in search for a suitable growing medium for callus tissue. They concluded that "Henk's medium" was more suitable than the wide variety of other media they tested.

 

Webster, 1966 -- Nature, London 212: 1472.

Various substances were investigated for the production of oat callus tissue on nutrient agar for culturing the chrysanthemum nematode Aphelenchoides ritzemabosi. After six weeks the number of nematodes in the culture tubes had increased fifty-fold.

  

Winkler & Pramer, 1961 -- Nature 192: 472-473.

A chamber for culturing and collecting the nematode Panagrellus redivivus is described and figured. The apparatus consists of a funnel with a ground lip and attached standard-taper stopcock. A Petri dish containing 25g of oatmeal and 45ml of water which is supported within the funnel is inoculated with the nematode. Water is added to the funnel to reach 3-4mm below the open end of the dish. The nematodes multiplied rapidly, moved out over the edge of the dish into the water and were

collected by openig the stopcock.

 

CYST PRODUCTION -- CSP

                                                                                    

Foot, 1977 -- New Zealand Journal of Zoology 4: 183-186.

Globodera spp, were cultivated on potato roots growing in sand contained within closed, clear plastic containers. Such a system inhibits foliage growth and stabilizes moisture balance.

 

 Winslow, 1955 -- Journal of Helminthology 29: 49-54.

A new method for the production and recovery of cysts of root eelworms (Heterodera sp.) for use in bio-assay is described. An improvement which greatly increases the cyst content of infested soil is presented.  The resulting float contains a minimum of debris thus avoiding the laborious and time-consuming step of separating the cysts from the debris. An apparatus for quick recovery of the float is described.

                                      

CYST RECOVERY -- CSR       

                                                                                 

Cooper, 1955 -- In  Soil Zoology, Kevan, Proceedings of the University of Nottingham Second Easter School in Agricultural Science, Academic Press, N.Y, pp.385-389..

A mechanical device for concentrating Heterodera cysts is described. Its development, advantages, and limitations are evaluated and the possiblity of its use in other fields is discussed. The apparatus, named the Kirton apparatus, is depicted and described as the prototype experimental model for concentrating or separating nematode cysts.

 

Fenwick, 1940 -- Journal of Helminthology 18: 155-172.

A method of recovering cysts from soils with less than 10% moisture is described. The yield had a slightly lower percentage of cysts  than that obtained by the dry floatation method. A new method of recovering cysts from the float is described, as is a new counting slide.

 

Fidler, 1963 -- Annals of Applied Biology 51: 269-275.

For working with cysts of Heterodera rostochiensis, a revolving plate with spring-loaded stop was devised whereby subsamples can be examined and the contained cysts picked off accordingly.

 

Seinhorst, 1975 -- Nematologica 20: 367-369.

The method described by Seinhorst, 1964 is altered. The previous danger of inhalation of poisonous vapors is eliminated by the use of 96% ethanol.. A better recovery of cysts was also obtained. Correction of a resulting problem is also described.

 

Shepherd, 1963 --- Nematologica 9: 647.

In order to avoid tedious sieving by hand to obtain Heterodera cysts,  a centrifical lawn  sprinkler with a small revolving disc and involute vanes was used. To prevent splashing and subsequent loss of cysts, the sprinkler was inverted over a sieve inside a closely fitting metal cover.

 

Winslow, 1955 -- Journal of Helminthology 29: 49-54.

A new method for the production and recovery of cysts of Heterodera spp for use in bio-assay is proposed. Roots are washed in the usual manner to extract the cysts and then treated in a special brass apparatus composed of a funnel. two sieves. The cysts and debris caught are washed into a container.

 

CYTOLOGY -- CYT 

                                                                                              

Hesling, 1960 -- Nematologica 5: 322.

In cytological  investigations on Heterodera cysts, female reproductive organs need to be mounted in an unbroken extended piece. If the contents from a cut cyst are pressed into a drop of medium strength Hydrogen Peroxide, the gelatinous matrix will be affected and the eggs and organs are liberated. Unwanted eggs can be removed with "a mounted eyelash" leaving the reproductive organs in clear display.

 

                ----- D -----

 

DEHYDRATION & CLEARING  -- DIE

 

Berwick, 1933 -- Science 78: 312-313.

The author developed an apparatus for dehydrating nematodes, particularly those possessing thick integuments for which the dehydration process must be exceedingly gradual. Large gordiacians were satisfactorily cleared by deydration in an alcohol series at 15 minutes per step, with final clearing in xylol.

                                                                                                           

 Cobb, 1917 --  Contributions to a Science of Nematology 5: 123-124.

Cobb, the "master inventer",  described a special device which he called a "differentiator". In this apparatus delicate specimens could be exposed to a very gradually changing series of dehydrating, clearing, staining,  and embedding materials.

 

Elsea, 1951 -- Proceedings of the Heminthological Society of Washington 18: 53-63.

A procedure is presented for histological work with root knot nematodes. Specimens are dehydrated and cleared by the use of cellusolve and isobutyl alcohol in a 2-day schedule, with final embedding in paraffin.

 

Hague, 1958 -- Nematologica 3: 149-153.

Two methods were presented to achieve concentration of potato root diffusate. The first was by leaching potatoes growing in soil  with tap water (ordinary diffusate). The second method by leaching potatoes growing in a pure silica sand with distilled water 'pure' root diffusate.

 

Hirschmann, 1959 -- Proceedings Heminthological Society Washington 26: 73-90.

Nematodes were first killed by heat and then tranferred to a 2% agar solution. After solidifying, the agar was trimmed into small blocks and fixed in one of three different ways. They were then dehyrated with an alcohol series and infiltrated with paraffin.

 

Minckler, 1944 -- Stain Technology 19: 63-64.

Formalin-fixed specimens of pin worms were satisfactorily cleared in a 6-step procedure of dehydration in dioxan followed by clearing in carbolxylene.

 

Oliveira Castro, 1935 -- Revista Dept. Nacional Prod. Animal 2: 131-136.

The author used a clearing agent of 60ml absolute alcohol, 60ml of water, and sufficient phenol to give a refractive index of 1.453 at 25C. It was found to be more rapid and to cause less distortion than glycerine, and it permits transfer of specimens to alcohol and back.

 

DETECTION -- DIC  

                                                                                                

Balan, Krizkova, Nemec and Kolozsvary, 1976 -- Nematologica 22: 306-311.

A quantitative method for detecting nematode attracting substances is described. Thin agar blocks measuring 10mm in diameter are cut and 0.2ml of a filtrate of the medium on which predacious fungi had grown are pippetted on to the surface of the blocks and allowed to diffuse into the agar. The blocks are transferred in an up-side-down position around the perimeter of a dish containing nematode suspension. The blocks are pressed down and after one hour nematode accumulation under the blocks is microscopically checked.

 

Bedding & Akhurst, 1975 -- Nematologica 21: 109-110.

The authors present a simple technique for the detection of insect parasitic rhabditid  nematodes in soil. They found that the larvae of the the greater wax moth Galleria mellonella when buried in soil was far more susceptible to parasitism by rhabditid

nematodes than are the usual hosts. This provided an excellent means of detecting insect parasitic rhabditid nematodes either in situ or in soil samples brought to the laboratory.

 

Davies, 1973 -- Nematologica 19: 570-571.

A rapid method for assessing nematodes in roots involves boiling the roots in boiling lactophenol-cotton blue stain, then washing in water to remove the excess stain. The roots are then placed in a solution of 1g zinc chloride oer 1,7ml of 12N Hydrochloric acid dor destaining.

 

Heald, Thames & Wiegand, 1972 -- Journal of Nematology 4: 298-300.

Detection of Rotylenchulus reniformis infestations by aerial infrared photography was attempted. The authors  were able to prove that differences in growth, after a period of almost four months, were not apparent but could be indicated by the use of aerial infrared photography..

 

Lung, 1989 -- Nematologica 35: 248-258.

A method for screening of nematicidal and  behaviour-influencing substances is described which not only allows for the screening of the above-mentioned substances but also of compounds affecting the behaviour of plant-parasitic nematodes. It can be used for the direct observation of nematodes and for a rapid test using the damage symptoms in the host tissue.  Plants and nematodes are kept in thin layers of Sephadex in small Petri-dishes.

 

DIAGNOSIS -- DIA  

                                                                                                

Cranston & Newton, 1965 -- Plant Pathology 14: 74.

A trouble-free test for the presence of eelworms on chrysanthemum is based on the fact that Aphelenchoides ritzema-bosi emerges in large numbers from infested leaves under moist conditions. Accordingly, suspected leaves are inclosed  in a clean dry polyethylene bag which has been made air tight. After 12 hrs.at a temperature of over 18C, 'trails' will be seen in the condensed droplets on the inside of the polyethylene.

 

Esser, 1989 -- Proceedings Soil and Crop Science Soc. of  Florida 48: 173-179.

An illustrated diagnostic compendium of members of the Dolichodoridae comprising 3 genera

is presented in an arrangement of increasing magnitude of stylet length..The paper presents a novel method of comparing 8 measurements of each of the species, as well as showing the lateral lines and the tails of the animals.

 

Fielding, 1951 -- Proceedings of the Heminthological Society of Washington 18:110-112.

The recommendation was presented to cut the nematode specimens in an effort to determine whether they were living or dead.  When living nematodes are treated thus, their body contents have a tendency to gush out. This does not occur in the case of dead nematodes.

 

Fortuner, 1985 -- Revue de Nematologie 8: 175-177.

A proposal is made for authors to present better diagnoses in describing nematodes. Among the problematic subjects proposed are "We don't know what the author is talking about", "Does the author really know what he is talking about" and "Guidelines for a good diagnosis". Fortuner concludes his presentation with the admonition that a new species should not be described unless the describer adhers to certain rules. "Only then can an objective diagnosis and reliable relationship be proposed that will be acceptable by all readers".

 

Granek, 1976 -- Journal of Nematology 8: 91-92.

The author points out that it is difficult and time-consuming to determine if juveniles of  Heterodera rostochiensis juveniles are living or dead. He proposes the technique of applying an electric current through the  nematode using stainless steel electrodes set 1mm apart. At 15-20V and a maximum of 10amps, juveniles which were alive projected their stylets whereas dead nematodes did  not.

 

Stoller, 1957 -- Plant Disease Reporter 41: 531-532.

An improved test for nematodes in the soil is reported bearing close affinity with the method reported by stoller,1957  but differs in several respects primarily in the use of plastic tubes affixed to the funnel stems. Differences between the two techniques are described. A detailed procedure using the new method is given.

 

Tarjan & Cheo, 1956 -- Misc. Publ. 47, Agric. Expt. Sta, Univ. Rhode Island, 27pp.

The nematocide screening program of the University of Rhode Island was a unique

program, commercially sponsored, using six different tests to evaluate the nematocidal efficacy of 556 proprietary chemicals supplied by the Mallinckrodt Chemical Works.

 

DRAWING -- DRA         

                                                                                     

Cobb, 1920 -- Transactions of the American Microscopical Society 39: 231-242.

The first of several methods presented, consistes of making a camera lucida drawing or diagrammatic chart of all the objects to record.  Each object is repreented by a simple characteristic diagram which is numbered in series. The chart is made using a camera lucida.

which shows the exact position of nematodes, and other objects, using a numbering system

identifying the subjects to the side of the chart showing positions

 

Goodey(J.B.), 1957 -- Tech. Bull. 2, Ministry of Agric., Fish. and Food, London, 47pp.                         

Use of a vertical or incident illuminator for examining surface structures on nematodes is described in a number of steps amoung which are that the distance from the light filament to the object should theoretically be equal to the tube length of the microscope. Also that the source of incident light should be as powerful as possible owing to the poor light reflecting  power of nematodes.

 

Goodey (J.B.), 1962 -- Nematologica 8: 80-83.

The tedious but very necessary process of figure preparation for publication in NEMATOLOGICA is described. It is pointed out that the original drawing should be three to four times larger than the published size. The dimensions are given of a

page in the journal and it is stated that a full page drawing should be a suitable multiple of the dimensions. Construction of histograms is discussed and illustrated.

 

Goodey (T.), 1949  -- Technical  Bulletin No. 2, His Majesty's Stat. Office, pg 18.

A method for making line drawings for black-and-white reproductions is credited by the author to workers at the Nematology Div., U.S. Bureau Plt. Ind., Beltsville, Md. where it is used regularly. Pencil sketches are made on white drawing board using a sheet of thin

Japanese paper into which a powdered dye, Prussian Blue, has been rubbed. The resulting faint blue drawing is completed by use of a hard thin blue lead pencil.

 

          ----- E -----

 

ELECTRICITY/ ELECTROPHORESIS-  -- ELC

                                                                                    

Daulton & Stokes, 1952 -- Empire Journal of Experimental  Agriculture 20: 271-273.                                                                

Root-knot nematodes  in dry soil, damp soil or water were destroyed when subjected to a pulsating electrostatic field set up, using a simple induction coil,  across suitably spaced electrodes. Tobacco plants showed no detrimental effects when subjected to the electrical treatment necessary for the destruction of the nematodes.   

 

Dickson, Huisingh & Sasser, 1967 --  Phytopathology 57: 458.

To prepare acrylamide gel electrophoretic protein profiles of  Heterodera glycines and Meloidogynej spp., 200 adult females of each species are homogenized in  0.001m NaHCO3 at 0C. Proteins are put in a characteristic band patterns by disc  electrophoresis at a pH of 8.3. Proteinpatterns are analysed with a microdensitometer.

 

Ellenby & Smith, 1968 -- Compte Biochem. Physiologie 26: 359-363.

Dry preparations  of  Ascaris cuticle gave an electrical output on vibration which varies with the energy input and with the direction in which it is applied in relation to the orientation of the collagen-fibre network; loading and unloading gives "on-off" responses.

These piezoelectric properties may play a role in nematode coordination.

 

Ferris & Siegel, 1957 -- Nematologica 2: 16-18.

Ultrathin sections of the cyst wall of Heterodera rostochiensis  were placed on collodion-coated grids for examination by the electron microscope. Both the exocuticle and the endocuticle were visible and more detail was seen than previously reported from light microscope studies. Detailes of the  exocuticle described in light microscope studies were not imaged in the electron microscope.

 

Granek, 1976 -- Journal of Nematology 8: 91-92.

Electrical stimulation was applied to second-stage juveniles of  Heterodera rostochiensis to determine viability. A variable transformer with a range of 0-140 volts, with a maximum of 10 amps and a pair of stainless steel electrodes embedded in plastic were used to straddle the juveniles in a drop of tap water. After voltage was applied, stylet ejection was observed as a measure of viability.

 

Heald, Menges, & Wayland, 1974 --  Plant Disease Reporter 58: 985-987.

Efficacy of ultra-high frequency electromagnetic energy (UHF) and soil fumigation on the control of the reniform nematode and common purslane among Southern peas, along with an increase in yield was reported. Combination of UHF and fumigation shows excellent control of the reniform nematode and common purslane with pea yields slightly higher than yields in fumigwated plots.

 

Laurence, 1968 -- Dissertation Abstracts 16: 1968-1969.

Electrophoretic patterns were analysed to interpret developmental changes from single-celled ascarid eggs to fully mature adults. It was theorized that such electrophoretic characterization  of changes in protein content through the developmental stages will aid in determining shifts in antigenic structure of  Ascaris lumbricoides var suum, and help to explain the production of a dual antibody response which occurs in experimentally infected animals.

 

Scott & Riggs, 1971 -- Phytopathology 61: 751-752.

The authors used imunoelectrophoretic analysis to compare three plant-parasitic nematodes. Reciprocal tests showed that two races of the soybean cyst nematode are serologically identical.  Immunoelectrophoresis of nematode extracts resulted in a greater number of precipitin bands.

 

Viglierchio & Goss, 1962 -- Jour. American Society Sugar Beet Technologists 12: 100-105.

The principal of electrostatic separation of cysts of the sugar beet nematode was proposed with the introduction of a constructed electrostatic separator. Seven figures accompany

this technical article. It was concluded that there was an induced axial polarization achieved with the cysts.

 

EMBEDDING -- EMB

 

Bird, 1958 -- Nematologica 3: 203-218.

In a study conducted on the Meloidogyne cuticle and egg sac, the author used frozen specimens as well as wax-embedded materials using the iso-propanol method of de Guisti & Ezman, 1955 (cf reference citations).

 

Bird & Deutsch, 1957 -- Parasitology 47:  319-328.

Using a modification of the layer stripping technique of Reed and Rudall, 1948, the authors studied the ultrastructure in layers of the cuticle of  Ascaris and described a technique for embedding in a poymerized monomer for ultrasectioning.

 

Coil, 1958 -- Proceedings Helminthologicsal Society Washington 25: 137-138.

 A technique was used for trematodes which may prove applicable to embedding nematodes. Living material was placed in acetone at a temperature below 0C then tranferred to a refrigerator for 24 hrs. Embedding occurred in wax at 56C using a

lamp adjusted so that the tissues were at the interface of the melted and solid wax.

 

Demke, 1952 -- Stain Technology 27: 135-139.

A description of a technique for mounting helminths in presented.  The specimens are embedded in celloidin and are dehydrated, cleared and mounted on slides in xylene balsam.

 

Ferris & Siegel, 1957 -- Nematologica 2: 16-18.

In working with the  Heteroderidae, the authors preparation of cyst walls for examination, the embedding in methylacrylate, and the technique of ultrasectioning.

 

Krusberg, 1959 -- Nematologica 4: 187-197.

Satisfactory section of both plant material and nematodes were obtained by removing sections of agar from cultures which contained both roots and nematodes. After fixation in CRAF III and dehydration  in an ethanol series, the tissues were embedded in TISSUEMAT, a comercial paraffin-wax medium.

 

Maneely, 1956 -- Annals Tropical Medicine and Parasitology 50:101-164.

Nonex 63B, a stearate of polyethylene glycol, was recommended as an embedding medium for invertebrates. It was found to obviate much of the shrinkage and distortion occurring with parafin wax and celloidin, especially where hard and soft components are side by side.

 

Smyth & Hopkins, 1948 -- Quarterly Journal of  Microscopical Science 89: 431-436.

It is pointed out that glycogen in tissue may be very impervious to the introduction of paraffin wax and with some rapid embedding methods it is easily lost during the subsequent cutting and  manipulation of sections  They recommend replacing the paraffin wax with Stedman's ester wax rather than enduring prolonged embedding times with paraffin.

 

Wright & Jones, 1965 -- Nematologica 11:125-130.

Techniques for the orientation, embedding and sectioning of several species of soil and marine nematodes were investigated. Orientation of the nematodes was possible after embedding them in agar after fixation, but prior to dehydration.The agar flats with nematodes were then infiltrated with "Maraglas 655" and the final block was formed in gelatin capsules.

 

ENZYMATIC ASSAY -- ENZ

                                               

Atkinson & Ballantine, 1977 -- Annals of Applied Biology 87: 407-414.

Measurements of adenosine triphosphate (ATP)  in nematodes were made by virtue of the bioluminescence produced by ATP in a luciferin/luciferase extract from fireflies. The ATP is extracted by maceration of the nematodes in 0.1 M arsenate buffer @ pH4 and at 4C. 

 

Goffart & Heiling, 1962 -- Nematologica 7: 173-176.

Investigations of  Ditylenchus destructor from potato, D. dipsaci from sugar beet, Heterodera rostochiensis  from potato and H. schachtii from sugar beet proved that they produce amylase, invertase and pectin dissociating  fermenting materials in the secretion of their salivary glands. These materials were liberated not only in living cells but also in cell-free substrates such as oatmeal agar. The action of the enzymes was detected by determinations of mono- and di-saccharide, by study of filtration velocity and by turbidity measurements of precipitations caused by enzymatic activity.

 

Krusberg, 1960 --  Phytopathology 50: 9-22.

Homogenates and extracts of Ditylenchus triformis, D. dipsaci and Pratylenchus zeae were assayed for hydrolytic, respiritory and terminal oxidative enzymes by techniques utilizing viscosity, titration,  colorimetric and spectrophotometric measurements.

 

Morgan & McAllan, 1962 -- Nematologica 8: 209-215.

In a study of hydrolytic enzymes in nematodes, pectinase and cellulase were demonstrated quantitatively by a modified viscometric method in high concentrations of Pratylenchus penetrans and Heterodera trifolii.  Turbatrix aceti, a non-parasitic species possessed no discernable cellulase.

 

 

ESTIMATION OF CYST CONTENTS -- ESC   

                                                     

Bijloo, 1954 -- Journal of Helminthology 28: 123-126.

A CEKA-homogenizer, Type UM, 15,000 rpm manufactured by E. Buehler, Tuebingen, Germany which rotates from 8,000 to 15,000 times a minute was used. The stirring spindle is placed in a  bottle with three verticle ridges. When dissected cysts in water were introduced, the material was thrown outwards to the glass wall of the bottle and at every rotation hurled against the ridges. Thus it was possible to shake all of the eggs and juveniles away from the cyst walls.

 

Bijloo, 1955 -- Meded. Landbou. Opzoeking.Staat Gent 20:291-300.

A simple method for estimating the contents of large numbers of cysts of Heteroders species

described a "second homogenizer technique' whih takes less time to use then originally was

suggested with a previous technique. .Specific details are presented in the English summary.

 

Byrd, Ferris & Nusbaum, 1972 -- Journal of Nematology  4: 266-269.

Using a novel procedure for extracting eggs of Meloidogyne spp.from soil, the egg masses were elutriated from the soil, gelatinous matrices of the egg masses were dissolved, and the dispersed eggs were stained to facilitate counting.

 

Fenwick, 1942 -- Journal of Helminthology 20: 50-66.

In a study of the degree of  Heterodera infectivity of soil and its determination, a technique for measuring the infectivity of soil with Heterodera schactii in terms of the number of larvae potentially capable of infecting plants per pound of soil is described.

Use of calcium hypochlorite solution soaking cysts for four hours was effective in liberating larvae.

 

Fenwick, 1951 -- Journal of Helminthology 25: 173-176.

A simple method of  assaying cyst contents, after preparing the aqueous suspension of eggs, is to agitate the suspension by blowing down a pipette which is then used to withdraw the subsample that is placed into a modified McMaster slide.

 

Fenwick, 1952 -- Journal of Helminthology 26 :55-68..

In dealing with the estimation of the cyst contents of the potato-root eelworm, Heterodera rostochiensis, the author proposes three general principles --- 'Taking the soil sample', 'treatment of cysts prior to dissection', and 'dissection of cysts'. In investigating the viability of cysts following  immersion in calcium hypochlorite solution, the author concluded that no valuable information on viability was obtained.

 

Fenwick & Reid, 1951 -- Journal of Helminthology 25: 161-165.

Tests were conducted using a capillary microbalance to estimate the errors introduced as a result of weighing out replicate cyst batches instead of counting.  It was suggested that if replication is increased by 50 -100%, then errors due to inequality in cysts numbers are adequately counteracted.

 

Fenwick & Reid, 1951 -- Nature, London 167: 534.

A method is presented for obtaining a quantitative measure of numbers of cysts

present on roots. The roots are thoroughly brushed in water, the "brushings" and remaining soil are collected on a 100-mesh sieve. The washings are transferred to a  a special 100-ml cylinder and elutriated with the cysts floating up and being caught in another container.

 

Oostenbrink, 1950 -- Vers. Meded. van den Plantenziek.Dienst,Wageningen 115: 1-230.

Oostenbrink estimated contents of the cysts by crushing the cysts using a thick cover glass affixed to a handle and estimating the halos thus created by comparing them to a set of prepared standards.

 

Petherbridge, Stapley & Thomas, 1938 -- Journal Ministry of Agriculture  45: 226-236.                                                                        

The authors crushed nematode cysts and conducted estimations of the number of eggs contained  using a squared micrometer microscope eyepiece and by counting the "halos" of eggs and larvae arount the crushed cysts.

 

Seinhorst & den Ouden, 1966 -- Nematologica 12: 170-171.

The method described by Bijlou for crushing nematode cysts was modified by placing wire spirals on the plunger. The required time for treatment of a sample was reduced to

7-10 seconds without the risk of destruction of nematode larvae.

 

ESTIMATION OF INFESTATION -- ESI    

 

Davies, 1973 -- Nematologica 19: 570-571.

A rapid method for assessing nematodes in roots begins with boiling the roots in lactophenol-cotton (blue), washing, then placing in an acid solution comprising 1g zinc chloride per 1.7ml of 12N hydrochloric acid (cf Holloway & Baker, 1968, Plant Physiology 43: 1878-1879) for destaining.

 

Fenwick & Reid, 1948 -- Nature 167: 534.

A rapid method for estimating the density of white cysts of  Heterodera rostochiensis

on potato roots involves a five-minute elutriation of samples.with the elutriate being stirred with a saturated magnesium or zinc sulfate solution in a measuring cylinder. The white cysts float to the surface, are collected, then washed, and 1-mm samples are withdrawn for the cyst counts.

    

 

Goffart, 1952 -- Zuckerruebenbau 14: 315-317.

The critical populaton level for sugarbeet nematode, Heterodera schactii, was estimated by the author to be about 100 x 10,000,000 cysts with contents per acre.

 

Johnson & Thompson, 1945 -- Journal of the Ministry of Agriculture 52: 266-270.

The critical populaton level for the potato root eelworm, Heterodera rostochiensisi, was estimated by the author to be about 500 x 10,000,000 cysts with contents per acre.

 

Lloyd, 1946 -- Annual Report, Agric. Research Station., Univ.of Bristol, pp 153-156.

 A method assessing the level of intensity of infestation  by Anguillulia (Ditylenchus) dipsaci on red clover was proposed. Several tests involving different kinds of soil were conducted. It was concluded that the tests gave evidence of soil infestation by nematodes and could be used to forecast the intensity of nematode attack on clover.

 

Umaerus & Videgard, 1967 -- Nematologica 13: 473-474.

The authors propose a simplified method for testing the 'infestivity' of Heterodera rostochiensis whereby the plants are grown in polyethylene bags which holds 200cc of soil. So as to obtain a suitable culture medium, perlite, vermiculite, sand and a standardized soil mixture were tested. Only the soil mixture proved suitable.

 

Waid, Capstick & Twinn, 1957 -- Pedologie 7: 159-161

A quantitative method of estimating fungal infections of natural populations of free-living nematodes uses a technique modified by the two junior authors for extracting free-living nematodes from samples. The conclusion reached by the authors was that their methods gave very different results from those obtained by conventional methods.

 

Williams & Winslow, 1955 --  in Kevin, Soil Zoology, Proc. Univ. Nottinghame, Second Easter School in Agric. Science, Butterworth, London, ,  pp 375-384.

The authors methodically list each step of their proposed maceration  procedure to determine the degree of  eelworm infestation of plant roots. Drawings are presented. They propose that the precedure can be used for determining the presence of nematodes in plant roots without dissecting them individually out of the roots.

 

ESTIMATION OF  NEMATODES -- ESN

                                                           

Anderson & Yanagihara, 1955 -- Phytopathology 45: 238-239.

A method for estimating numbers of motile nematodes in large  numbers of soil samples employed a modification of the funnel technique. Paper cups of 12-oz capacity were coated with hot paraffin and used as containers as depicted.. This method is empirical and its usefulness appears to be limited to relative estimates of motile forms.in large numbers of samples.  It is clean, cheap, rapid, relatively accurate, and easily carried out.

 

Ayala, 1961 -- Journal of Agriculture of the University of Puerto Rico 45: 265 - 296.

In a study entitled an analysis of the quantitative and qualitative composition of the nematode

populations in pineapple fields,  the author describes several simple methods which preclude the need for more elaborate collection equipment. A modification of a method earlier described by Cobb was used.. In isolating nematodes a combination of Cobb's sieving method and the Baermann funnel method was used, with few modifications.

 

Blake, 1958 -- Proceedings of the Linnean Society of New South Wales 83: 241-244.

A turbidimetric method for estimating the number of nematode larvae in an aqueous suspension stabilized with 0.5% carboxymethylcellulose is presented. The light absorption due to the turbidity of the suspension is measured with a Hilger "Spekker" absorptiometer.

 

Byrd, Ferris & Nusbaum, 1972 -- Journal of Nematology 4: 266-269.

A method for estimating numbers of eggs of Meloidogyne spp. in soil was developed by modifying and combining certain existing techniques. Eggs dispersed from egg masses were stained to facilitate counting. It is suggested that the data thus obtained facilitates the study of population dynamics and the analysis of root-knot epidemics.

 

Cobb, 1918 -- Agricultural Technology Circular, U.S.Dept.Agric.,Bur.Pt. Ind.1: 1-48.

Soil sampling tubes which were open cylinders of thin metal with the rim of one end reinforced and of the other end sharpened were used were used to obtain soil samples from which estimation of nematodes was made.

 

Everard, 1962 -- Nematologica 8: 321.

The principle of absorptiometry was applied to estimating the number of nematodes in a mixed culture. Optical density readings of several concentrations of nematode batches were taken and the results compared with the corresponding  microscopic counts. Those results indicated a close correlation between the absorptiometric readings and the microscopic counts.

 

Willams & Winslow, 1955 -- in Kevin, Soil Zoology, Proc. Univ. Nottingham, Second Easter School in Agric. Science, Butterworth, London,  pp 375-384.

Nematode cysts are gently squashed between a metal and a glass slide then washed into a graduate cylinder and made up to required volume with water. The suspension is homogenized with blown air and 10 ml is placed into a Perplex counting slide. Counting methods are discussed.  An additional counting method is covered using a Peters 1-ml eelworm counting slide.

 

EXSHEATHMENT -- EXS

                                                                                              

Bird, 1954 -- Nature 174: 362.

The cuticle of several parasitic nematodes from sheep was investigated. As soon as exsheathment occurred, cuticles were separated from the larvae by differential centifugation in saturated sodium chloride and then hydrolysed in hydrochloric acid. The hydrolysate was examined by two-dimentional paper chromatography. Nine amino acids were demonstrated in the hydrolysis.

 

Lapage, 1935 -- Journal of Helminthology 13: 103-114.

The second ecdysis of the infective larvae of certain Trichostrongylidae in solution of sodium sulphide and of organic compounds containing sulphur was part of a study made on parasites of sheep. In the course of the investigations, the following were studied:  infusions of garlic, effects of allyl sulphide, sodium sulphide, cystein hydrochloride, sodium taurocholate, saccharin, 0.2% sulphonal, and sodium thiosulphate.

All of the solutions named could be used for artificial production of ecdysis.

.

Myers & Krusberg, 1965 -- Phytopathology 55: 429-437.

The authors mainly studied organic substances discharged by the plant-parasitic nematode, Ditylenchus triformis in a number of tests. Also included in these studies were

D. dipsaci, D. myceliophagus, Meloidogyne incognita & Pratylenchus penetrans.

It was concluded that this was the first report that stylet-bearing nematodes can metabolize chemicals taken from solution in which they were incubated.

 

 

EXTRACTION, BAERMANN FUNNEL -- EXBF

                                                     

Adams, 1965 -- Plant Disease Reporter 49: 662-664.

Temperature was found to influence the quantitative recovery of nematodes with a modified Baermann funnel. The optimum temperature was found to be between 15 and 25C. Higher or lower temperatures yielded smaller numbers of nematodes. Evidence was presented that temperature can change the character of  the nematode populations recovered. Temperature was found to influence the quantitative recovery of nematodes with a modified Baermann funnel. The optimum temperature was found to be between 15 and 25C. Higher or lower temperatures yielded smaller numbers of nematodes. Evidence was presented that temperature can change the character of  the nematode populations recovered.

                                             

Anderson & Yanagihara, 1955 -- Plant Disease  45: 230-239.

A modification of the Baermann funnel technique was described and illustrated.

Conical paper cups were coated on the inner serface with hot paraffin.  For strength,

a coated cup was placed inside an uncoated cup with the point cut off and the two supported upright in a 12oz tin can. A shallow cup about 1 inch deep with a flat bottom

made out of stainless steel or aluminum window screen fits within the paper cup. Facial cleansing tissue with the soil sample is lowered onto the screen and water is added to cover the soil sample. After 4 days, a long needle is introduced so as to puncture the bottom of the cup allowing 12 to 15ml of nematode suspension to be drawn off.

 

Baermann, 1917 -- Tijdschrift Geneesk. Nederland Indie 57: 132-137.

This very basic method allows for the immersion of nematode material in water on porous material in a funnel the bottom of which is fitted with a flexible extension closed off by a clamp. The nematodes leave the material, sink down to the tube with its extension , and are drawn off to be examined. Numerous modifications and embellishments have been proposed.

 

Christie & Perry -- Proceedings of  Helminthological Society of Washington 18:106-108.

The authors state that neither sieving soil nor the Baermann Funnel method result in the collection of nematodes in clear water reasonably free from debris. They propose a combination of the two methods which they have been using with good success.

 

Esser, 1957 -- Plant Disease Reporter 41: 269-270.

An improved post Baermann funnel technique draws water from the funnel into a centrifuge tube and allowed to settle. The tube is then placed under a dissecting microscope and the collected nematodes at the bottom are draewn off with a pipette discharged on to a slide, and enclosed with a sealed cover slip.

 

Kerr & Vythilingam, 1967 --  Nematologica 12: 511-517.

In an unusual study of factors influencing extraction of nematodes from soil, the authors, using a modified Baermann funnel technique, processed duplicate soil samples at 1370m elevation and at sea level.  The numbers of nematodes extracted at sea level was only 14% of those extracted at the higher elevation. Experimental results indicated that this was due to differences in temperature between the two locations.

 

Linford. 1941 -- Phytopathology 31: 634-648.

The Baermann funnel technique was further modified by the author who eliminated the stem from the funnel.  A folder piece of filter paper was then placed within the funnel into which the sample was placed. The funnel was then placed into a small beaker so that the tip of the filter cone contacted the water in the beaker.

 

Miller, 1957 -- Plant Disease Reporter 41: 194.

A quick method for the separation of nematodes from soil samples used the centrifical floatation technique but employed a 325-mesh sieve through which the supernatant was passed instead of sedimenting it in water.

 

Overgaard-Nielson, 1947 -- Natura Jutlandica 1: 271-278.

A set of nine small Baermann funnels were mounted within a box. Attached to the lid of the box was an electric light which was able to heat the interior of the box to about

30C.

 

Rohrbacher, 1957 -- Proceedings Helminthological Society Washington 24: 24-25.

Recovery of nematode larvae by the Baermann Apparatus was increased by the addition of a nonionic detergent applied at the rate of 0.5ml per liter of water. Infective larvae from cattle nematodeswere obtained from Bermuda-grass, orchidgrass, and crimsom clover.

 

Schindler, 1961 -- Plant Disease Reporter 45: 747-748.

A simple substitute for a Baermann funnel was proposed. A 3-3/8 inch hole is cut  in a board. A plunger is devised using a dowel affixed to the circular piece removed from the board., and 4-1/4 inch diameter circles are cut from aluminum window screening, placed over the hole in the board and forced down through it using the plunger.  The resulting screen is placed in a Petri dish top and two-ply Kleenex tissue paper is placed on the screen. Screened residue from soil samples in placed on the tissue paper and covered with water.

 

Staniland, 1954 -- Journal of Helminthology 28: 115-117.

A modification of the Baermann funnel technique was used to collect nematodes from plant material. A drawing of the apparatus is is presented  and its use is described, as is removal of the nematodes from the equipment.

 

Stoller, 1957 -- Plant Disease Reporter 41: 531-532.

An improved test for nematodes in the soil is rep           `orted bearing close affinity with the method reported by Miller, 1957 but differing in several respects, primarily in the use of plastic tubes affixed to the funnel stems. Differences between the two techniques are described. A detailed procedure using the new method is given.

 

Tarjan, 1949 -- Phytopathology 39: 24.

Standardized weights of infected Boxwood (Buxus sempervirens) roots were immersed in water contained in Baermann funnels. Pratylenchus sp. evacuated the roots for as long as 9 weeks. Most satisfactory results were obtained by drawing off aliquots 3X weekly for three weeks. At that time 95.4% +/- 1.1 % of the total number of nematodes in the roots had evacuated.

 

Tarjan, 1950 -- Phytopathology 40: 1111-1124.

Using a modification of Goffart's quantitative method which was applicable to free-living nematodes,  2-g. root samples were immersed in water contained in Baerman funnels.  It was found that a 3-week immersion of the roots gave a valid indication of the nematodes contained in those roots.

 

Tuppen, 1975 -- Nematologica 21: 263-164.

Extracting Longidorus and Xiphinema from soil in a further modification of Cobb's decanting and sieving technique, the author increased the aperture of the nylon sieves used in the final Baermann funnel extraction from 90 micrometers to 106 micrometers.  This significantly increased the recovery of adult L. macrosoma without loss of the first instars.

 

Zuckerman, 1960 --Proceedings of the Helminthological Society of Washington 27: 37-39.

A method for the concentration of nematodes for mounting from the Baermann apparatus

 is described. The nematode population is sampled without bias. This method is proposed as an adjunct to other soil and plant tissue extraction techniques in which the nematodes are separated from large quantities of liquid.

 

EXTRACTION, CENTRIFICAL FLOATATION -- EXCF  

                                  

Caveness & Jensen, 1955 -- Proceedings  Helminth. Soc.of Washington 22:87-89.

50cc aliquots of soil are mixed with water and centrifuged in tubes for approximately 5 minutes. The resulting supernatant fluid is gently poured off.. A water-sugar syrup mixture (484.5g of sugar in a liter of water) is added and the tubes again centrifuged for 5 minutes. After spinning, the syrup liquid is poured off and the remainder immediately diluted with 500cc of tap water to avoid excessive harmful effects to the nematodes. After 20 minutes, the excess water is decanted leaving the nematodes which had settled to the bottom in a minimum of water.

 

Dickerson, 1977 -- Plant Disease Reporter 61: 1054-1057.\.

An evaluation of the direct centrifical-floatation method (DCF) of recovering nematodes from soil was developed and its efficiency compared with three other standard methods. It was found that the method was not as affective as the other methods.

 

Gibbons & Grandison, 1966 -- Nematologica 12: 642-643.

An improved centrifugal-floatation technique for the isolation of  Ditylenchus dipsaci is based on the sedimentation of the nematodes in sucrose solution of graded densities. The procedure described is quick, large numbers of nematodes can be processed in a few tubes, the procedure is not detrimental to the nematodes, and it can be used for the purification of other nematode species preparations.

 

Jenkins, 1964 -- Plant Disease Reporter 48: 692

A rapid centrifugal-floatation technique previously reported  for separating nematodes from the soil was modified by using a combination of the sieving and the centrifugal-floatation methods. The combination of the two techniques recovers non-sedentary, soil-dwelling nematodes of all types.

 

Leiper, 1937 -- Journal of Helminthology 15: 153-166.

Whereas the centrifical-floatation method for obtaining contained nematodes had been applied to nematodes in feces,  the author realizing its utility applied it for the extraction of nematodes from plants and soil.

 

Stoller, 1957 -- Plant Disease Reporter 41: 531-532.

An improved test for nematodes in the soil was presented. The author claims that the novel feature of the test is the use of polyethylene plastic tubes 0.002 inches thick which permits the collected nematodes to remain alive in contrast to those collected in rubber tubing.

 

Whitehead & Flemming, 1965 --  Annals of Applied Biology  55: 25-38.

The technique of layering a water extract on a denser solution and centrifuging was used by the authors. The resulting suspensions are briefly centrifuged, rid of floating organic matter, shaken, mixed with sucrose solution, poured into a centrifuging container, balanced and spun at 2100g. The supernatant is then  mixed with water and then poured through a bank of sieves from which the nematodes are washed.

 

EXTRACTION, CYST METHODS -- EXCM

                                                      

Andersen, 1956 --  Nematologica 1: 303-306.

Collected debris and cysts were spread on a rolling belt of paper or rubber which accomplished removal of 80 to 90% of the debris. The author further proposed drying the soil at 110-120C prior to processing which helped to float heavy cysts.

 

Carroll, 1933 -- Journal of the Irish Department of Agriculture 32: 183-201.

The author desired to expedite the tedious and time-consuming method of direct examination and segregation of cysts from accompanying debris. He placed the floated material in small quantities on a sheet of supported paper.  The material was spread out, the paper tilted and tapped slightly which accomplished separation of the cysts from most of the accompanying unwanted material.

 

Curtis, 1962 -- Nematologica 7: 25-31.

An apparatus for the quantitative extraction of cysts of all stages from wet and dry soil is described.The apparatus exploits the principle of differential sedimentation under controlled flow. Evaluation tests. and performance of the apparatus are descibed

 

Dunn, 1969 -- Journal of Nematology 1: 7.

The extraction of cysts of Heterodera spp. from undried soils by centrifugation in high density solutions was accomplished by washing and sieving  the soil suspension. The residues were suspended in sucrose solution, centrifuged then decanted. A "Vibromixer" was used to break up soil aggregates.

 

Faulkner & Greet, 1984 -- Nematologica 30: 99-102.

A machine which allows for the  rapid and efficient separation of nematode cysts from dried root debris is described.  It incorporates the moving belt principle but has several additions and refinements which overcome most of the difficulties encountered in the machine described by  Hesling (particularly the effect of electrostatic charging).

 

Fenwick, 1940 -- Journal of Helminthology 18: 155-172.

In a paper dealing with the recovery of cysts from soils with a water content of less than 11%, the author also proposed a new method of recovering cysts of Heterodera schachtii from the float by using the principle of differential floatation.

 

Green & Parrott, 1966 -- Nematologica 12: 601-609.

In order to extract nematode cysts from a large volume of soil, the authors suggest the following treatment. The soil is dried, crushed, and poured into a hopper. A vibrating chute takes the soil to a water tank from which the cysts float onto screens.In addition, a

re-appraisal of the extraction of  Heterodera cysts from large volumes of soil resulted in a redesign of the floatation apparatus and automation of the process where possible. In efficiency, the resulting process was equal to a Fenwick can used for 200 grams of soil, and better than a can used for larger samples.

 

Hesling, 1952 -- Journal of Helminthology 26: 69-70.

An apparatus was devised for the extraction of nematode cysts from soil. The device, which is illustrated,  is an application of the entolomologist's aspirator and is connected to a filter pump attached to a laboratory tap.

 

Jones, 1955  -- in Kevin, Soil Zoology, Proc. Univ. Nottingham, Second Easter School in Agric. Science, Butterworth, London,  pp 394-401.

Within a paper dealing with quantitative methods for the estimation of cyst-forming nematodes (Heterodera sp.) in soil, the author refers to water floatation methods for cyst extraction from the soil and alludes to imperfections in technique. It is suggested that for research purposes cyst counts of 100 or more are desirable and that the sample size should be varied accordingly.

 

Kirchner, 1954 -- Nachrichtenblatt des Deutschen Pflanzenschutzdientas 5: 81-85.

A system is described using a glass funnel with roughened inner surface into which are placed  dry soil samples.  The funnel is closed and a stream of water agitates the soil sample causing the mixture to swirl around within the funnel. Once the agitation ceases and the suspension settles, the bottom plug is removed allowing the soil and water to pass out. Cysts and other material are collected from the sides of the funnel.

 

Kort, 1960 -- Plantenziektenkundige Dienst , publ. 233; 7 pp

A technique for the extraction of  Heterodera cysts from wet soil and for the estimation of their egg and larval content is described. The author admits that the technique, which was developed from techniques published by other nematologists, permits quantitative extraction

of cysts from wet soil.  Illustrations and drawings accompany the article.

 

Morgan, 1925 --Journal of Helminthology  3: 185-192.

In order to obtain a relative indication of the degree of  nematode infectivity a soil infested with the potato root eelworm, Heterodera rostochiensis, might have, counts of the cysts were made. Jones, 1955 , however, felt that "cysts are a better measure of the age rather than the strength of a population".

 

Reid, 1955 --  Plant Pathology  4: 28-29.

A Hagedorn-needle method for the dissection of cysts was proven to be reliable It has enabled one, with practice, to achieve the dissection of 100 cysts per minute A special

metal or perplex slide is used with this method.

 

 Shepherd, 1964-- Nematologica 9: 647.

A water sprinkler designed to separate Heterodera cysts from debris in hatching experiments is proposed. A 20-mesh sieve retains as much larger debris as possible which is then washed on a 40-mesh sieve for about an hour with a powerful swirling water current.

 

Williamson, 1975 -- Nematologica 25: 372-374.

Rapid and simple extractions of cyst nematodes from clay soil were made. During the use of standard techniques, excluding dispersants, for extraction of cyst nematodes from soil, it was found that by using hot water instead of cold, or frozen or defrosted soil samples, extraction from clay soils were quicker and easier to perform.

 

Yuhara & Aihara --  Research Bulletin of Plant Protection, Japan 18:1-5.

This pulication in Japanese describes a cyclonic seed sorter which successfully separates cycsts from soil particles and cysts.

 

 

EXTRACTION, EGGS -- EXEG

 

Byrd, Ferris & Nusbaum, 1972 -- Journal of Nematology  4: 266-269.

The authors describe a technique for obtaining egg masses from soil by using a self-cleaning., funnel-shaped elutriator in addition to a technique that can process up to four samples at one time.

                                                                                

Chuang, 1962 -- Nematologica 7: 317-330.

A new technique for handling nematode eggs using garden snail's mucous was described. Ferlilized eggs in different stages of development were placed in water on a slide and mixed with fresh snail's mucus.The mucus with the eggs was fixed in 'Petrunkewitch' fluid, dehydrated and embedded in ester wax. Sections were stained in Mayer's haemalum and counterstained in Mayer's carmalum .

 

Dunn, 1973 -- Journal of Nematology 5: 73-74.

The extraction of eggs of  Pratylenchus penetrans from alfalfa callus tissue was accomplished by processing for 30 seconds in a food blender the tissue  immersed in 200ml of 30% sucrose solution, with subsequent centrifugation in a food blender. The technique also has been used to extrract eggs of other species cultured in vitro. and, with modifications, the extraction of eggs from field soil.

 

Flegg & McNamara, 1968 --  Nematologica 14: 156-157.

Soil is shaken for 2 min in sucrose solution (s.g. 1.18), placed into centrifuge tubes and spun for 6 min at about 1700g.. The supernatant is then immediately poured into water.After 1 to 2 hrs of settling, all but the bottom 100ml of water is removed and that remaining water is examined for eggs.

 

Gooris & D'Herde, 1972 -- Publication of the Ghent State Agricultural Research Center.

 In a process which extracts Meloidogyne juveniles and eggs, soil is mixed with water, sieved, and made up in volume to 2.5 liters with water. The mixture is then mixed by a current of compressed air. A 1/2 liter sample is withdrawn and divided into organic and mineral fractions. The organic fraction is processed in a Waring Blendor for 2 min, then centrifuged at 1800g for 5 min. The supernatant is discarded, sucrose solution (sp.gr.1.15) is added, contents are mixed and again centrifuged. The supernatant is poured through onto a sieve. The supernatant retained on the sieve is rinsed with water and washed into a beaker for examination.

 

Jones, 1945 -- Annals of Applied Biology 32: 351-380. 

In working with soil populations of the beet eelworm, Heterodera schachtii Schmidt,  the author mixed a suspension of eggs by bubling compressed air into the suspension.. Using a siphon, he forced over drops of  known size in which the eggs and larvae could be counted.

 

McClure, Kruk, & Misaghi, 1973 -- Journal of Nematology 5: 230.

A method for obtaining  quantities of clean Meloidogyne eggs is described. The procedure combines treatment using sodium hypochlrite with differential centrifugation. Eggs treated by this method (0.4% available chlorine) hatched normally with the hatched larvae readily penetrating and developing  in the roots of tomato seedlings.

 

Zacheo & Lamberti, 1974 -- Nematologia Mediterranea 2: 55-59.

The supernatant from a settled soil water mixture is decanted into a centrifuge tube, root tissue similarly treated is placed in another centrifuge tube. Two grams of Kaolin are mixed in each tube which, after balancing, are centrifuged at 5000g for 2 min. The supernatant is poured off and the plug is mixed with 1 molar sucrose solution and recentrifuged for 2 min. Nematode eggs are recovered when the supernatant is poured on to a 5 micon aperture sieve in a funnel connected to a suction pump.

 

 

EXTRACTION,  ELUTRIATORS -- EXEL

                                                                      

Byrd, Barker, Ferris, Nusbaum, Griffin, Small & Stone, 1976 -- Jour.Nematol. 8: 206-212.

Two semi-automatic elutriators for assaying soil samples for nematodes are described A four-unit elutriator combines conventional extraction methods with "turbinate" sample splitters for collection of nematodes, and 250-425 micron sieves are used with a variable speed motorized sample shaker. Several secondary features are described.

 

D'Herde & van den Brande, 1964 -- Nematologica 10: 454-458.

A method for the quantitative extraction of Xiphinema and Longidorus spp from strawberry fields in Belgium is presented. The extraction method allows a quantitative recovery of the nematodes within a few hours. A excellent schematic diagram of the successivew stages in the  separation process is given.

 

Durie, 1959 -- Journal of Helminthology 33: 189-196.

A new technique for the recovery of Trichostrongyle larvae from soil and pasture samples is described. The technique is based on the difference in density of larvae and debris under the influence of an upward stream of water flowing at a gradually decreasing rate. The results from trials with known numbers of larvae  gave an average recovery of of 74% from pasture, and 78% from soil.

 

Seinhorst, 1956 -- Nematologica 1: 249-267.

Two types of apparatus are described and figured for the extraction of nematodes from soil by use of the Baerman Funnel, by elutriating and by sieving.

 

Seinhorst, 1964 -- Nematologica 10: 87-94.

In a report dealing with methods for the extraction of Heterodera cysts from not previously dried soil samples, an elutriator for the separation of cysts from soil is described and illustrated.  Cysts mixed with organic debris are obtained which are further processed using three parts acetone and one part carbon tetrachloride or by using a modification of Buhr's method.

 

Tarjan, Simanton & Russell, 1956 -- Phytopathology 46: 641-644.

A soil washing apparatus essentially including a system of spray jets , a glass tube for observing the washing procedure, and a system of inclined sieves for  selectively trapping the nematodes in a minimum of debris was illustrated. Results of three experiments indicated that approximately 96 % of the nematodes in a given sample can be obtained by washing for 10 minutes.

 

EXTRACTION, ERLENMEYER FLASK -- EXES

                                                          

Guenther, 1967 -- Nematologica 12: 641-642.

The author illustrates a simple set-up of potted plants and collection funnel for obtaining  Heterodera larvae.  The article is written in German.

 

Hooper, 1961 -- Nematologica 6: 336.

The author proposes a modification to the double Erlenmeyer flask method proposed by Seinhorst, 1955 in which the funnel was attached to the flask by a rubber sleeve. A description of the modification and illustrations make up the short ommunication.

 

Seinhorst,1955 -- Tijdschrift voor Plantenziekten 61: 188-190.

A system of  inverted 2-liter Erlenmeyer Flasks, funnels and beakers for separating nematodes from soil is described and figured. 

 

EXTRACTION, FILTRATION --EXFT

                                                                      

Brodie, Blow, Brace, and King, 1976 --  Plant Disease Reporter 60: 936-938.

A cylindrical fiber filter of 3.5cm diameter is closed at one end so as to form a "filter sock".  This is placed in a 600ml beaker. Nematode cysts and debris collected on a 60-mesh sieve are washed through a funnel into the "filter sock".  Once the sock has dried, the collected cysts may be removed.

 

Godfrey, 1935 -- Phytopathology 19: 611-629.

In a series of 3 tests attempting to determine the influence of sieve mesh size on the numbers of Ditylenchus dipsaci extracted from soil samples, Godfrey first used an automatic stirrer to break up clods of soil in water.. He then passed the suspensions through two 50- micron, two 75-micron and two 105-micron sieves, then through a

bank of four 50-micron sieves.

 

Kleiburg, 1960 -- Nematologica Supplement II: 22-27.

The effect of drying nematodes on cotton wool filters was evaluated. It was concluded that a high recovery of nematodes can be obtained only when the filter is dried gradually at 20C  and a relative humidity of 40-50%.

 

Lewis, 1960 -- Phytopathology 50: 240.

A satisfactory method was developed for extracting living preadult larvae of Ditylenchus dipsaci in large numbers from dried infected onion scales.  The dried scales were placed iin a sieve suspended in water.

 

Miller, 1957 -- Plant Disease Reporter 41: 192-193.

A quick filtration technique is described using 5-ounce paper cups and "Scotties" facial tissue. The bottom is cut out of the paper cup leaving the rim for strength. Filter pads are made from the facial tissue are affixed to the bottom of the paper cups and secured with rubber bands. Either an appropriate portion of the soil sample under investigation or its washed residue are placed in the cups.

 

Oostenbrink, 1954 -- Mededelingen van de Landbouwhogeschool en de Opzoekingsstations van de Staat te Gent, Belgium 19: 377-408.

The author proposes the use of  cottonwool filters for eliminating excess water and fine soil particles in a very short time after washing soil for extracting nematodes. This process is also claimed to be useful for extracting nematodes from plant tissue.

 

Stemerding, 1964 -- Verslagen Mededelingen Plantenziektenkundigan Dienst 141: 170-175.

A blender-cottonwool filter method for collecting migratory endoparasitic nematodes from roots is a combination of the essential parts of different techniques. Roots are macerated in a electrical blender and placed on a cotton-wool filter through which the nematodes pass into water.

 

Townshend, 1963 -- Nematologica 9: 106-110.

The Oostenbrink direct cottonwool filter extraction method was modified so as to preserve laboratory space. The method employs aluminum cake pans, plastic embroidery hoops and cottonwool filters on cheesecloth which are clamped taut within the plastic rings. Soil samples are spread on the sieves in the pans, water is added, the pans are stacked and covered with a hood.

 

EXTRACTION, FLOATATION -- EXFL

                                                                      

Bird, 1959 -- Journal of Helminthology  3: 185-192.

The author is believed to be the investigator who introduced floatation of nematode cysts from the soil.  This procedure proved to be an improvement over the laborious and tedious method of direct examination of the sample.

 

Buhr, 1954 -- Nachrichtenblatt Deutschen Pflanzenendienst 8: 45-48.

The soil sample is placed within a glass cylinder with a strip of filter paper fitted arond the wall. When water is added and stirred, cysts and other debris rise to the surface and adher to the filter paper which is removed, unrolled.  Cysts can be seen within the debris that clings to the paper.

 

Byrd, Barker, Ferris, Nusbaum, Small & Stone.-- 1976. J. Nematology 8: 206-212.

Two semi-automatic elutriators for extracting nematodes and certain fungi from the soil are described. The first is a 4-unit elutriator which combines conventional extraction methods with several major features. The second elutriator operates on similar principles but is cheaper to build and requires greater operator participation.

 

Byrd, Nusbaum & Barker, 1966 -- Plant Disease Reporter 50: 954-957.

A rapid floatation-nematode extraction procedure is reported involving the use of flocculating agents instead of a centrifuge."Separan", an organic compound, was found to be the most suitable flocculating agent used for most soils. The method requires only 1-3 minutes per sample and compares favorably with other standard procedures.

 

Cagnolo, de Doucet, Doucet & Rienzo, 1999 -- Nematology 1 : 757-758.

A method is described which is based on the floatation technique for nematode exraction from the soil  using a modified decantation container. 

 

Carta & Carta, 2000 -- Nematology  11: 201-210.

The standard sugar technique used to isolate nematodes from soil and debris is refined by centrifuging nematodes in several solutions of increasing specific gravity. The data is mathematically analysed.

 

Caveness & Jensen, 1955 -- Proc. Helminthological Society Washington 22: 87-89.

A modification of  an earlier method of extraction by centrifical-floatation is proposed. The modification makes possible nematode egg isolation and recovery of more nematodes, in addition to other advantages.

 

Cobb, 1924 -- Journal of Parasitology 11: 103-105.

An apparatus is illustrated for removing nematodes from soil by floatation. Soil is introduced into the top of a long revolving tube. Water from the bottom of the tube fills the tube "at a rate slightly in excess of that at which nemas sink". The nematodes float off a spout near the top of the tube into a receptacle.

 

Crofton, 1954 -- Parasitology  44: 313-324.

A flotation technique was employed by the author in  which he use zinc sulphate with a variety of collecting techniques for obtaining nematodes from grass samples.

 

Durie, 1959 -- Journal of Helminthology 33: 193-208.

A new technique for the recovery of Trichostrongyle larvae from soil and pasture samples is described. The technique is based on the difference in density of larvae and debris under the influence of an upward stream of water flowing at a gradually decreasing rate. The results from trials with known numbers of larvae  gave an average recovery of of 74% from pasture, and 78% from soil.

 

Minderman, 1956 -- Nematolgica 1: 216-226.

The author separated nematodes in samples from soil and humus by immersion in a strong magnesium sulfate solution ( specific gravity 1.25) followed by centrifuging.

 

Miller, 1957 -- Plant Disease Reporter 41: 194.

A method for the quick separation of nematodes from soil samples follows the same procedures as described by Caveness & Jensen, 1955 except that the nematodes in the sugar solution supernatant are not allowed to settle but are poured directly on a 325-mesh sieve, rinsed with water and examined.

 

Oostenbrink,  1960 -- In Nematology, Sasser & Jenkins eds., University of North                                                                                    Carolina Press, Chapel Hill, Chapter 6, pp. 85-102.

Modified floatation apparatuses are described and figured. As in other apparatuses of this general type,  an  introduced soil sample is agitated by an upward stream of water that delivers nematodes, and light debris through an outlet tube onto a nest of screens.

 

Rodriguez-Kabana & King, 1972 -- Plant Disease Reporter 56: 1093-1096.

The authors substituted sugarcane molasses for sucrose in the preparation of the solution used for the extraction of nematodes from the soil by the floatation-sieving method. In paired extractions involving over 10 different soil samples, the molasses solution yielded equal  or higher nematode numbers than the standard sucrose solution usually used.

 

Rodriguez-Kabana & King, 1975 -- Journal of Nematology 7 : 54-59.

The efficiency of extraction of nematodes by floatation-sieving using molasses and sugar and by elutriation was studied.  Blackstrap molasses was investigated as an economical substitute for sucrose used for extracting nematodes from soil by the floatation-sieving technique. It was found that the molasses solution extracted greater numbers of nematodes than the standard 1.0 M sucrose solution.

 

Seinhorst, 1962 -- Nematologica 8:  117-128.

Modifications were suggested for the elutriation method for extracting nematodes from soil.

Various aspects of the illutriation method such as width of tubes of the elutriator, methods for the extracton of nematodes from soil, and use of special banks of sieves are covered. Numerous photos and drawings accompany the article thus enabling the reader to more accurately understand the technicalities in the descriptions.

 

 

Talame, 1965 -- L'Italia Agricoltori 102: 3-8.

A new type of floatation apparatus for the recovery of nematodes from soil samples (in Italian with English summary) is described with a reference to the nonavailability of such devices which are constructed in Italy. The author further points out that devices often are constructed for specific needs of each particular laboratory, and often not useful for Italian soils.

 

Trudgill, Evans and Faulkner, 1960 -- Nematologica 18: 469-475.

A perspex plastic tube 38 cm long and of 63 mm inside dameter uses a controlled water current passing through a sintered plate to segregate nematodes from soil particles. The apparatus is useful for extracting all types of nematodes, and Heterodera females in particular.

 

Wallace, 1956 -- Nematologica 1: 227-238.

For purposes of obtaining accurate extimates of cyst population, Wallace proposed use of a dispersing agent in the wash water to breakup soil crumbs which might be containing cysts. Calgon was added to the water so as to achieve the desired results.                

 

Willams & Winslow, 1955 --  in Kevin, Soil Zoology, Proc. Univ. Nottingham, Second Easter School in Agric. Science, Butterworth, London, pp 375-384.

In this floatation method for obtaining nematode cysts, material caught on a 60-mesh sieve is washed onto filter paper in a funnel. The cysts are then microscopically recovered  from the filter paper using a turntable, and then transferred to a watch glass containg a little water.

 

Winslow, 1955 -- Journal of Helminthology 29: 49-54.

Towards the goal of obtaining uniformly-sized cysts, small samples of heavily infested soil were processed by floatation in a conical flask  The floated material was then decanted into a suction funnel containing removable sieves of the different mesh sizes desired.

 

EXTRACTION,  INCUBATION -- EXIN

                                                                      

Dolliver, 1959 -- Phytopatology 49: 537.

While investigating  Pratylenchus infection of grass roots, the author found that increasing the incubation temperature to 24C increased the rate of emergence.  He also found that more nematodes emerged from roots in distilled water than from roots in water containing dilute salt solution.

 

Godfrey, 1931 -- Phytopathology 21: 323-329.

Large quantities of root-knot nematode larvae were collected by incubating chopped infected roots on a wire screen within covered Petri dishes.  There was sufficient water within the dishes to keep the roots adequately moistened by capillary action.

 

Tarjan, 1960 -- Plant Disease Reporter 44: 31-35.

It was found that use of 1-mil thick plastic bags would serve well as incubation chambers for obtaining maximal numbers of burrowing nematodes, Radopholus similis, from infested soil and root samples. Storage of collected samples in the sealed plastic bags for 6-7 days at 75F can result in increase yield as compared to yields from non-incubated samples.

                                                                      

 Tarjan, 1972 -- Plant Disease Reporter 56: 186-188.

A plastic bag incubation technique was developed and statistically shown to be effective for evaluating the presence and relative number of citrus nematodes, Tylenchulus semipenetrans, in excised Citrus jambhiri roots. It was found that a sample of infested roots weighing at least 5 grams, unwashed  but from which adhering soil had been gently shaken, incubated at 21.3C in thin plastic bags to which had been added about 10ml of 3% hydrogen peroxide yielded the highest number of nematodes.

 

West, 1957 -- Plant Disease Reporter 41: 600-602.

The method proposed was actually a modification of the method proposed by Young, 1954. It was reported that incubating burrowing nematode-infected roots directly in water gave quicker results with higher recovery rates.

 

Young, 1954 -- Plant Disease Reporter 38: 794-795.

The very useful and convenient method of incubating roots containing nematodes in closed, moist  containers such as Mason jars was proposed.  The utility of this method became quickly apparent in the rapidity of the process and the low cost of materials needed. After a period of one to several days incubation the evacuated nematodes are washed from the roots

 

EXTRACTION,  MACERATION -- EXMC 

 

Dropkin, Smith & Myers, 1960 -- Nematologica 5: 285-288.

Roots infected with Meloidogyne spp are immersed in flasks containing pectolytic enzymes at room temperature and are kept at constant agitation in a wrist-action shaker resulting in maceration of the roots..The nematodes are killed but their bodies remain intact. Other chemical agents such as periodic acid or a mixture of chromic and nitric acids also macerate the infected roots.

 

Stynes, 1976 -- Nematologica 22: 376.

A routine method for extracting nematodes from fixed plant roots stipulates that after storage in FAA fixative, the roots are heated to 65C for 24 hours in 0.07 M ethylene diamine tetra-acetic acid adjusted to a pH of 10.5.  Mechjanical maceration liberates the nemas from the roots.

 

Taylor & Loegering, 1953 -- Turrialba 3: 8-13.

During investigations of nematodes associated with root lesions in abaca, the authors placed about 5 grams of washed infected roots, cut  into centimeter-long pieces, into about 80ml water in a Waring Blender. The blender was operated for 10 - 20 seconds and the resulting mixture was washed through 60- and 200-mesh sieves. The residue on the 200-mesh sieve was washed into a beaker and examined for nematodes.

 

Willams & Winslow, 1955 -- in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pp 385-389.

Washed infested roots are stained in acid fuchsin for one minute, washed, and partially dried. They are then cut into 1 cm lengths, weighed and placed in an M.S.E. 'Atomix' homogenizer (12,000 rpm) with 80 ml water for 20 seconds. The resulting suspension is passed through 60, 100- and 300-mesh sieves and then microscopically examined.

 

 

EXTRACTION,  MISCELANEOUS --  EXMI    

                                                   

Adamo, Madamba & Chen, 1976 -- Journal of Nematology  8: 178-179.

Extracting the rice white-tip nematode, Aphelenchoides besseyi, with match sticks was proposed. Presoaked, wet matchsticks were placed in a vertical position in dishes of agar. Recovery of nematodes was compared to other methods. Whereas high numbers of nematodes were recovered from all methods, the greatest number was obtained by using the match-stick method.                

                

Ayala, Roman & Tarjan, 1963 -- Jour.Agric., University of Puerto Rico 47: 219-225.

 Four methods for extracting nematodes from a range of soils were evaluated.The sugar-floatation technique was more effective with sandy soils while the othere three methods (using two Oostenbrink elutriators and a sieving Petri-dish method) were more effective with clay soil.

 

Beaver, 1953 -- American Journal of Tropical Medicine and Hygiene 2: 102-108.

In order to trap hookworm larvae, the author place damp absorbent cotton pads in direct contact with the soil. The larvae migrated up into the moist pads which then were placed in water in flasks or a Baemann funnel.

 

Buecher, Yarwood & Hansen, 1974 -- Nematologica 19: 565-566.

A screen to separate young larvae of Aphelenchus avenae from adults and eggs envolved a sieving method designed to allow easy separation of a continual supply of newly hatched larvae. Over a period of nine days, 336 L-2 juveniles were collected using a specially

designed screen that separated L-2 and L-3 larvae from eggs and L-4 larvae and adults.

 

Chapman, 1957 -- Plant Disease Reporter 41: 836-841.

Investigating the effects of aeration and incubation of roots infected with Pratylenchus, the author determined that aeration was more important than temperature, particularly with large amounts of roots or large roots.

 

Cobb, 1898 -- Misc. Publ 215, Dept. of Agriculture of New South Wales, 62pp.

Using the principle of differential sedimentation of soil particles with careful decantation of the wash water, the author successfully collected nematodes in New Zealand soils.

 

Dropkin, 1959 -- Phytopathology 49: 18-23.

The efficiency of extracting juveniles of the root-knot nematodes from roots was increased by the simple expedient of holding plants at the wilting point for two weeks. The juveniles were prevented from hatching from the eggs but were not prevented from developing normally.

 

Dropkin & Smith, 1960 -- Phytopathology 50: 634.

Recovery of nematodes from infected roots by enzyme preparations reported the use of juice from potato rotted by Erwinia carotovora which was used to macerate nematode galls.

Commercial enzyme preparations (1% pectinol 10M at pH 4) macerated roots more slowly than the undiluted supernatant.

 

Freckman, Kaplan & Van Gundy, 1977 -- Journal of Nematology 9: 176-181.

Nematodes found in desert soils are fixed before extraction from the soils by adding 5% formaldehyde @40C or 4% glutaraldehyde @22C (with Dow 'Separan' added) to the fixative. After mixing of the soil with the fixative and settling for 30 seconds, the supernatant is decanted thru a 26um-aperature sieve.

 

Goodey, 1945 -- Journal of Helminthology 21: 45-59.

The simple and quite practical method of soaking seeds in water for an extended period was employed by the author who recovered specimens of  Ditylenchus by soaking grass seed overnight in an incubator.

 

Guenther, 1966 -- Nematologica 12: 641-642.

A simple method for obtaining larvae of  Heterodera is described and depicted. It is a modified Baermann method that involves the entire potted plant over a specialized funnel apparatus.  The paper is written in German.

 

Hohmaier & Meyer, 1937 -- Science 86: 568.

Since Trichinella larvae in the stomach move to the small intestines of the host, this was used in proposing a filter method for clean isolation of the larvae. A glass funnel supported in a stand has its neck inserted into a hole in a rubber stopper. The stopper is inserted securely into a centrifuge test-tube. A cylindrical jar, a fitting glass cover, 4 layers of gauze and a rubber stopper are also used. Directions are given for use of these materials. The end result is the collection of living larvae free from coarse particles.

 

Hussey, 1971 -- Journal of Nematology 3: 99-100.

Quantities of living Meloidogyne females were obtained by placing excised galled roots in a 50% Pectinol 59L solution and agitating in a reciprocal shaker at 180 oscillations/min for approximately 10 hrs at 25C.  The treatment insured the collection of dislodged nematodes which were then caught in a 60-mesh sieve, suspended in 20% sucrose solutions and centrifuged at 1000g for 10 min.

 

Kauzal, 1940 -- Australian Journal, Council Scientific Industrial Research 13: 95-106.     A simple device for the extraction of nematodes from plants or soil relies upon lateral movement of nematodes from the sample in an inner Petri dish to an outer one which is filled with water and acts as a nematode trap. The assembly is covered to insure high humidity.

 

Krusberg, 1960 -- Phytopathology 50: 9-22.

Homogenates and extracts of Ditylenchus triformis, D. dipsaci and Pratylenchus zeae were assayed for hydrolytic, respiratory and terminal oxidative enzymes by techniques utilizing viscosity, titration,  colorimetric and spectrophotometric measurements. The author  used the principal of centrifical force to accelerate the separation of sinking and floating components that previously had been incompletely separated using another method.

 

Minderman, 1956 -- Nematolgica 1: 216-222.

Nematodes were isolated from oak leaf litter by placing a sample in water on a piece of cottongauze in a beaker , shaking for one minute followed by 4 minutes rest, during a 24-hour period. The number of nematodes obtained during a 24-hour period had to be multiplid by the factor 1.25 to get approximately the number of nematodes originally in the sample.

 

Myers, Chen and Balasubramanian, 1971 -- Journal of Nematology 3: 85.

Two methods for separating larvae of uniform length from mixed adults and larvae of  Panagrellus redivivus are presented by the authors who propose that sometimes it is advantageous to initiate inoculated cultures with larval stages of uniform size. The article does not contain any illustrations.

 

Potts, 1910 -- Quarterly Journal of Microscopical Science 55: 433-484.

 A small piece of meat was placed in soil which successfully attracted  large numbers of  saprobic nematodes which then could be cultured.independently.

 

Rohrbacher, 1957 --  Proceedings Helminthological Soc. of Washington 24: 24-25.

The author increased the efficiency of extracting nematodes from the surface of plant fragments by the addition of a non-ionic emulsifier, Triton X-100 (Rohm and Haas) added  at the rate of 0.5  ml/liter of water in the funnel

 

Wallace, 1956 -- Nematologica 1 : 227-238.

For purposes of obtaining accurate extimates of cyst population, Wallace proposed use of a dispersing agent in the wash water to breakup soil crumbs which might be containing cysts. Calgon was added to the water to achieve the desired result.

 

Webb, 1971 -- Nematologica 17: 173-174.

An apparatus for the extraction of  nematodes from sterile culture is described and illustrated. Nematodes extracted by the apparatus were alive and did not seem to lack oxygen.

 

Webb, 1975 -- Nematologica 21: 408-409.

An apparatus essentially based on one reported by the same author four years earlier is described and figured. This modified apparatus extracted Pratylenchus  spp.from excised sweet corn roots  and from alfalfa callous more efficiently than did the earlier model.

 

EXTRACTION,  MISTIFIERS  -- EXMS

       

Seinhorst, 1950 -- Tijdschrift voor Plantenziektenkundige Onderzoek 56: 289-384.

This work described the principle of spraying  water on tissues suspended on a screen in a funnel within a funnel. The runoff water with nematodes is caught in a shallow tray. The author also illustrates two simple instillations using a spray mist.

 

Sturrock, 1961 --  Journal of Helminthology 35: 309-314.

This paper on the quantitative use of the Seinhorst "mistifier" to recover nematodes from soil, faeces and herbage introduces a modified form of the apparatus.  Results are presented indicating the high rate of recovery in a relatively short time.as well  as the recovery from a known number of nematodes and a moderate degree of variation between recoveries from a replicated series of mistifier units.

 

Webster, 1962 -- Nematologica 8: 245-251.

In studying the quantitative extraction of Ditylenchus dipsaci  from plant tissues, the author points out that nematode loss from the collecting tray of a modified Seinhorst mistifier increased with time. Nematodes were successfully extracted from chopped narcissus bulbs and leaves. Particle size of the  chopped leaves was not critical. Extraction for 48 hours resulted in recovery of  at least 85% of the nematodes.

 

EXTRACTION,  SIEVING -- EXSV 

 

Anderson & Yanagihara, 1955 -- Phytopathology 45: 238-239.

An apparatus for extracting free-living nematodes from the soil is described. Its components consist of a 6-mesh screen which fits into a paper cup which is supported directly above a tin can.

 

Bravo, 1973 -- Agronomia Lusitania 34: 293-297.

A simple method of making sieves for the extraction of nematodes from soil debris, free of metallic components, is described. Nylon gauze of the appropriate mesh is pressed in between two rings obtained from a cylindrical polyethylene box.

 

Buecher, Yarwood & Hansen, 1973 -- Nematologica 19: 565-566.

The authors investigated various pore-size screens in order  to separate young larvae of  Aphelenchus avenae from adults and eggs. They found that a screen made from a metal sheet with holes of 15 micrometers diameter placed 120 micrometers apart separated L-2 and L-3 larvae from from eggs, L-4 larvae and adults.

                                                                      

Christie & Perry -- Proceedings Helminthological Society of Washington 18: 106-108.

The authors state that neither sieving soil nor the Baermann Funnel method result in the collection of nematodes in clear water reasonably free from debris. They propose a combination of the two methods which they have been using with good success.

 

Cobb, 1898 -- Misc. Publ 215, Dept. of Agriculture of New South Wales, 62pp.

The author employed the use of differential sedimentation accompanied by decantation for obtaining nematodes.

 

Cobb, 1918 -- Estimating the nema population of soil. Agric.Technology Circ. 1, pp 1-48.                

For the purpose of using sieves for collecting nematodes from soil, the use of Miller's bolting silk is suggested for constructing the finest sieve. This type of sieve is compared to wire sieves and is found to be superior because it can be dismantled and the cloth washed. With special weaving, the strands one way can be double and the other way single resulting in a mesh that is oblong and more efficient than can be obtained with wire.

 

Dalmasso, 1966 -- Rev. Ecol. Biol. Soil III: 473-478. (In French)

A simple method of exracting nematodes from the soil is described, depicted and tested for nematodes belonging to the orders Tylenchida, Dorylaimida, Rhabditida and other orders of nematodes.  The efficiency of extraction was tested against the Seinhorst illutrator using different types of soil.

 

Dickson & Struble, 1965 -- Phytopathology 55:497

A sieving-staining technique for the extraction of egg masses of  Meloidogyne incognita

from soil is described whereby the soil is washed and the separated eggmasses collected.

These are then stained with a 2% Phloxine B for at least 5 minutes

 

Flegg, 1967 -- Annals of Applied Biology 60: 429-437.

 For extracting species of Xiphinema and Longidorus from a variety of soils,  the Cobb decanting and sieving technique was modified using a bank of sieves each of a 150 micron aperture before final separation from inert debris in a Baermann funnel.

 

Hellinga, 1942 -- Mededelingen Instituut Rationale Suikerproductie 12: 163-182.

The author first processed soil by washing the soil sample and passing through a course sieve in order to remove the coarser soil components. The mixture was then passed through a finer sieve. The retained cysts were then washed off into a dish and the

floating cysts were collected.

 

Kleijburg, 1960 -- Nematologica Supplement II: 22-27.

A series of tests were conducted evaluating the mesh size of sieves for recovery

Ditylenchus dipsaci from a lighter soil as well as soil which had been fortified with nematodes from diseased onion.

 

Mishra, Vijayalakshmi & Seshadri, 1977-- Indian Jour. Nematology 5:259-260.

After sieving soil extracts into a beaker, the authors decanted the supernatant through a sieve with 50 micron openings and the process was repeated  3 times on the residues left in the sieve.

 

Moskaluk & Hawn, 1973 -- Journal of Nematology 5: 72.

A mechanical sieving device for the extraction of nematodes from aqueous suspensions of soil is described and illustrated. The novel feature of the apparatus is the attachment of a variable speed, electric stirring motor fitted with a short crank barthat strikes the top rim of the sieve thus agitating it.

 

Pitcher & Flegg, 1968 -- Nematologica 14: 123-127.

An improved final separation sieve for the extraction of plant-parasitic nematodes from soil debris is proposed by the authors. The sieve is free of metallic components and adhesives. Use of such sieves prevents the mortality, especially  of Dorylaimid nematodes, caused by metal sieves.

 

Seinhorst, 1956 -- Nematologica 1 : 249-267.

Two types of apparatus are described and figured for the extraction of nematodes from soil by use of the Baerman Funnel, by elutriating and by sieving.

 

Seinhorst, 1962 -- Nematologica 8: 117-128.

Clay soil was dispersed in water without any damage to the nematodes present by using a "vibromixer"  Methods are described for the extraction of nematodes less than 2mm from soil and photos of special banks of sieves for nematode suspensions are presented.

 

Simon, 1957 -- Nematologica 2: 434-440.

A simple, portable apparatus consisting of two sieves and specifically designed for work in the field was used for the isolation of  Heterodera cysts.

 

Twinn, 1962 -- in Progress in Soil Nematology, Butterworths, London pp. 261-267.

In an article on the extraction of free-living nematodes from forest soil and litter, the author modifies Minderman's original shaker technique by washing each of the subsamples at hand in about 40ml of water by mechanical stirring for one minute, when the leal pieces were filtered off by pouring the suspensions on to 30 mesh nylon gauze circles.

 

Yeates, 1968 -- Nematologica 14: 456-458.

A simple method of making sieves, using nylon gauze and PVC tubing, is described . .  PVC pipe and nylon guaze are the two materials used.  The illustrations accompanying the article

shows how the components are fitted together.

 

 

EXTRACTION,  SUCTION -- EXSU

 

Alam, 1976 --Nematologica Mediterranea  4: 245-247.

A modified device for the rapid selection of soil and water nematodes from mixed suspension is described and figured. The main principle is use of suction pressure to capture nematodes with a needle bearing a  fine point.

 

Cobb, 1918 -- Estimating nema population of soil.Agricul.Technol. Circular 1, pp 1-48.

A suction apparatus for separating organisms and organic particles from mineral matter in the soil is described and illustrated. Gravity is used as the suction source.  Residues from the soil in an evaporating dish are agitated resulting in the nematodes being in one plane of descent due to their specific gravity and thus segregated from the other components.

 

Khan, Saxena & Alam, 1973 -- Indian Journal of Nematology 2: 79-80.

A suction-type nematode picking apparatus is described with an accompanying labeled photograph. The assembly allows for obtaining about 40-50 nematodes per minute.

 

Valleau & Johnson, 1947 --Phytopathology 37: 838-841.

The relationship of meadow nematodes, Pratylenchus spp, to brown root-rot of tobacco was investigated. In the course of their investigations, the authors proposed using a nematode-collection method that has become a standard procedure in later years.  They found that nematodes could be collected more efficiently using a capillary pippette into which the nematodes were quickly drawn.

 

 

          -----  F -----

 

FIXATIVES -- FIX    

 

Cobb, 1918 -- Estimating nema population of soil. Agricult. Tech.Circular 1, pp 1-48.

Flemming's Mixture is referred to as one of the best fluids  for killing and fixing nematodes.  In addition to formulas for both a strong and a weak Flemmings mixture, formulas are presented for Gibson's mixture, Bouin's mixture, acetic alcohol sublimate,

and alcoholic formol.

 

Courtney, Polley & Miller, 1955 -- Plant Disease Reporter 39: 570-571.

TAF, an improved fixative in nematode technique is described using an improved fixing and preserving fluid, triethanolamine formalin. The fluid remains unchanged for more than two years after becoming stabilized. By using this improved fixative and the described technique, it is possible to rapidly prepare nematodes of near-life resemblance in mounts that will last for at least 14 months.

 

Franklin & Goodey, 1949 -- Journal of Helminthology 23: 175-178.

A cotton blue lactophenol technique for mounting plant-parasitic nematodes is proposed. The nematodes are first "relaxed" in heated water, then fixed overnight in F.A. 4:10. They are then heated in lactophenol containing 0.01% cotton blue dye and finally mounted in slightly tinted lactophenol.       

 

Freckman, Kaplan and Van Gundy, 1977 -- Journal of Nematology 9: 176-181.

Fixation before extraction of nematodes found in desert sands can be accomplished by adding 5% formaldehyde at 40C or 4% glutaraldehyde at 22C.  The soil sample is slowly poured into the fixative while being continually stirred. 7.5micrograms/ml of Dow 'Separan' is added to the fixative , allowed to settle for 30 sec, then passed through a 26 micron aperture sieve.

 

Hoffman, 1954 -- Transactions of the American Microscopical Society 73: 328-329.

A modification is proposed of a polyvinylalcohol fixative originally proposed by Brooke and Goldman. Instructions are presented by Hoffman on how to use the fixative for small helminths and protozoa.

 

Thorne, 1925 -- Transactions of the American Microscopical Society 44: 171-210.

Discussing, in part, the subject of fixation of nematodes, the author proposed the use of hot Ditlevsen's fixative  prior to permanent preparation.

 

           ----- G -----

 

GROWTH & DEVELOPMENT --GRD         

Arant & Knapp, 1932 -- Science 75: 495-496.

A simple method for rearing and mounting hookworm larvae is described. Larvae were reared in a mixture of  2 parts charcoal and 1 part feces. Juveniles were isolated through the use of two general methods and were mounted using alscohol as a killing and hardening agent. Larval structures were more distinct when haematoxylin was used rather than Congo red, Orange G or alum cochineal.

 

Benson, 1978 -- Plant Disease Reporter 62: 68-70.

A direct versus a photographic technique as nondestructive estimates of growth response on perennial ornamentals affected with nematode decline is presented. Highly significant correlations were obtained when either nondestructive technique was compared with weight on a log - log basis. The direct technique gave slightly better coefficients of  determination than the photographic technique when compared with weight.

                                                          

Bird, 1959 -- Nematologica  4: 31-42.

 The development of  the root-knot nematodes Meloidogyne javanica and M. hapla in tomato was studied. Because the third and fourth moults occur within the shed cuticle of the second moult, the larvae can not feed and are dependent on stored energy.  Taking advantage of such peculiarities, freshly dissected larvae were studied by a modification of the  "perfusion method".

 

Blake, 1966 -- Nematolgica 12: 129-137.

The histopathological changes in banana roots caused by Radopholus similis  and Helicotylenchus multicinctus  were described.  Interactions with Fusarium oxysporum and Rhizoctonia sp. and the burrowing nematode were found to be non-pathologinic.to the banana roots.

 

Bryant, Nicholas & Jantunen, 1967 -- Nematologica 13: 197-209.

Some aspects of the respiratory metabolism of  Caenorhabditis briggsae (Rhabditidae) are presented. A method is described for the production of large numbers of the nematode free from culture medium. Various aspects of the respiratory metabolism of these worms are examined. Preparations of worms were subjected to sonic disruption as well as spectrophotometric studies.

 

Endo, 1959 -- Phytopathology 49: 417-421.

Reproduction of  Pratylenchus brachyurus on host plants for 5 months indicates that high populations can develop on corn, soybean and peanut plants. Norfolk sandy loam was found to be the most favorable soil type for infection and reproduction of the nematode on suitable host plants. Migration of the root-lesion nematode P. zeae was influenced by soil type and the presence of a suitable host plant.

 

 

          ----- H -----

 

HATCHING , USING CHEMICALS-- HMC

 

Clarke, 1966 -- Nature 211: 546.

Picrolonic Acid was found to be a hatching agent for the potato cyst nematode,Heterodera rostochiensis. The acid, at concentrations of 0.4 to 4.0 mM hatches eggs of the nematode as effectively as potato root diffusate (hatch rating 102).

 

Clarke & Shepherd, 1964 -- Nematologica 10: 431-453.

From 238 compounds tested for their hatching ability for Heterodera schachtii,  only 31

of these gave hatches equal to or greater than obtained with beet root diffusate. No correlation was found between hatching ability and redox potential for the 25 redox compounds examined.

 

Clarke & Shepherd, 1966 -- Nature 211: 546

After testing the ability of more than 400 compounds to hatch eggs of  Heterodera rostochiensis using techniques which already had been described, except for anhydrotetronic acid, we found only one compound, namely picrolonic acid, which at concentrations of 0.4-4 mM hatches eggs as effectively as potato root diffusate (hatch rating 102).

 

Clarke & Shepherd, 1967 -- Nature 213: 419-420.

Flavianic acid effectively hatched eggs of  5 different species of cyst nematodes in the genus Heterodera. A 0.6-3 mmolar aqueous solution of  flavianic acid hatched more eggs from 4 of the species than appropriate root diffusates. With H. schactii & H. rostochiensis, chain length between terminal polarizable atoms seems an important determinant of hatching activity.

 

Clarke & Shepherd, 1968 -- Annals of Applied Biology 61: 139-149.   

Of the 444 compounds tested as stimulants for hatching of the eggs of the potato root eelworm,  45 did so to varying extent.  The most effective compounds were found to be picrolonic acid, anhydrotetronic acid, and vanadates.                                                                      

 

Hartwell, Dahlstrom & Neal, 1960 -- Phytopathology 50: 612-615.

A crystalline hatching stimulant for the golden nematode was obtained from lyophilized leachings. It was extracted with absolute ethyl alcohol and precipitated in an oil-like state by adding diethyl ether. Additional preparation resulted in an active crystalline substance.

 

Massey & Neal, 1953 -- Journal Washington Academy Sciences 43: 396-401.

The authors concentrated the hatching factor by freeze-drying at high vacuum, extracting using various solvents, and also by use of chromatographic technique.

 

Rogers, 1958 -- Nature 181: 1410-1411.

The chemical stimulation to hatch of infective eggs of  Ascaris lumbricoides is reported.

The process is initiated by a hatching fluid and the eggs subsequently hatch. Optimal conditions to induce the hatching are presented.

 

Shepard, 1962 -- Nature 208: 391-392.

In a quest for artificial hatching agents for Heterodera schachtii cysts and eggs, 70 dyes from 16 dye-groups were tested on replicated batches of 100 cysts. Those dyes with a desirable hatch rating from 121 to 91 were methyl red alcohol soluble, Janus green, picric acid, Nile blue sulphate, Rivanol, Auramine O, and New Blue R.  Sugar beet root diffusate rated 100.

 

Winner, 1957 -- Nematologica 2: 126-130.

Some amino-acridines were tested for their hatching activity on Heterodera schactii eggs Stimulation of the juveniles within the cysts could not be explained by the surface-activity or redox-potentials of the compounds. The stimulating effect was not restricted to 9-amino-acridines.

 

Winslow, 1959 -- Nematologica 4: 231-238.

Tests were conducted to determine if anhydrotetronic acid stimulates larval emergence from duplicatesamples of cysts of four species of the genus Heterodera  The results suggested that chemical may be no more related to the potato nematode hatching factor than to the beet nematode hatching factor.

 

 

HATCHING, PHYSICAL METHODS -- HMP

 

Cobb, 1914 -- Journal  of  Agricultural Research 2: 217-230.

The author buried citrus nematodes, Tylenchulus semipenetrans, within small glass containers so that the nematodes could be exposed to the effects of the plants in order to study hatching and development under conditions very nearly natural.

 

Curtis, 1965 -- Nematologica 11: 213-217.

An improved method of hatching larvae of cyst forming nematodes involves an apparatus in which cysts or cysts and debris lie on a sieve in a flow of recirculating hatching medium.  The greatly improved rate of emergence and total emergence compared with standard hatching methods is thought to be caused by the increased aeration under existing conditions, and also due to the more frequent replacement of the spent hatching medium.

 

den Ouden, 1954 -- Mededlingen Inst. Rationale Suikerproductie 2: 24,101.

One of two methods dealing with Heterodera schactii describes single cysts in solution. The other suggests that up to 1500 cysts for each test solution can be used. The cysts are on filter paper which is wetted from below and thus allows for adequate ventilation.

 

den Ouden, 1963 -- Nematologica 9: 225-230.

A new method of hatching egg suspensions was proposed in a comparison between the use of free and encysted eggs in hatching and pot experiments with Heterodera rostochiensis. The method described was a modification of a method earlier described  (see Shepherd, 1959) A description and illustration of the needed apparatus is presented.

 

Ellenby & Gilbert, 1960 -- Journal of Helminthology 34: 99-102.

A convenient modification of the Dilution Hatching Trial for the potato-root eelworm proposed that the number of cysts used for each replicate be reduced to 10 instead  of a larger number.  Log. total hatch is used, instead of total hatch itself, and the mean for each dilution determined from the log. values. Reduction of the total number of cysts used permits the use of commercially available utensils to form an easily handled set-up.

 

Johnson & Shamiyeh, 1968 --Phytopathology 58: 729.

An agar-slide technique is reported  for studying hatching of Meloidogyne incognita eggs in soil. Eggs of the nematode were combined with 1.7% agar and 2 drops were spread on a microscope slide. After additional procedures, including staining, numbers of hatched and unhatched eggs were recorded.

 

Moriarty, 1963 -- Nematologica 9: 157-158.

The author depicted a nylon sieve with taut mesh so that even immersion in water would not trap air beneath the sieve.  The sieve is easy to clean and cysts are not trapped at the sides. The sieves fit conveniently into 10 cm Petri dishes.

 

Shepherd, 1959 -- Nematologica 4: 161-164.

A simple apparatus to be used for hatching tests has been found to increase the rate of larval emergence of  Heterodera schachtii, presumably due to better moisture and aerobic conditions.

 

Steele, 1976 -- Journal of Nematology 8: 23-25.

Rate of hatching of  Heterodera schachtii was greatly increased by placing cysts in sieves enclosed by small disposable cups (obtained from Hykro Pet Industries, Denmark and Thunderbird Container Corp., Texas).

 

Viglierchio, 1961 -- Phytopathology 51: 330-331.

The author proposes a simplified technique for hatching tests of Heterodera schachtii. Stating that a desirable feature would be the incorporation of the best features of several hatching test techniques with a minimum time expenditure per replicate, he proposes a method which is "a step in the right direction". The method is described and an illustration shows the equipment used.

 

 

HATCHING , ROOT  DIFFUSATES-- HMR

 

Robinson & Neal, 1956 -- Phytopathology 46: 665.

In studying the influence of hydrogen ion on the emergence of golden nematode juveniles, it was found that the optimum pH for emergence was between 2.0 and 3.0 for aqueous solutions of hydrochloric, fumaric or citric acids. When tomato plant leachings were diluted 10-fold there was a stimulation of hatch at pH 2.5.

 

Schmidt, 1930 -- Archiv fuer Pflanzenbau 3: 420-464.

As a test to distinguish between various strains of the potato root eelworm,Heterodera rostochiensis ,  root exudates were used and is a procedure still being used.

 

Steele & Fife, 1969 -- Journal of  Nematology 1: 223-226.

The authors tested the effect of liquid nutrient culture on the hatching activity of sugar beet root diffusate for  Heterodera schachtii. They concluded such a method provides a way of obtaining hatch-stimulant free of soil contaminants. Root diffusate, concentrated fifty-fold or dried by vacuum distillation  retained hatching ability.

 

Viglierchio & Lounsberry, 1957 -- Phytopathology 47: 536-537.

There was an increase in larval emergence when egg masses of Meloidogyne hapla were exposed to excretions from  Rutgers tomato seed radicles. The hatching stimulus was statistically significant and evident within 24 hours. It was noted that the directional movement of the larvae may be a reaction to a cheotactic agent coming from the roots of the tomato seedlings.

 

Widdowson, 1958 -- Nematologica 3: 108-114.

The author investigated various aspects of root diffusate production in water culture. He took into consideration the pH, how frequently the nutrient solution should be changed, and the length of time to allow the root diffusates to accumulate.

 

Willams & Winslow, 1955 -- in  Kevin, Soil Zoology, Proc. Univ. Nottingham Second Easter School in Agric. Science, London, Butterworth,  pp 380-382.

"Hatchability" of Heterodera eggs may be determined by incubating cysts in root diffusate collected from a host plant known to stimulate larval emergence. A sample of 100 or more cysts in one ml of root diffusate is incubated at 25C for 3 weeks or

longer. At intervals, the spent hatching fluid containing  larvae is drawn off with a fine tipped pippette leaving the cysts to which fresh hatching fluid is added. 

 

 

HATCHING,  MISCELLANEOUS -- HMS

 

Dahlstrom, 1956 -- Dissertation Abstracts 16: 17-19.

Biochemical studies on the golden nematode (Heterodera rostochiensis) hatching factor involved experiments in which the author  found that the fungus, Aspergillus awamori, responded to concentrations of the hatching factor by increased mold pad weight and increased acidity of the medium.

 

Ellenby, 1943 -- Nature 152: 133.

The author modified the "single cyst technique" described by Gemmell, 1940 by placing vaseline circles in a Petri dish. Each circle contained the test solution described by Gemmell and a single cyst. Moist paper in the lid maintained the necessary moist atmosphere.

 

Ellenby & Gilbert, 1957 -- Nature 180: 1105-1106.

A technique in which the hatching factor is assayed by its cardiotonic activity is described. Within limits, the response of perfused frog heart agreed with dilution and results of the usual larval hatching technique.

 

Fenwick, 1943 -- Journal of Helminthology 21: 37-41.

The author made additional improvements to the technique proposed by Gemmell, 1940. It was suggested that the individual dishes made by heating coverslips over a carbon rod could be otherwise used. These miniature dishes which hold about 0.2cc are fixed to glass plates and are placed 50 dishes to a 15cm Petri dish during the tests.

 

Fenwick, 1949 -- Journal of Helminthologyy 23: 157-170.

From an experiment counting the total hatch from each individual cyst of the potato-root eelworm,

it was concluded that hatching of bulk samples of cysts would be preferable, the size of the

bulk sample being as large as possible.

 

Fenwick, 1951 -- Journal of Helminthologyy 25: 49-56.

In ongoing investigations on the emergence of larvae from the cysts of the potato-root eelworm, a shortened method for the conduct of  hatching tests was developed. The method was tested out several times and a satisfactory degree of accuracy was consistently attained.

Graphs and tables accompany the article.

 

Fenwick, 1952 -- Annals of Applied Biology 39:  457-467.

A technique for estimating the strength for hatching ability of any given sample was offered. The final standardization is expressed as "L.A." value or units and represents the logarithm of the concentration of the active factor in any given sample and is proportional to its activity.

 

Fenwick & Widdowson, 1958 -- Journal of Helminthology 32: 125-134.

The authors published a detailed and illustrated paper with descriptions of techniques for collection of  large quantities of cysts, collection and storage of diffusate,  setting up randomized batches of prepared cysts, conduct of hatching tests and the interpretation of results.

 

Fassuliotis & Feldmesser, 1952 -- Phytopathology 42: 466.

In an attempt to concentrate golden nematode-stimulatory elements from potato-root leachings, leachings were separated on the basis of differential freezing points into fractions which resulted in significantly different larval emigration rates when in contact with cysts of the golden nematode, Heterodera rostochiensis.

 

Germmell, 1940 -- Bulletin of the West Scotland Agricultural College 139: 1-15.

The author described "the single cyst technque" which was the placement of single cysts in drops of root exudate on coverslips supported on glass rings in Petri dishes. Moistened filter paper located at the bottom of the dish provided a humid atmosphere.

 

Janzen & van der Tuin, 1956 -- Nematologica 1: 126-137.

Procedures for obtaining, processing and purifying root diffusates, as well as leaf extracts, were described.

 

Shepherd, 1959 -- Nematologica 4: 161-164.

The rate of larval emergence from cysts was increased in tests with the beet eelworm, Heterodera rostochiensis,  by providing increased aeration and using small sieves made from nylon material and polyethylene tubing. By using these materials, the rate of emergence of the larvae from the cysts can be materially increased.

 

.Triffitt, 1934 -- Journal of Helminthology 12: 1-12.

An explanation was presented justifying the use of at least 300 cysts of the potato root eelworm for conducting quantitative experiments that are even roughly accurate

 

 

HISTOLOGY --HST  

 

Seshadri, 1964 -- Nematologica 10: 519-539.

A comprensive study of the morphology and histological anatomy of  Criconemoides xenoplax was reported. The text gives ample details and is well illustrated.

                                

          ----- I, J -----     

 

IDENTIFICATION -- IDE  

 

Akhurst, 1975 -- Nematologica 21: 267-272.

The author proposed a method of cross-breeding to facilitate the identification of  Deladenus spp.which are nematode parasites of wasps. The procedure using simplified methods are described and reputed to greatly reduce the time and effort involved in such experimentation.

 

Bae, Robbins & Szalanski, 2004 -- Journal of Nematology 36: 305.

The authors identified lance nematodes (Hoplolaimus) with PCR-RFLP of RTSI. Hoplolaimus columbus, H. galeatus, and

H. magnistylus were used for DNA sequencing and PCR-RFLP analysis to characterize the first internally tramscribed spacer region (ITSI) of these 3 species.

 

Boag, Topham, Smith & Fong San Pin, 1988 -- Nematologica 34: 164-172.

Within an article on advances in computer identification of nematodes, the authors propose 3 procedures, --matching--, -- modified Gower's coefficient --, and a statistical technique for comparison using a small microcomputer.  Results from all three of the procedures were statistically highly correlated in their ranking of species, but the time taken by the matching

method was less than a third of that taken by the statistical procedure.

 

Chitwood, 1949 -- Proceedings Helminthological Society of Washington 16: 90-104.

In this "pioneer" report, the author showed that perineal patterns of female root-knot nematodes can have distinct and differing patterns.  He further postulated that typical patterns can be recognized that are of value in the identification of populations. Numerous illustrations are presented to support his statements.

 

Cobb, 1919 -- Contriutions to a Science of Nematology, VIII:, pp 213-340.

Under the title "The orders and classes of nemas' the author proposes a classificationof nematodes based on the study of several hundred genera. The classification proposes relationships based on and correlated with the mouthparts.

 

Coomans, 1979 -- Annales Soc. r. Zool. Belg.108: 115-117

A proposal for a more precise terminology of the body regions in the nematode was made.

It develops further schemes earlier proposed in  the summary of discussions made at an  earlier symposium.  It recognizes two basic symmetries of the nematode body, a bilateral and a triradial symmetry. The bilateral symmetry is composed of 4 quadrants whereas the

triradial symmetry is composed of 3 sectors.

                                                                                     

Diederich, Fortuner & Milton, 2000 --  Nematology 1: 17-30.

Various systems of identification and the approach used for each are discussed and generally evaluated. The GENYSIS  (General Identification System) developed at the University of California is discussed.

 

Esbenshade & Triantaphyllou, 1985 -- Journal of Nematology 17: 6-20.

Enzyme phenotypes were obtained for 291 populations from 16 species of Meloidogyne

which originated from 65 countries. The authors concluded that multienzyme phenotypes represented by two or more enzymes often offered biochemical profiles more valuable for specific characterization of species than single enzymes.

 

El-Sherif & Mai, 1968 -- Nematologica 14: 593-595.

Working with Aphelenchus avenae, Panarellus redivivus, and Diplogaster sp.,the serological relationships of these three genera were studied. The results indicated that the first and the last named nematodes are closely related serologically. The data indicate that future detailed experiments may demonstrate the feasilbity of using serology in nematode taxonomy.

 

Flegg, 1967 --  Plant Pathology 16: 167-169.

A rapid identification method is proposed for identifying British Longidorus species. The lengths of functional and replacement odontostyles of the various stages are used as well as other easily observed characters to separate  morphometrically similar species.

 

Franklin, 1940 -- Journal of Helminthology 18: 63-84.

The author tested 3 methods for differentiating between strains of  Heterodera schachtii. One attempt at stimulating larval hatching by means of root secretions from host plants proved unsuccessful for the oat and pea strains of the nematode. The potato strain was stimulated by secretions from solanaceous plants. Infection of host plants by the nematodes should be the final test for differentiating between strains. Measurements of larvae also revealed significant differences between some strains.

 

Goodey, 1959 -- Nematologica 4: 211-216.

Data to be considered, observed and where possible, reported upon when presenting descriptions of new species is suggested. The specific areas covered in this article are: Figures, Source and reatment of Material, Measurements of Specimens, and Details to be Observed.

 

Joyce, Griffin & Burnell, 1994 -- Nematologica 40 : 601-602.

A comparison of isoelectric focusing (IEF) in conjunction with silver staining and sodium dodecyl sulphate-polyacrylamide gel electrophoresis  (SDS PAGE) for the identification of Heterorhabditis species showed IEF to be the more sensitive method.

 

Kort, Ross, Rumpenhorst & Stone, 1977 -- Nematologica 23: 333-339.

The authors proposed a new scheme for naming pathotypes of the potato cyst nematodes. Pathotypes of Globodera rostochiensis are designated  as "Ro1 to Ron" whereas those of Globodera pallida are designated  "Pa1 to Pan".

 

Luc & Dalmasso, 1975 -- in Nematode vectors of plant viruses, Plenum Press, pgs 53-70.

A "lattice" for the identification of species of  Xiphinema is modified from the one given by Stegarescu, 1966. The lattice is in accordance with the principles of polytomous

determination..  So as to offer the most simple and useful possible instrument of determination, only 12 characters have been chosen. It should be regarded only as a working instrument which is open to improvement.

 

Mulvey, 1972 -- Canadian Journal of Zoology 50: 1277-1292

Identification of Heterodera cysts by terminal and cone top structures was accomplished by detailed examination on cysts of 39 of the 53 described species within the genus . Five morphologically distinct groups are recognized based on differences in vulval slit length, fenestral length, underbridge structures, and bullae.

 

Platt, 1981 -- Cahiers de Biologie Marine 25: 83-91

Pictorial taxonomic keys: their construction and use for the identification of freeliving nematodes is suggested for the identification of marine nematodes because the more widespread availability of such keys would facilitate identification of, and interest in, this group of organisms. Numerous illustrations are presented.

 

Rey, Andres, & Arias, 1988 -- Revue de Nematologie 11: 129-135.

A computer program to assist in the identification of nematodes is described. The program utilizes an index which measures the probability that a character is common to the unidentified  specimen and the reference species. The computer output lists the five species most similar to the specimen being investigated.

 

Subbotin, 1999  -- Nematology 1 : 195-207.

The author identified  Heterodera avenae by morphometric analysis and also by employing the more advanced techniques of rDNA-RFLP analysis.

 

Tarjan & Vinton, 1986 -- Journal of Nematology 18: 632.

A novice-oriented computer program for identifying the more common marine nematodes is proposed in which only the morphology and anatomy of the neatodes being studies is required. The program was written in Turbo Pascal and is based on identification using only one to 3 characters.

 

Vrain, Wakarchuk, Levesque & Hamilton, 1992 --  Fundamental Applied.Nematology 15: 563-573.

Populations of the X. americanum group were separated using restriction froagment length differences in the 5.8s gene and the internal transcribed spacers of ribosomal DNA.

 

Windrich, 1970 -- Netherlands Journal of Plant Pathology 76: 93-98.

A rapid method for distinguishing the tulip and daffodil races of Ditylenchus dipsaci (Kuehn) modifies an earlier method proposed by Kooistra. An aqueous suspension of  50 to 100 nematodes was introduced under the epidermis of the outer bulb scale. Inoculation of leaves also is covered.

 

ILLUMINATION -- ILM           

 

Cobb, 1917 .. Notes on Nemas in Contrib. to a Science of Nematology V, pp 117-128.                                                        

For accuracy in microscopy, the most perfectly corrected lenses should be used, both as condenser and objective, The light used must be as nearly white as possible such as bright sunlight reflected from a plane, white matte reflector. A 2-mm apochromatic objective is suggested as the condenser and a 2- or 1.5-mm apochromat as the objective lens.

 

Ellenby, 1958 -- Nematologica 3: 81-90.

A study of day length, and cyst formation in the potato root eelworm was made. It was thought that differences might not be due to factors affecting plant growth. Instead, it was suggested that they might be a secondary consequence of differences in varietal response to environmental conditions, posibly differences in photoperiodic reactions.  Accordingly, experiments were conducted demonstrating the effect of day-length on cyst formation. The results showed that growing conditions may influence the number and size of cysts, the egg concentration within, and presumably the hatching response.

 

Eren & Pramer, 1968 -- Phytopathology 58: 644-646.

The use of a flourescent brightener greatly facilitated direct tests of fungistasis.  The ease  and precision with which measurements were made of spore germination and growth of the nematophagous fungus Arthrobotrys  conoides in soil were increased. The fungus was susceptible to fungistatic factors in untreated soil.

 

Feldmesser, 1967 -- Nematologica 13: 141-142.

An in vitro technique for nematode inoculation and culture of plants whereby sterile plants in Petri dishes are maintained at 1800-2000 foot candles of light at 21.1-23.3C. Inoculation, fertilization and watering are accomplished via injection ports in tapes used to anchor cotton plugs in holes in the dish tops.

 

Gillard & van den Brande, 1956 --Nematologica 1: 184-188.

The authors published an article completely in French and most unfortunately lacking a summary in English    The reported research deals with the effect of seven colored lights on tomato plants infected with Meloidogyne arenaria.

 

Mathews, 1953 -- Transactions of the American Microscopical Society 72: 190.

It was found that hollow cone or annular bright field illumination gave contrast and resolution superior to that obtainable with narrow pencil illumination and , under favorable conditions, comparable to that obtained with phase optics. Variable hollow-cone illumination was produced with an ordinary Abbe condenser  (N.A. 1.25) and microscope lamp with a diaphram. The performance of hollow cone illumination suggested it is a system of variable color phase contrast microscopy using standard optics.

 

Simon, 1956 -- Nematologica 1: 274, Plate XXV.

The author presents two illustrations of illuminating, with reflectors, a worksite dealing with cyst-forming nematodes, notably Heterodera rostochiensis. The article is written wholely in German.

 

Tarjan & Hopper, 1953 -- Plant Disease Reporter 37: 313-314.

The effect of increased photoperiod on egg mass production by Meloidogyne incognita

was investigated.  Tomato seedlings were inoculated with 5 egg masses each and grown in average greenhouse temperatures of 66F and 82F with differences in photoperiod. Differences in reproductive activity were significant for the plants exposed to the higher temperature. Length of photoperiod had no significant effect on nematode reproduction.

 

Winfield & Southey, 1986 in Southey (ed.) Laboratory methods for work with plant and soil nematodes, Ministry of Agriculture......Reference Book 402: 95-97.

A description of Koehler microscope illumination is presented by the authors. They claim that method of illumination provides the best resolution and minimal glare which are the principal technical objectives of brigt field microscopy.  The excellent illustration presented is from Fidler, 1976

 

 

 INFECTION, INDEXING INFECTION -- IND        

 

Baines, Miyakawa, Cameron & Small, 1969 -- Journal of Nematology 1: 150-159.

The infectivity and development of two biotypes of the citrus nematode (Tylenchulus semipenetrans) were compared on highly-resistant Poncirus trifoliata, "Troyer" citrange, and sweet orange selection "Homosassa". The observed differences in infectivity were interpreted to reflect differences in the ability of the nematodes to penetrate the epidermis and hypodermis and become established in host roots.

             

Brock & Giles, 1949 -- Journal Australian Institute Agricultural Science 15: 154-157.

The authors conducted tests at Red Cliffs, Australia in an area of  Barmera Sands infested with Heterodera marioni  and on which tomatoes had been grown for several sucessive years. On six occasions plants were given a health rating using a rating scale from 0 (dead plants)  to10 (healthy, vigorous plants). After the last picking, the root system of each plant was rated for the seveity of nematode attack using a rating scale of  0 to 5.

 

Drolsom & Moore, 1958 -- Plant Disease Reporter 42: 596-598.

 Root-knot resistant breeding lines of flue-cured tobacco were grown with two or more species of the root knot nematode, Meloidogyne spp. After 60 to 70 days, roots were rated for gall deveopment, root decay, and eggmass production.  The authors presentred data only for egg mass production because it denotes reproduction in the host, whereas gall development does not necessarily indicate completion of the life cycle.

 

Edwards, 1950 -- Journal of Helminthology 24: 123-136.

In working with the root-knot nematode and tomato plants on the Gold Coast of West Africa, Edwards used 4 ratings for infected plants. These were: 1. Roots free of infestation; 2.Roots with light infestations, 3. Roots with moderate infestations, and 4: Roots with severe infestations. He then summarized the results on a percentage basis.

 

Endo & Sasser, 1958 -- Phytopathology 48: 571-574.

The authors proposed using a "white cyst" index in experiments involving the soybean cyst nematode, Heterodera glycines.. Plants dug from each plot were evaluated as to the number of white cysts attached to the roots from 1=trace to 5=severe infection.

 

Giles & Brock, 1949 -- Jour. Australian Inst. for Agricultural Science 15: 154-157.

In controlled studies on root-knot nematodes, the authors expressed their findings using yield data with two kinds of ratings. Plant health ratings were on a scale of 0 to 10, whereas root gall ratings were expressed on a scale of 0 to 5. A set of photographic standards served as references.

 

Kushman & Machmer, 1947 -- Proc. Helminthological Society Washington 14: 20-23.

The relative susceptibility of 41 sweetpotato varieties, introductions and seedlings to attack by the root-knot nematodes (Heterodera marioni)  was evaluated. A novel infection index was used  in which the 'Sum of scores' was divided by the number of plants x 5 and multiplied by 100.

 

Miles & Turner, 1928 -- Journal of Helminthology 6: 59-76.

The authors used a root-gall index by which they measured the results of their experimental treatments.  There were four classifications for their treatments.Photographs were used as examples of standards.

 

Sasser, 1954 -- Bulletin Maryland Agricultural Experiment Station A-77: 1-31.

The author used several criteria to index infection. Four species and one variety of the root-knot nematode, Meloidogyne, were inoculated to a variety of plants. Susceptibility ratings, average numbers of larvae in roots, and counts of nematodes showing development beyond the first larval stage were recorded.

 

Shibuya, 1952 -- Memoirs Faculty Agriculture, Kagoshima 1: 1-22.

The author worked with root-knot nematodes attacking sweet potato plants in Japan. In his conclusions Shibuya suggests that the most hopeful line of attack in the determination of the cause of giant cell formation is an analysis of the proteins of resistant and susceptible varieties by immuno-chemical methods. The proteins from the resistant variety probably possess a serological peculiarity which is absent in the susceptible varieties.

 

Smith & Taylor, 1947 -- Phytopathology 37: 85-93.

Asystem for indedxing root-knot resistance was developed. Six replicate plots for each variety were located in soil heavily contaminated with the root-knot nematode. Plants are grown to maturity, roots are removed and grouped. Disease indexes are then calculated using the Horsfall and Heuberger modification of the McKinney system.

 

Zeck, 1971 -- Pflanzenschutz-Nachrichten Bayer 24: 141-144.

A rating scheme for field evaluation of root-knot nematode infections is presented that has 10 infestation levels. Infestation level 0 is a complete and healthy root system with no infestation.

Infestation level 10 applies to a dead plant with dead root system.

 

 

.INOCULATION , FOLIAGE  -- INOF

 

Christie, 1936-- Proceedings of the Helminthological Society of Washington 3: 69-72.  

The experimental infection of strawberry plants with a strain of the bud and leaf nematode, Aphelenchoides fragariae, was reported on. The results indicate that under greenhouse conditions, placing the parasites in a drop of water at the crown of the plant near the bases of the leaf petioles is usually effective in establishing an infestation.

 

Christie & Crossman, 1936 -- Proc.Helminth. Society of  Washington 3: 69-74. 

The reseach reported gives notes on the strawberry strains of the bud and leaf nematode, Aphelenchoides fragariae, related to the experimental infestation of strawberry plants.

It was found that, under greenhouse conditions, placing the nematodes in a drop of water at the crown of the plants near the bases of the leaf petioles is usually effective in establishing an infestation.

 

Felber, 1947 -- Science 106: 251.    

The method proposed by the author of introducing substances into plants by inserting a wick that is suspended into a container of inoculum with the other end inserted into the plant, is used by Cairns and Davis, 1956 (cf entry under INO).

 

Hastings & Newton, 1937 -- Canadian Jour. Research, Sec. C, Botanical Sci. 15: 168-174.

The authors induced foliar infections with Ditylenchus dipsaci by clamping glass rings containing the nematode mixed with moist peat moss directly on to the leaves. In addition, seeds were germinated in sterilized soil which had been infested with the nematode.

 

Korsten, 1953 -- Tijdschrift over Plantenziekten  59: 27-28.  

The author used the same method of inoculation of plants as previously reported by Williams, 1936. His technique differed, however, in that the trays of plants were then kept at high humidity at a temperature of 15-20C.

 

Powell & Moore, 1961 -- Phytopathology 51: 201-202.

A technique for inoculating leaves with root-knot nematodes employs a hypodermic syringe

containing larvae of  Meloidogyne icognita acrita which are injected into the leaf midribs of tobacco plants.  The technique was found suitable for studying infection by the nematodes, and also for subsequent pathogenesis.

 

Williams, 1936 -- Gardeners Chronicle, 3d Ser. 99: 316.    

Test Plants were inoculated with the Bulb and Stem Nematode, Ditylenchus dipsaci, by spraying the plant parts with an aqueous suspension of the nematodes.

 

 

INOCULATION, MISCELLANEOUS--  INOM  

 

Baker, 1948 -- Rept. of the Entomological Society of Ontario, Canada 78: 32-38.  

Tubers of potatoes were inoculated by the 'flap' and 'core' methods where the tuber is cut with a cork borer and the inoculum inserted, The "flap" or "core" is then replaced and the cut covered with celophane tape.  Using the "pad" method, inoculum is applied to the outside of the tuber by means of chiropodist's "corn pads".

 

Baker, Maurer & Maurer,1967 -- Phytopathology 57: 662-666.     

A study of the ecology of plant pathogens in soil with emphasis on mathematical models and inoculum density was conducted. The models were constructed assuming perfect distribution of inoculum in three-dimentional space for four host-parasite interactions. Slopes of the curves resulted from when successful infection was plotted against inoculum density. Such slopes may represent abstract systems, but may be useful in providing information in analysis and interpretation of data from experiments                                                                                                                    

Bingefors, 1953 --Proceedings of the Sixth International Grassland Congress: 1591-1596. 

Seedlings about 1 cm long are laid along filter paper strips which then are rolled up so that the seedlings are at one end. The plants are then infected by dipping the roll in a concentrated solution of Ditylenchus dipsaci. The rolled cylinder is then placed in a beaker of water and the plants covered with a bell jar. After 2-3 weeks at 20-25C, plants with swellings are removed

 

Cairns & Davis, 1956 -- Final Rept., U.S. Atomic Energy Comm.Cont. AT-(40-1)-2164. 

Radioactive isotopes were injected into plants using inoculation methods proposed by Felber, 1947 and Tarjan, 1959.

 

Cobb,1914 -- U.S. Journal of Agricultural Research 2: 217-230.

In an article written exclusively about the "Citrus Nematode", Tylenchulus semipenetrans,

the prolific author proposed a special "brood capsule" devised for incubation experiments. The glass capsules were easily constructed and manipulated, thus offering few difficulties

in studying hatching and rearing of the nematode. The material surrounding the egg placed in the capsule consisted of soil particles and citrus root detritus.

 

Ellenby, 1955 -- Journal of Helminthology 29: 77-80.   

For the purpose of infecting plants with Heterodera rostochiensis, the author placed cysts in sterilized sand within a stainless steel sterilized gauze basket,  in the form of a cylinder placed in soil, which then was planted .

 

Harrison, 1959 -- Phytopathology 49: 540.  

Nematode inoculum contained in thin glass bubbles formed at the end of a thin glass tube is placed at the desired depth in soil. When necessary, the bubble containing the inoculum is broken by applying pressure to the stem protruding above the soil.

 

Hesling, 1957 -- Nematologica 2: 285-299.   

A procedure that would aid the selection of viable nematode cysts was proposed. By sieving the material through a wire mesh with apertures of 0.556 sq. mm., cysts of proper size were collected.  These were then inspected to insure egg content.

 

Marks & McKenna,1981 -- Annals of Applied Biology 97: 119-121.

The Felden dispenser is used for the rapid delivery of standard inocula of the cyst nematode Globodera rostochiensis.  Standard volumes of cysts of mixed sizes contain titres of infective eggs and larvae which are largely independent of cyst numbers. A  rotating spindle within a steel dispenser distributes cysts at each full turn of the of the spindle.  The method reduces inoculum variability when compared with other cyst accumulation techniques.

 

Oostenbrink, 1952 --  Tijdschrift over Plantenziekten 58: 84.   

A single seed of a test plant and a single cyst of  Heterodera spp. are placed in glass tubes filled with a fine gravel and a top layer of steamed sand. Air is blown into the tubes weekly; the tubes are sprayed with nutrient solution when necessary.

 

Ross & Brim, 1957 -- Plant Disease Reporter 41: 923-924. 

In working with Heterodera glycines,  the lack of uniformity of nematode infestation in field experiments was overcome by using a double row planting method. Only  6 inches separated the susceptible check plants and the test plants.

 

Sasser, 1951 -- Phytopathology 41: 564. 

A gelatine capsule of small size was used for containing a single root-knot nematode egg mass. It was encased in a small quantity of moisened soil. Once placed in position and immediately watered, the juveniles could be released within 24 hours.

 

Seinhorst, 1952 -- Tijdschrift over Plantenziekten 58: 103-108.  

Seeds are inserted into thick filter paper pads which are kept moistened  with plant nutrient or water.  They are then placed in a speciali boxng 0.5% Aretan to control contaminants. After storage in a special box,  when the seedlings are about 2 cm long, they are inoculated  with a suspension of nematodes in carboxymethylcellulose and injected with a hypodermic syringe.

 

Sergeev, 1957 -- Meditsinkaya Parazitologiya i Parazitarnie Bolezi 26:194-195.    

The author constructed  a specialized cylinder made of longitudinal sections of noncorrosive wire which was covered with wire mesh and is placed in the ground.The apparatus is designed to determine the effects of varying depths on ova carried within the cylinder.

 

Slootweg, 1956 -- Nematologica 1: 192-201.   

For use in pathogenicity tests with fungus/nematode complexes,  it was found possible to destroy nematodes and their eggs by grinding the  root inoculum in a colloid mill for 1 hour at 800 rpm.

 

Stanton, 1951 -- Phytopathology 41: 476-477.  

Using  polyethylene bags as moist chambers for inoculation studies was investigated successfully.

 

Tarjan, 1959 -- Plant Disease Reporter 43: 451-458.  

An specialized container capable of being pressure-charged  was used to inoculate small trees by injection. The method is referred to by Cairns and Davis, 1956 as a means of inoculating larger plants.

 

Yuksel,1960 -- Nematologica 5: 289-296.

Observations on the life cycle of Ditylenchus dipsaci on onion seedlings were conducted by the author. Using fourth-stage larvae obtained from dry garlic, Allium cepa, a hole was made with a needle in young seedlings 2cm above the beginning of the roots. Water was added and the opening was widened and nematodes in a droplet of water were inserted, after which the needle hole was sealed with clay.

 

 

INOCULATION, ROOTS & TUBERS -- INOR    

 

Bergeson, Athow, Laviolette & Thomasine, 1964 -- Phytopathology 54: 723-728     .

Aspects of tobacco ringspot virus being transmitted by Xiphinema americanum from infected cucumber or soybean to cucumber were investigated. It was determined that pin-pricking soybean roots immersed in inoculum resulted in more infection than rubbing the roots with inoculum or cutting and dipping them in inoculum.

 

Bingefors, 1950 -- Kungliga Landbruksakademiens Tidskrift.89: 420-434. 

The author employed the basic method for inoculation of plants with Ditylenchus dipsaci by growing the seedlings between two sheets of moistened filter paper and then inoculating the plants by applying drops of nematode suspension to the roots.

 

Bingefors, 1953 -- Proc. Sixth Internat. Grasslands Congress 6:1591-1596.

Swedish nematode-resistant red clover strains were bred by selection on naturally infected fields. In order to make intensified selection possible, an artificial infection method was developed. Seedlings about 1cm long which were germinated on filter paper were laid along filter paper strips which are then rolled forming a cylinder with the seedings at one end. The cylinder is then dipped into a concentrated suspension of nematodes. It is then placed in a beaker of water and covered with a bell jar. After 2-3 weeks at a temperatures of 20-25C, all plants with root swellings are removed and planted.

 

Cairns, 1953 -- Thesis, University of Maryland, 102 pp. 

Ditylenchus dipsaci inoculum was placed into moist soil or vermiculite in a glass tube or vial which was then fastened with adhesive tape in an inverted position over the surface of the tubers.

 

Dropkin, 1957 -- Nematologica 2: 72-75.     

So as to meassure the ability of root-knot nematode juveniles to enter plant roots, larvae were placed  in a drop of water on a cover glass within a Petri dish. A root tip of a seedling was placed in the drop of water. Sand was then sprinkled over the entire root, slightly moistened, after which the  cover of the dish was replaced.

 

Seinhorst & Dunlop, 1945. Tijdschrift over Plantenziekten 51: 73-81.  

Tubers were inoculated by means of inserting the nematodes into an incision ,whereas stems were inoculated by putting the nematodes between young tissues  at the top of the plant.

 

Wilson, 1943 -- Annals of Applied Biology 30: 369-370.  

Bulbs and corms were directly inoculated with a nematode suspension placed in sterilized glass tubes, each of which contained a finely drawn out tip.  Once the tip was inserted into the plant, the nematodes were able to enter the plant singly. Another type of  inoculation  was using a drip method whereby the nematodes contained in water dropped down through the drawn out ends of the tubes onto foliage or other surfaces, or dripped into the axils.

                                                                                                              

 

INOCULATION, USING ANIMALS -- INOI                                                                                                             

Christie, 1936 .. Journal of Agricultural Research 52: 161-198.    I

A study of the life history of  Agamermis decaudata, a nematode parasite of grasshoppers and other insects was conducted. A device for experimentally infesting grasshopper nymphs was devised. Illustrations are presented of the materials used.

 

Hsu, 1951 -- Journal of Helminthology 25: 131- 160. 

During experimental studies on egg development, hatching and retrofection in Aspiculuris tetraptera, infective eggs of the nematode were applied to the anal opening of clean mice which were immobilized in cages specially designed  to prevent the possibility of oral infection. Of 12 mice thus treated, evidence was collected from 6,  by the finding of immature or adult worms in the intestine, that retrofection had occurred. It consituted the first experimental proof of the occurrence of retrofection in a non-human Oxyurid.

 

Payne, 1923 -- American  Journal of  Hygiene 3: 584-597.        

In experiments infecting dogs with Ancylostoma caninum, larvae washed on to pieces of bread were fed to dogs.  So as to avoid accidental loss of the larvae before they reached the stomach, the first capsule containing the the larval-impregnated bread was inclosed in a larger capsule which was fed to the dogs.

 

 

ISOLATION --  ISO                                                                                                     

 

Cairns, 1958 -- Annual Rept. for 1958, Project 534, Alabama Agricultural Expt. Station.

Cairns investigated the efficacy of a treatment reported by Shorb, 1937 (see below) specifically for separating plant parasitic nematodes from associated free-living nematodes. He did not find the technique useful but felt that the principle involved was sound.

 

Cairns, 1960 --  Methods in Nematology: A Review. Chapter 5. pgs 33-84. In Sasser & Jenkins (eds.) Nematology Fundamentals ...soil forms. Univ. of North Carolina Press.

It is suggested that slow drying of nematodes capable of surviving the rigors of anabiosis is a good way of separating them from accompanying unwanted species that are incapable of surviving the rigors of dessication.

 

Hollis, 1958 -- Nature 182: 956-957.

The swarming nature of  Tylenchorhynchus martini, whereby the nematodes in a mixed culture are induced to form a mass of this one species, was investigated. When the mass of nematodes was transferred to another container,  they were again induced to swarm for a final transfer. By this procedure,  a 99% pure suspension of the nematode was finally isolated..

 

Joshi & Wilt, 1973 -- Proceedings Helminthological Society of Washington 40: 167-168.

A simple technique for isolation of  Pelodera chitwoodi, bacteriophagous nematode, from soil uses Agarbacterium sp. and a Vibrio sp. as food for P. chitwoodi. A mud sample infested with seven different genera of nematodes and Pelodera were placed in the center of the dish and the cultures were incubated at 25C. Just 3 to 4 hrs afterwards, 99% of the nematodes accumulated around the bacterial colonies were identified as Pelodera chitwoodi.

 

Krigar & Samoiloff, 1974 -- Journal of Helminthology 6: 217.

A rapid method for separating larvae from a mixed population of  Panagrellus redivivus

involves washing and centrifuging the collected nematodes. A "unichem filter sampler" is used

as part of a device which displaces the fluid containing a high concentration of nematodes.

 

Kumar, 1971 -- Nematropica 17: 601-602.

A modified Hesling aspirator for the selection of active nematodes from aqueous suspensions is described and illustrated. In essence, the short article merely seeks to improve by modification the Hesling aspirator.

 

Schuurmans Stekhoven, Berends, & Soelistio, 1954 --

   Mitteilungen Biologischen Bundesanstalt Land- Forstwirtschaft 30:11-16.

A new method for the isolation of small numbers of nematodes is reported in which the living and the dead nematodes can easily be separated. In addition, a large number of nematodes can be caught in a very short while thereby saving time.         

 

Shorb, 1937 -- Proceedings of the Helminthological Society of Washington  4: 52.

The author introduced a dilute acid treatment for isolating certain animal parasitic nematodes from saprophagous nematodes.

 

          ----- K, L -----

 

KILLING BEFORE FIXATION -- KIL                                                                      

                                                             

Berland, 1961 -- Nature 191: 1320-1321.

Towards the goal of killing nematodes in an extended position, several specimens of  Contracecum aduncum were killed in glacial acetic acid and subsequently transferred to 70% ethanol. No adverse effect on cleared specimens was observed after treatment. In addition, anatomy was studies satisfactorily in stained sections.

 

 Esser, 1974 -- Proceedings of the Helminthological Society of Washington 41: 10-13.

Specimens of  Xiphinema macrostylum placed  in 2% formaldehyde die slowly. Physical changes are not obvious until 20 to 30 minutes following the cessation of movement. Distortion or shrinkage of the specimens was not observed.

 

Luyet & Hartung, 1941 -- Biodynamica 3: 365- 367.

In an article titled "Death by devitrification in the nematode Anguillula aceti" the authors

referred to previous work where it was found that the nematode could be revived after solidification in liquid air. Exposures of the solidified worms to temperatures from -39 to -5C for one minute was always fatal. Exposure of the nematodes to -43 to-50C for durations as long as 30 min did not prevent revival. These facts are interpreted as being due to devitrification of some essential constituent of the bodies of the animals.

 

Netscher, 1970 -- Nematologica 16: 603.

A rapid technique for mass-killing of nematodes with hot fixative requires vacuum-filtering the suspension. When the filter appears dry,  hot f.p 4:1 or f.a. 4:1 solution is poured on.  After drawing off the liquid, the nematodes are washed from the filter with 4% formaldehyde. After fixation, the nematodes may be processed into glycerine.

 

Netscher & Seinhorst, 1969  -- Nematologica 15: 286.

For purposes of killing specimens with heat in a fixative solution, the authors found that use of a 4% formaldehyde was desirable following killing  in FA 4:1 or FP4:1.

 

Seinhorst, 1962 -- Nematologica 8: 29-32.

It is suggested by the author that killing nematodes by means of a hot solution of 0.5% acetic acid in water will preserve the animals in a better condition than heating them in a drop of water.  A pipette is described for the heating and transferring of the acetic acid solution as well as a specialized dish.

 

Seinhorst, 1966 -- Nematologica 12: 178.

The use of hot  f.a. 4:1 (formalin acetic acid alcohol) is proposed as a considerable improvement to the previously suggested use of hot 0.5% acetic.acid. The procedure for use is the same as previously described by Seinhorst, 1961.

 

Staniland, 1950 -- Journal of  Helminthology 24: 91-99

The author proposes the use of iodine for killing nematodes prior to fixation and mounting. Nematodes are killed in two seconds by iodine in a solution of 1 part in 2000 (a 0.1% 'Lugol's' solution.

 

LIFE PROCESSES -- LIF

 

Allen & Raski, 1950 -- Plant Patholgy 40: 1043-1053.

A method of distinguishing between living and dead nematodes by enzymatically induced fluorescense was proposed using a solution of fluorescein diacetate and acetone stored at -10 degrees. A 1-25 dillution of the solution was prepared in buffer and a drop of this was added to living and dead nemas giving a final concentration of 0.01% fda. A cover slip was placed over the drop and sealed, and the slide was examined under ultra-violet light. Living nematodes exhibited fluorescense in discrete particles in the gut, whereas dead nemas exhibited diffused fluorescense throughout the whole animal.

 

Croll, 1966 -- Nematologica 12: 610-614.

The phototactic response and spectral sensitivity of the marine nematode, Chromodorina

viridis were reported on and conclusions on the nature of the paired pigment spots were presented. A description was given of the directional response of the nematode to light which showed a positive phototaxis at most visible wavelengths and a negative phototaxis in the near ultraviolet The chemical nature of the pigment spots was investigated and it was postulated that the spots were a haemoglobin derivative.

 

Shepard, 1962 --Tech.Commun.32, Commonwealth Bureau of Helminthology.

The emergence of larvae from cysts of  Heterodera is very adequately covered in a 90-page booklet.which deals with diverse, and yet related, subjects such as techniques, maturation, moisure, aeration, temperatur, light, etc. Within its pages several unreported methods are discussed or referred t.  An extensive bibliography adds credence to the value of the publication.

 

LYOPHILIZATION -- LYO                                                                                           

 

Bosher & McKeen, 1954 -- Proceedings Helminthological Soc. Washington 21:113-117.

 The effects of  lyophilization and low temperatures were studied by freezing  Ditylenchus dipsaci to -80C. Additionally, they found that nematodes kept in solutions with high osmotic values had higher survival percentages.

 

Luyet & Hartung, 1941 --  Biodynamica 3: 75.

Death of Anguilulla aceti by the process of devitrification was investigated by the authors. They found that the nematode can be revived after having been solidified in liquid air if it is rewarmed rapidly within a fraction of a second. If the nematode remains frozen for just a few seconds, however, it can not be revived.

 

Luyet & Hartung, 1941 -- Biodynamica 3: 363-367.

Although frozen Anguilulla aceti can be revived under certain conditions, exposure of the solidified nematodes to temperatures from -39C to -5C for one minute was always fatal. These facts are iterpreted as due to a devitrification of some essential constituent of the body of the animal.

 

Weinman & McAllister, 1947 -- American Journal of Hygiene 45: 102-120.

The authors worked with a variety of protozoa, and some helminths, and found that they could be successfully stored at -70C. Microfilariae of  Wuchereria bancrofti, Dirofilaria immitis and Litomosoides carinii survived in great numbers.  Infective larvae of the hookworm, Ancylostoma caninum,  and the whipworm, Stongyloides stercoralis, have been stored successfully

 

          ----- M -----

 

MANIPULATION -- MAN                                                                         

 

Abdel-Malek, 1951 -- Journal of Parasitology 37: 321.

It was found that one drop of a solution of 24g of menthol dissolved in 10ml of  95% alcohol was adequate, when added to 100ml of water, to make motionless various helminths including nematodes.

 

Barboza, 1948 -- Anais da Academia Brasiliera de Ciencias 20:277-280.

The author discovered the very simple expedient of rendering helminths motionless prior to fixation -- he immersed them in ice water.

 

Bijloo, 1954 -- Journal of Helminthology 28: 123-126.

The aspirator used by Hesling, 1952 was modified so that the  selection of unwanted debris accompanying the cysts could be averted.

 

Brodie, Blow, Brace, and King, 1976 --  Plant Disease Reporter 60: 936-938.

A cylindrical fiber filter of 3.5cm diameter is closed at one end so as to form a "filter sock".  This is placed in a 600ml beaker. Nematode cysts and debris collected on a 60-mesh sieve are washed through a funnel into the "filter sock".  Once the sock has dried, the collected cysts may be removed.

 

Chuang, 1962 -- Nematologica 7: 316-330.

For a study of the embryonic and post-embryonic development of  Rhabditis teres, a new technique using garden snails mucus was employed for handling nematode eggs. Fertilized eggs in different stages of development were placed in a drop of water on a slide and mixed with fresh snail's mucus. The egg-containing mucus was fixed in "Petrunkewitch", dehydrated and embedded in ester wax.

 

Clapham, 1947 -- Journal of Helminthology 22: 37-46.

The author proposed a practical method of isolating spicules and gubernacula by placing the nematodes in a 4% solution of NaOH in a covered watch glass and to incubate several hours at 37C.  At the proper time, the hydroxide is neutralized with HCl and the spicule pairs and gubernacula transferred to a slide for examination.

 

Cobb, 1920 -- Transactions American Microscopical Society 39: 231-242.

A "compressorium for nematodes" is a simple metal spring device for preparing flattened sections of nematodes for studies on chromosomes or cell contents.A thorough discussion of the principles involved and illustrations are presented

 

Fenwick & Franklin, 1942 -- Journal of Helminthology 20: 67-114.

The authors were able to render nematodes motionless prior to making measurements by placing them in a drop of water exposed to a strong concentration of chloroform vapor for 30 to 60 minutes.

 

Ford,  1957 -- Plant Disease Reporter 41: 81-90.

The author devised a method of collecting nematodes contained in water by using a pippette with a finely drawnout tip. The pippette is connected by rubber tubing to a large test tube in which a descending column of water creates a suction which is transmitted to the glass pippette.  The pippette then selectively picks up the desired nematodes.

 

Franklin, 1937 -- Journal of Helminthology 15: 61-68.

Franklin found that cysts of the potato cyst nematode could effectively be picked up using a fine, moistened paint brush.

 

Hargis, 1953 -- Journal of Parasitology 39: 224-225.

Two g. of chloretone diluted in 500ml of water serves as a relaxing agent when working with various helminths.  Exposure of an inverted slide with nematodes in a drop of water over.osmic acid or Flemming's solution also is effective in producing motionless nematodes.

 

Hesling, 1952 -- Journal of Helminthology 26: 69-70.

Cysts of the potato root nematode effectively were picked up from debris using an entomologist's aspirator.

 

Hooper, 1977 -- Nematologica 23: 126-127.

Spicule and stylet protrusion was found to be induced in some plant and soil nematodes by exposure to an ammonia solution. The author postulated that the specific action of ammonia on spicule and caudal muscles on many of a list of given species suggests that females possibly produce ammoniacal secretions that induce a copulatory response in the male.

 

Nazmi, 1932 -- Journal of the Egyptian Medical Association 15: 83-84.

An improvement is suggested over use of the platinum loop manipulator for purposes of skimming floating ova from concentrated solutions that are used specifically for ova extraction.

 

Seymour & Doncaster, 1972 -- Nematologica 18: 261-264.

Techniques for manipulating small nematodes gave details of construction and operation for an observation cell.  Glass suction cannulae of 20 - 50 microns aperture, connected to syringes , hold nematodes securely. Finer microcannulae of 5 to 10 microns aperture can be inserted into the nematode for pressure recording.  Microdissection knives are made from plate glass sliverfused or glued to glass handles.

 

van Someren, 1939 -- Journal of Helminthology 17: 83-92.

It was discovered that nematodes, which had been immersed for 2 or 3 hours in a dilute Neutral Red solution, would protrude their buccal stylets when they were killed by the addition of cold 70% alcohol

 

MEASURING --  MEA

 

Anadon, 1955 -- Boletin Real Soc. Espanola Hist. Nat, Biol. Sec.52: 137-145.

Calculations were made of the volume of nematodes using mean values for length and breadth.

 

Boag & Smith, 1985 -- Nematologica 31: 360-363.

A technique for measuring and identifying live nematodes uses video equipment similar to that used by Seymour et.al., 1978 and the computer and digitising platen previously suggested by

the authors. The advantages of use are that the technique does not interfere with the behaviour of the nematode, and the measurements of the nematodes are immediately available.

 

Cobb, 1890 -- Agricultural Gazette of New South Wales 1: 131-136.

The basic form of a nematode formula with accompanying details is first proposed

with examples and illustrations.

 

Cobb, 1902 -- Agricultural Gazette of New South Wales Misc. Publication 601, 8 pp.

Details for use of a Nematode Formula are presented, as well as the description and illustration of an improvised  measuring instrument. The formula, referred to as a decimal formula, gives relative measurements of the length from the anterior end of various parts of the nematode body. These mesurements are then placed in a mathematical formula. Use of a map measurer for determining lengths also is suggested.

 

Dropkin, 1954 -- Phytopathology 44: 43-49.

The infectivity and gall size in tomato and cucumber seedlings infected with Meloidogyne incognita var. acrita were studied with gall areas being measured by a photographic and planometric technique.  It was concluded that gall size  depends not only upon the number of larvae per gall, but also on the species of the plant and of the nematode.

 

 Fenwick & Franklin, 1942 -- Journal of Helminthology 20: 67-114.

In an effort to obtain consistent and accurate estimates of the length of  Heterodera juveniles, the authors contemplated the form of sample which would give the most reliable estimate  of the mean length of the whole population.  Although the studies were made only on juveniles of  H. rostochiensis, the ultimate purpose was to find out whether juveniles of various species could thus be identified.

 

Fenwick & Franklin, 1951 -- Journal of Helminthology 25: 57-76.

Using standard techniques, juveniles of  8 populations of   Heterodera from known host plants of 86 different species and varieties were measured.  The work indicated that the populations usually fell within certain parameters of measurement. The summary made by the authors concludes with the statement " If more than one species of Heterodera is present in a population, the distribution of lengths of the larvae will not fall into a normal curve, and the methods given will only serve to identify the species present under very special conditions."

 

Roggen & Asselberg, 1971 -- Nematologica 17: 187-189.

The authors contend that the uncritical use of  ratios for nematode descriptions can easily lead to unjustified conclusions.  A method is described which they state makes it possible to decide whether a ratio is statistically acceptable or not.

 

Storey & Atkinson, 1979 -- Annals of Applied Biology 93: 299-304.

A method for measurement of the adenosine triphosphate (ATP) content of cysts for Globodera rostochiensis as a method for assessing the efficacy of fumigant nematocides is given. The technique used is based on bioluminescent photometry. The ATP technique clearly offers a reliable and relatively inexpensive method for estimating the efficacy of fumigant nematocides and is much more rapid that the alternative hatching tests or egg counts.

 

Zuckerman, Nelson & Kisiel, 1972 --   Journal of  Nematology 4: 261-262.

It was found that the specific gravity of the nematode Caenorhabditis briggsae increased with age in comparison to young nematodes. In their discussion they suggest that the results of the experiments may explain the decrease in ability of the nematode to withstand osmotic stress as physiologic aging progresses.

 

MICROPLOTS -- MCP                                                                     

 

Bijloo & Boogaers, 1956 -- Nematologica 1: 20-30.

A study was made of the population decrease of  Heterodera rostochiensis in the absence of a host plant after DD treatment of the soil. It was found that DD does not hatch larvae in the cyst in the absence of a potato crop, but kills the larvae within the eggs.

 

Caswell, Nelsen and Thomason, 1985 -- Revue de Nematologie 8: 90-92

A microplot technique is described for establishing individual plants under near-field conditions using a limited soil volume which allows recovery of entire  plant root systems and the endoparasitic or semi-endowparasitic nematodes associated with them.

 

Jones, 1955 -- in Soil Zoology, ed D.K.M. Kevan, London, Butterworth, pp390-393.

A technique is described which combines the advantages of using large pots with conditions found in the field. Microplots are constructed from standard concrete paving stones and slotted concrete posts jointed together with bitumastic   

        

Mai, 1958 -- Phytopathology 48: 263.

The author presents a description of small field plots for experiments involving plant pathogenic nematodes. Plots measuring 5 x 8 ft. bounded by 12-inch redwood boards and separated by alleys at least 2 ft. wide have been successfully used for reseach with the golden nematode, Heterodera rostochiensis.

 

MICROSCOPY -- MIC                                                                                 

 

Abu-Gharbiah & Smart, 1967-- Nematologica 14: 1.

A plastic bottle applicator for ringing microscope slides with ZUT Slide Ringing Compound was proposed in addition to supplement use of a brush for ringing slides. It was felt essential that the orifice of the mouthpiece be approximately i mm in diameter.

 

Bird & Deutsch, 1957 -- Parasitology 47: 319-328.

A modified layer stripping technique was used to study layer ultrastructure in the cuticle of  Ascaris.  The authors also described a technique for embedding in a polymerized monomer for ultrasectioning.

 

Cobb, 1898 -- Misc. Publication 142, N.S. Wales Dept. Agriculture, 5pp

A method of using the microscope with transmitted sunlight shows the entire arrangement that the microscopist would use, then a drawing of a section of the head of the microscope camera, and finally the outside large mirror that is located outside a short way from the microscope. The mirror is positioned through the use of  guy wires which were controlled by the microscopist.

 

Cobb, 1916 -- Transactions of the American Microscopical Society 35: 7-22.

Masonry bases for the instillation of microscopes and their accesories including the camera lucida and the microscope camera are proposed and illustrated in part. Sections of the work were entitled: m`asonry as a base, the use of girders, carriers for the accessories, camera lucida, camera lucda drawings of difficult opaque objects, etc.

 

Cobb, 1917 .. Notes on Nemas in Contrib. to a Science of Nematology V, pp 117-128.

The use of a apochromatic microscope objective suggested by the author necessitates the use of a special slide which carries the object to be examined,  essentially consisting of a metal slide carrier and two cover glasses, the bottom one square and the top one round,  between which the specimen is mounted. A drawing of the arrangement is shown.

 

Cobb, 1920 -- Transactions of the American Microscopical Society 39: 231-242.

An article dealing with microtecnique and offering suggestions for methods and apparatus is presented.  The specific subjects offered are "Systematically examining a large series of microscopical objects", " Object support for a freezing microtome",

"To obtain an end view of a nema, rotifer, or other similar small object", " Destaining of nemas and other small objects in the Differentiator", and a  "Compressorium for chromosomes".

 

Ellenby, 1968 --  Experiencia 24: 84-85.

Using a variety of highly specialized equipment. the author determined the water content of  Panagrellus redivivus by interference microscopy. Photographs made using a fringe field eyepiece using monochromatic light are presented.

 

Ellenby, 1968 --  Proceedings of the Royal Society B 169: 203-213.

A study was conducted of the dessication survival in the plant parasitic nematodes, Heterodera rostochiensis Wollenweber and Ditylenchus dipsaci (Kuhn) Fillipjev.

Using interference microscopy, it is shown that the free second-stage larvae of the

potato-root eelworm, Heterodera rostochiensis, takes up water at the same rate as one still inside the egg-shell.  The enclosed larvae, however, lose water far less rapidly.

 

Esser, 1958 -- Turtox News 36: 16-17.

The author presents details of an attachment  to the stage of a stereoscopic dissecting microscope used for holding a Petri dish containing several smaller culture dishes. The Petri dish is rotated to conveniently permit examination of the contents of each of the smaller dishes.

 

Esser, 1974 -- Nematologica 19: 566-567.

The use of "ZUT" as a microscope slide cover glass support was suggested.  A 15mm round cover glass placed over the subject in liquid is then sealed with ZUT at the proper viscosity.

It is suggested that care be taken not to have the coverglass positioned too high so that the microscope objective swung into place does not affect the cover glass.

 

Green, Stone, Turner & Clark 1975 -- Journal of Microscopy 103: 89-99.

Nematodes from the orders Tylenchida and Rhabditida were fixed and processed in several different ways. Drying from acetone, critical point drying, infiltration with glycerine and a conducting agent, and infiltration with epoxy resin were tested.  Of these, the epoxy resin method was the most promising.

 

Hoegger & Estey, 1976 -- Journal of Nematology 8: 357-358.

A chamber for critical-point drying of nematodes and other biological specimens is described and figured.  Specific details are presented for the construction of a universal chamber and for the materials used. A diagram of the parts and a photo of the chamber connected to a vacuum flask likewise are presented.

 

Maggenti & Viglierchio, 1965 -- Hilgardia 36: 435-463.

The preparation of nematodes for microscopic study-perfusion by vapor phase in killing and fixing was reported. The study investigated vapor phase profusion for killing and, at least, partially fixing nematodes preparatory to mounting.  A number of the more common fixative agents were investigsated to illustrate the possibilities of vapor-phase profusion.

 

Mason & Bosher, 1963 -- Proceedings of the Helminthological Society of Washington 30: 19-20.

A combination die for the preparation of aluminum micro slides is described and illustrated. The apparatus punches and forms. Cobb metal slides with a single lever movement in contrast to similar equipment previously described that necessitated two movements. Cost and construction details are adequately presented.

 

McClure, 1969 -- Nematologica 15: 155,

Styrene divinylbenzene spheres are suggested as a convenient support for microscope slide coverslips. They are available in 4 sizes, from 6 to 100 microns. The spheres are immersed in absolute alcohol and are applied in a small drop to the microscope slide using a glas rod or a micropipette.

 

Taylor & Smiles, 1955 -- Trans. Royal Soc. Trop. Medicine & Hygiene 49: 303.

The authors studied various microfilariae using  advanced microscopical  equipment and techniques such as phase-contrast and ultra-violet light.

 

 

MICROTECHNIQUE -- MTE  

                                                            

Cobb, 1920 -- Transactions of the American Microscopical Society 39: 231- 242.

An object support for a freezing microtome is designed to reduce the metal parts to a minimum and to concentrate the effects of the freezing mixture on the object to be frozen.

The object is placed on a thin but rigid metal plate. Six supporting spokes , and a dome-shaped German Silver wheel are added  Two accompanying figures clarify the object.

 

Haarlov & Weis-Fogh, 1955 --  in Soil Zoology, ed D.K.M. Kevan, London, Butterworth, pp 428-432.

The successful use of a technique earlier proposed for staining microorganisms in the soil was proposed. Violamin acts selectively to stain living or newly dead cells deep blue and to contrast them to brown humus particles.

 

Tattar, Stack & Zuckerman,1977 -- Nematologica 23: 267-269.

A apparent nondestructive penetration of Caenorhabditis elegans by microelectrodes was reported. Glass micropipettes less than one micrometer tip diameter and filled with 3M KCl  were inserted for one min.into the esophagus between the median and basal bulb, or into the region of the mid-intestine. Twelve of  15 of the pierced nematodes survived for 96 hrs whereas 13 of 17 of the unpierced nematodes survived for the same period. Both groups oviposited and both fed normally during the same period.

 

Taylor & Netscher, 1974 -- Nematologica 20: 2.

An improved technique for preparing perineal patterns of  Meloidogyne spp suggests that

a 45% lactic acid be used to rid the sections from adhering body tissues. Use of the lactic acid produced excellent results without any of the difficulties which were encountered with the acetic acid useage.

 

MODELS -- MOD

 

Baker, Maurer & Maurer, 1967 -- Phytopathology 57: 662-666.

Mathematical models and inoculum density were factors in studying the ecology of plant pathogens in soil. Models were constructed assuming perfect distribution of inoculum in

three-dimensional space for four host-parasite interactions.       

                                                                                  

Hollis, 1963 -- Nematologica 9: 475-494.

A theory of nematode-plant relations was developed. The theory predicts that (a) plant damage is related to diameter of roots attacked , (b)  parasites are capable of causing significant crop losses, (c) certain conditions must exist before crop losses can occur, and (d) resistance of small grasses to root-knot and reniform nematodes occurs  because their roots are of insufficient diameter to support development and reproduction of the females.

 

Hung & Yang, 1948 -- Chinese Review of Tropical Medicine 1: 7-8.

A new method for the preparation of paraffin models of helminths was presented by the authors.

 

MOISTURE -- MOI                                                                                      

 

Bloom & Couch, 1959 --- Phytopathology 49: 534.

The "split-root" technique was used to determine the importance of soil moisture on root-knot nematode development in the roots of tomato plants. One-half of the root system of each plant was grown in soil held at field capacity by frequent irrigations, The other half was in soil held at the permanent wilting percentage.

 

Couch & Bloom, 1960 -- Phytopathology 50: 319-321.

 Influence of soil mpoisture stresses on the development of the root-knot nematode were investigated using a split-root technique.The root systems of Pearson and Rutgers tomato plants were held at two soil moisture stresses, field capacity and permanent wilting percentage.

 

Daulton & Nusbaum, 1962 -- Nematologica 8: 157: 157-168.

In a study determining the effect of soil moisture and relative humidity on the root-knot nematode Meloidogyne javanica, it was found that two techniques for maintaining prescribed soil moisture levels (soil columns of varying height in glass cylinders and soil subjected to suction in scintered-glass funnels) gave comparable results.

 

de Guiran, 1975 -- Nematologica 21: 261-262.

A simple method for maintaining constant soil moisture stresses during experiments

on nematodes is proposed with a drawing of the principal components. The principal advantages of the method are : (1) moisture stresses between saturation and wilting point could be readily obtained, (2) moisture stress remained constant, and (3) the soil was normally aerated.

 

Dropkin & Martin, 1957  ---  Phytopathology 47: 519.

Inhibition of hatching of nematode eggs under conditions of moisture stress produced by various solutions were studied by the authors.

 

Kable, Zehr, & Mai, 1966 -- Nematologica 12: 175-177.

A simple apparatus for the study of soil moisture relations of plant parasitic nematodes under monoxenic conditions is described. It is suitable for a wide range of studies on nematodes involving the interaction of environmetal, host plant and soil microbial factors. The system basically is a modified Haines apparatus

which has the added advantage of sterilty.

 

Linford, 1941 --- Phytopathology 31: 859-865.

The author determinined soil moisture relationships of the root-knot nematodes by burying eggs of the nematode at varying soil moisture content .

 

Peacock, 1957 --- Nematologica 2: 114-122.

The effect of soil moisture content upon survival of root-knot nematodes was studied by applying calculated amounts of water to air-dried sterile soil.  The soils were held at known moisture content in air-tight jars.

 

Wallace, 1955 ---Annals of Applied Biology 43: 477-484.

The effects of soil moisture were investigated by the author who used a suction plate method which he had described in an earlier publication (Nature 173: 502. 1954).

 

Wallace, 1955 --- Journal of Helminthology 29: 3-16.

 Engaged in studies on Heterodera schachtii larval emergence, the author investigated effects of water saturation deficiency of cysts. This was accomplished by placing the cysts in trays suspended over solutions of caustic potash in secured in tubes.      

 

MOLECULAR BIOLOGY -- MOL                                                                 

 

Burroughs & Baffey, 1986 -- Revue de Nematologie 9: 199-200.

The authors proposed that both the size and number of restrictive fragments formed will reflect the geonomic base sequence which offers the potential of a unique "fingerprint' of a genome independent of phenotype.  Eggs and juveniles of two Globodera spp were used. Details are given of the extraction of  total DNA, restriction endonuclease digestion and  garose gel electrophoresis.   (Restrictive fragment length polymorphism)

 

Curran, Baillie & Webster, 1985 -- Parasitology 90: 137-144.

Geonomic DNA restriction fragment length differences were used to identify nematode species. Following fractionation by agarose gel electroforesis, repetitive DNA can be visualized as distinct bands in stained gels and the restriction fragment lengths used as diagnostic characters.

 

Fullaondo, Barrena, Viribay, Salazar & Ritter, 1999 -- Nematology 1: 157-163.

A method using  RAPD (Random Amlified Polymorphic DNA analysis) fragments specific for two Globodera species were used to derive two primer combinations for PCR (Polymerase Chain Reaction) which amplify only one specific band in each species.The use of primer combinations to identify species is highly sensitive.

 

Vrain, Wakarchuk, Levesque & Hamilton, 1992 --

                                                       Fundamental Applied Nematology 15: 563-573.

Populations of the Xiphinema americanum were separated using restriction fragment length differences in the 5.8s gene and the internal transcribed spaces of ribosomal DNA. Although some separations between species/populations were clearly resolved, others e.g. mixed populations of  X. rivesi and X. americanum from Pennsylvania and West Virginia could not be separated.

 

MOUNTS, BODY PARTS -- MNV                                                                                        

 

Anderson, 1958 -- Annales Parasit. Humaine et Comparree 33: 171-172.

It was proposed that the cut anterior end of the nematode be manipulated in a small drop of molten glycerine jelly on a thin cover slip until the nematode's anterior end touches the cover slip. The jelly hardsens and the cover slip is inverted and transferrd to a slide where it rests on two wire or glass supports of sufficient diameter so that the drop barely touches the slide. The edge of the coverslip is then sealed with glycerine jelly.

 

Brzeski, 1971 -- Nematologica 17: 490.

A technique for mounting vulval cones of  Heterodera cysts employs the use of castor oil , instead of clove oil , as the clearing agent  It specifically is pointed out that excellent results can be obtained by using the less costly castor oil.

 

Cooper,1955 -- in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pp 419-420.

Mounting technique for identification of Heterodera eelworm cysts describes in detail preparation of the cysts for diagnosis. After soaking in water for an hour,  the cyst is scraped clean externally, cut in half  but as far as possible from the vulval cone. Eggs and debris are cleaned out with a needle and finely sharpened forceps. The specimen is then placed in absolute alcohol for a few minutes, then into clove oil and finally into Canada balsam.

 

Franklin, 1962 -- Nematologica 7: 336-337.

After briefly reviewing the work of others, the author investigated several methods for the preparation of posterior cuticular patterns of Meloidogyne spp. In examining the patterns prepared, the striae were examined and the phasmids were searched for. No distortion or abnormality resulted from any of the treatments. Phasmids were seen more easily in specimens NOT fixed in formaldehyde. Of the  methods tested, none seemed to influence the characters used for identification.

 

Galli-Valerio, 1932 -- Zentralblatt Bakter., Parasit., Infek Hyg.. 125: 129-142.

The author discovered that some nematode eggs, if first fixed in 4% formaldehyde and then allowed to dry on the slide following fixation, were suitable for being effectively mounted in balsam.

 

Krull, 1934 -- Proceedings of the  Helminthological Society of  Washigton 1: 5-6.

In describing the use of fresh egg albumin as a mounting medium for the study of living nematodes, it was pointed out that the slide becomes semi-permanent as the albumin dries around the edge of the cover glass. The medium is excellent for making intra-vitam stains. Specimens which ordinarily do not take up the stain do so in the albumin.

 

Wilson & Markell, 1952 -- Journal of  Laboratory & Clinical Medicine 40: 145

It was found that if polyvinyl alcolhol is incorporated into a fixative, it would insure that ova preparations would show little or no distortion after several weeks.

 

MOUNTS,  EN  FACE  -- MNF 

 

Anderson, 1958 -- Annales de Parasitologie 33:171-172.

In an article written entirely in the French language, the author describes and illustrates the method of obtaining an apical view of a nematode.  Two illustrations, a top and a side view of the subject slide, adequately illustrates the anticipated results.                                                                   

 

Basir, 1949 -- Transactions American Microscopical Society 68: 123-126.

A modification of the Cobb technique for preparing head mounts was proposed. The head of the nematode is severed on a glass slide in a thin streak of solidified glycerine jelly. It is then moved to the upright  position in melted jelly or in glycerine.                                                            

 

 Buhrer, 1949 -- Proceedings of the Helminthological Society of  Washington 16: 3-6.     .

Buhrer proposed a technique for the beheading and en face examination of nematodes and similar animal types.The article is arranged under the following sections: Fixation, Washing, Dehydrating, Beheading, and Completing mount.

 

Cairns, 1960 --  in Cairns Sasser & Jenkins, Chapt. 5, pp.33-84, N. Carolina Press.

Holes about the diameter of a nematode can be made in thin pieces of celluloid or plastic with a squarely cut-off needle that makes wells into which the severed head ends of nematodes fit. Either dilute fixative or solid mounting material can be used. The location of the well containing the head can be indicated on the plastic at the time the perforations are made.

    

Chitwood & Wehr, 1934 -- Zeitschrift fuer Parasitenkunde 7:273-335.

The authors discussed the preparation of end views and gave their views on their value in taxonomic work. In addition they proposed a modification of the technique proposed by Cobb.

 

Cobb, 1920 -- Transactions of the American Microscopical Society 39: 231-242.

Descriptions were presented for preparing mounts of the heads of nematodes as well a other organisms. (Side Note: It is fortunate that Edna Buhrer (1949), a very precise worker in the USDA Division of Nematology,  presented complete descriptions  of the methods employed by Cobb.

 

Esser, 1974 -- Ptroceedings of the Helminthological Society of Washinngton 41:10-13.

Nematodes mounted by two permanent munting methods were compared after six years.

Body length decreased in both preservatives. Esophagus length increased in lactophenol and decreased in glycerine. The guide ring appeared double in specimens processed in glycerine, and single in those processed in lactophenol

 

Tarjan & Cairns, 1955 -- Proceedings Helminthological Society Washington 22: 32.

Methylcellulose was proposed as a medium for the rapid preparation of temporary head mounts. Not only could specimens be transferred directly from fixative or water into this

material, but after severing the head of the nematode, the head could be placed directly into this medium and a cover slide placed over the medium. Manual manipulation of the coverslide

brings the head into the proper position for viewing and application of molten paraffin-petroleum jelly cements the coverslip firmly in place.

 

Troma & Douvres, 1953 -- Proceedings Helminthological Society of  Washington  20: 59.

A modified en face technique for the preparation of nematode head mounts is described

with three illustrations of the special slide needed for the technique..

 

Webber, 1955 -- Trans. Royal Soc. Tropical. Medicine & Hygiene 49: 300-301.

A modification of the head mounting technique is proposed.  The head end of a whole nematode is threaded through a small hole which has been drilled in a microscope slide ade from Perplex.  The head projects upward and is covered by a cover slip whereas the remainder of the nematode rests in a shallow chamber beneath the slide.

 

 

MOUNTS, PERMANENT, MISC. PREPARATIONS -- MNP

 

Arant & Knapp, 1949 -- Science 75: 495-496.    

Nematodes are killed in 5% alcohol, transferred to 70% alcohol after 15 min. and then to a drop of alcohol on a slide. The alcohol is ignited and the nematodes, which become attached to the slide, are stained by adding a drop of Delafield's hematoxylin, then washed, dried and mounted in Canada balsam.

 

Bailenger & Neuzil, 1953 -- Annales Parasit. Humane Comparree 28: 392-398.    

Whole mounts of  helminths were stained with chlorozal black E  after fixation in hot alcohol,  The specimens were then transferred to a solution with hydrochloric acid  0.5ml, glycerol 10ml, and water to make 100ml.  This solution with the specimens slowly evaporated to glycerol at 37C. Final mounting was in sodium alginate gel.

 

Baker, 1953. -- Canadian Entomologist 85:77-78.

A rapid method for mounting nematodes in glycerine transfers nematodes fixed in 5% formaldehyde to a series of 6 solutions with increasing amounts of glycerine and also containg water, lactic acid, phenol, and formaldehyde.

 

Capstick, 1956 -- Nature 177: 896-897.       

For the preparation of mounts of nematodes, it is suggested that 80/20 nickle/chromium resistant wire can effectively be used as supports between the slide surface and the coverslip. The wire is made in standard diameters of 25, 30, 40, and 50 microns and can be cut to short lengths.

 

Dass, 1949 -- Stain Technology 24: 229-230.    

Specimens were mounted in acid fuchsin-lactophenol, kept at 60C for 6 hours after which the acid fuchsin was replaced with cotton blue counterstain.  The specimens were finally mounted in pure lactophenol.

 

Davis, 1924 -- Botanical Gazette 77: 343-344.

A slide-cover slip ringing material, called lactophenol gum, was proposed using gum arabic, freshly distilled water, glucose and lactophenol. The prepared solution is filtered through glass wool and is used cold.  It dries quickly and adequately seals lactophenol mounts.

 

deGrisse & Choi, 1971 -- Meded. Rijksfaculteit Landbouw.,Gent 36: 617-619    

A rapid method for transfering fixed nematodes to anhydrous glycerine was proposed. Water in tissues is removed and replaced by ethyl alcohol after the nematodes are killed and fixed. The specimen-containing vessels are placed in a dessicator which is evacuated using a water pump until the ethanol boils under reduced pressure. An aliquot of  "Solution II of Seinhorst" is added, and the container is placed in an incubator at 40C. After evaporation of the ethanol, the nematodes are in pure glycerine.

 

Esser & MacGowan, 1973 -- Journal of Nematology 5(1): 76.      

The problem of mountant not staying within prescribed limits on a glass slide has been overcome by use of a method that controls droplet size and prevents mounting preparations from flattening and creeping in an uncontrolled manner across the length and width of a microscope slide. Use of "Silicad", a silicone preparation is described.

 

Goodey, 1935 -- Journal of Helminthology 13: 173-190.     

Nematodes were first fixed in 5% formaldehyde, washed with water, and then placed in 70% alcohol  with 3% glycerine. The nemas in small vials were then placed in a dessicator containing calcium chloride.

 

Jutras & Tarjan, 1961 -- Journal of Parasitology 47: 369-371. 

A two-component apparatus for constructing "Cobb" metal slides for microscopy  is described. The formation of the aluminum slides is accomplished by three assemblies. A final shaping of the aluminum mount is performed by a fourth assembly. Nematodes mounted in glycerine and contained between two #0 cover slips slip into the metal mount and are positioned in place by a rigid vinyl plastic insert.

 

Loos, 1901 -- Records of the Egyptian School of Medicine  1901:27-138.    

Employing the principle of slow dehydration into glycerine, Loos placed small nematodes in 2% glycerol  in 70% alcohol (glycerol increased to 10% if worms were large). The mixture slowly evaporated  to pure glycerol in which the nematodes were mounted (or in glycerine jelly).

 

Morgan, 1928 -- Journal of Helminthology 6: 79-80.   

Nematodes were first inserted into hot Ditlevsen's fixative and then transferred to a mixture of  30% alcohol and 2% glycerine which was allowed to evaporate to almost pure glycerine.

 

Seinhorst, 1959 --Nematologica 4: 67-69.   

A rapid method for transfering  fixed nematodes into anhydrous glycerine is described.  Nematodes are placed in a mixture of 20ml of 96% ethanol, 1ml glycerine, and 79ml of distilled water.The nematodes are then gradually transferred into pure glycerine following a series of intermediate steps.

 

Tahmisian, 1945 -- Stain Technology 20: 26.      

It was found that if hookworms were pierced with a fine needle at the 70% stage of passage through the alcohol series, opacity of the worms was avoided when they were mounted in balsam.

 

Thorne, 1935 --  Proceedings of the Helminthological Society of Washingtton 2: 98.

A new slide-ringing material named "ZUT" was proposed by the author in his series

"Notes on free-living and parasitic nematodes". The material contains linseed oil, nitrocellulose, butyl acetate and toluene. It is usable as a material for ringing cover slips

in the preparation of "permanent" slides.

 

van der Vegte, 1959 -- Nematologica 4: 356-357.     

To save time and to combine the advantages of  a temporary water mount and a permanent glycerine mount, the fixative  F.A.A. (formalin acetic acid)  was used and to which gum arabic and pure glycerine were added.

 

Yetwin, 1944 -- Journal of Parasitology 30: 201.    

A procedure and medium were described for mounting nematodes from a glycerine or formalin solution. The final mounting medium was made of  Bacto glycerine, glycerine, and chromium and potassium sulphate which hardens into a permanent mount.

 

Zullini, 1972 -- A brief note on the rapid mounting of nematodes in glycerine (in Italian).                                                           

Bolletino di Zoologia agaria e di Bachicoltura, Ser II 11: 19-20     

A description is given of a method for the inclusion of soil nematodes in glycerine by slow evaporation directly on the slide.

 

 

 

MOUNTS, PERMANENT, MOUNTING FLUIDS-- MNR     

 

Baker, 1953 -- Canadian Entomologist 85: 77-78.        

A rapid method for mounting nematodes in glycerine was proposed. Specimens in water killed by gentle heat are transferred to 5% formalin/water solution and left at least overnight. They are then  transferred to lactophenol, warmed to about 65C, and then passed through 5 mixtures which essentially decrease the amount of water and increase the amount of glycerine.  The nematodes finally are mounted in pure glycerine.

 

Capstick, 1956 -- Nature 177: 896-897.

For the preparation of mounts of nematodes, it is suggested that 80/20 nickle/chromium resistant wire can effectively be used as supports between the slide surface and the coverslip. The wire is made in standard diameters of 25, 30, 40, and 50 microns and can be cut to short lengths.

 

Chatterji, 1935 -- Zoologischer Anzeiger 109: 134-138.    

The author foud the chemical substance  Dioxan to be effective when used in a technique for the permanent mounting of nematodes. Nematodes after storage in 70% alcohol were left overnight in Dioxan , cleared in clove oil for 1 - 2 hrs., and then mounted in Canada Balsam.

 

Esser, 1973 --  Plant Disease Reporter 57: 1045-1046.      

A four-minute lactophenol fixation method for nematode preparations is proposed. Nematodes are heated in water up to 35C by use of a timer-operated hotplate at 370C for 2 minutes. Stained lactophenol is then added to the water and heat continued for two additional minutes.

 

Gallego, 1947 -- Revista de Iberica de Parasitologica 7: 117-182. 

Small nematodes were first fixed with formaldehyde or Flemmings solution and then mounted in a medium consisting of distilled water 50g., gum arabic 30g., chloral hydrate 200g and glycerine 10g.

 

Hesling, 1960 -- Nematologica 5: 322

In transferring delicate specimens from clearing agent to mountant on a glass slide, the transferred clearing agent may dilute the mountant  Specimens  will usually adher to  a tiny drop of mountant on the tip of a finely drawn glass rod and thus can be transfered to the mountant on the slide. The small amount of clearing agent also transferred will have no effect on the mountant.

 

Li & Yang, 1945 -- Nature 156: 297-298.     

A medium for mounting nematodes was prepaired with much tolerance for water thus making subsequent dehydration unnecessary. the material was an aqueous medium containing gum arabic, chloralhydrate,and glycerine.

 

Lippens & Grootaert, 1974 -- Nematologica 19 (year 1973): 562-563.       

A routine method for mounting nematodes in resin with high refractive index employs "ERL", an epoxyresin with low viscosity.Other materials used are paraldehyde and osmic acid. The authors present a list of six advantages which this method offers.

 

Reed, Hutchinson & Race,  1957 -- Plant Disease Reporter 41: 25-26.       

A new, permanent method for mounting phytoparasitic nematodes uses a modified Berlese Fluid composed of distilled water, gum Arabic, chloral hydrate, and glycerine. Details of the cuticle as well as the glandular systems are well-defined. The slides made by the authors have  remained permanent for more than two years.

 

Rubin, 1951 -- Stain Technology 26: 257-260.       

A procedure was described in which nematodes are cleared and then permanently mounted in a mixture of  polyvinyl alcohol, phenol and lactic acid. Larvae and eggs can be transferred directly from formalin, water, alcohol or glycerine alcohol.

 

Scott, 1955 -- Journal of Parasitology 41: 219.      

A description is presented regarding the preparation and use of glychrogel medium, composed of glycerine, camphor, chrom alum and gelatin.  In addition, a procedure for working with small filarial nematodes was given in which they are processed from alcohol to increasing concentrations of glycerine, and finally into glychrogel.

 

Smith, 1966 -- Nematologica 12: 177.    

Dimethy hydantoin formaldehyde resin was found to be a useful mounting medium for nematodes in two main ways. First, easily prepared mounts which are of some value for weeks can be prepared ; second, the preparations made are of more permanent value, and after 18 months have shown very little shrinkage..

 

Tarjan, 1967 -- Nematologica 13: 153-154.                

An abbreviated method for preserving nematodes is described whereby a minimum of 35 minutes is required to transfer fixed specimens into anhydrous glycerine. Nematodes from 53 genera were processed over a period of three years. Only partial collapse in some specimens of Trichodorus and Criconemoides were evident.

 

 

MOUNTS, PERMANENT, SPECIAL SEALS  -- MNS         

 

Abu-Gharbieh & Smart, 1969 -- Nematologica 15: 615-618.                                                     

A technique is described and illustrated for the purpose of sealing microscope cover glasses

with the sealing compound  "Zut"., while using a polyethylene bottle applicator. The technique provides fast, effective and uniform seals.

 

Boparai & Chabra, 1968 --  Jour.Research Punjab Agricultural University 5: 70-71. 

"Araldite" was reported as a suitable ringing material for preparing nematode slides.

"Cutex", wax, wax + grease mixture, and "Quickfix" were also tried as sealing materials for glycerine-mounted nematode slides. The slides ringed with "Araldite" can be stored for a long time.

 

Caveness, 1969 -- Journal of Nematology  1: 96.   

Two silicone rubber preparations, Dow Corning's Silastic and General Electric's Silicone Seal were evaluated as slide ringing materials during a period of 26 months. Most water-formalin mounts sealed with silicone rubber or zut dried in 2-6 months. Glycerine slides ringed with silicone rubber or zut showed no signs of evaporation after 26 months at  room temperature.

 

Esser, 1974 -- Nematologica 19:  566-567.

The special nematode slide ringing material 'ZUT' is proposed usage as a cover glass support for nematode slides to replace fingernail polish which has received wide usage.  Nematode specimens were adequately supported without body flattening.  The chief disadvantage of its useage lies in the adequate recovery and remounting of specimens from other slides.

 

Porter, 1939 -- Turtox News 17:14.         

A way for preserving glycerine jelly slides was proposed by. treating the water in glycerine jelly with formalin. Thus treated, the jelly becomes bound, and even exposed glycerine jelly will remain unchanged in consistency when so treated.  A drop or two of formalin is placed at the edge of the coverslip and allowed to stand for several hours while the formalin diffuses into the gelatin. After the surplus jelly is removed, the slide can be sealed.

 

 

MOUNTS, TEMPORARY -- MNT   

 

Abe, Kito, Aryuthaka, & Tamura -- Nematologica 39:135-137

A photographic slide mount was used for mounting nematodes. Using double cover glass preparations allows larger nematodes to be observed.  The method is adequately illustrated using 4 diagrams. The authors point out that the type of slide used does not fit the standard

microscope slide holders or microslide storage containers         

 

Cairns & Tarjan, 1955 -- Proceedings of Helminthological Society of  Washington 22: 32.

Methylcellulose for the rapid preparation of nematode head-mounts envolves a technique using a thick methylcellulose preparation as an aid in the preparation of temporary nematode head mounts. This method is specifically applicable as a rapid routine diagnostic procedure when an en face view is wanted within minutes after the living specimen is examined. The head is not distorted by this treatment                                     

 

Cobb, 1918 -- Estimating the nema population of the soil, Agricultural Technology

Circular, Bureau Plant Industry, United States Departmentof Agriculture, pp 1-48.

One method that is useful for a great variety of purposes is mounting temporarily in water under cover glasses cemented in place by means of very hot melted wax applied from wax candle of small size.

 

 Doncaster, 1962 -- Nematologica 7: 258.

The author describes the sealing the cover slip of a temporary water mount of nematodes with candle wax mixed with Vaseline (petroleum jelly), 1-2 parts of  Vaseline with 4 parts of parrafin. A tube for applying the mixture to slides is described.

 

Langeron, 1905 -- Compte Rendu Seances Societe Biologie 58: 749-750.

Glycerine-2 parts, lactic acid-1 part, phenol-1 part, and water-1 part is used at half strength as the temporary mounting fluid after fixation in dilute formalin. The nematodes are afterwards put in full strength medium for mounting.

 

Moorthy, 1937 -- Journal of Parasitology 23: 100-102.

Moorthy worked with guineau worm larvae which were obtained from infected cyclops which had been killed in cold mercuric chloride - sodium chloride solution. The larvae were dissected out into the fixative where they were 'relaxed' by gentle heat. They were then stained with methylene blue and Giemsa for 24 hrs. and finally mounted in 2% formadehyde.

 

Peters, 1955, --  in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pg. 417.

A note on handling and processing nematodes  proposes the creation of a temporary mount by supporting the coverglass on three small pieces of glass fibre which has the same diameter as the nematode specimen and sealing with hot parafin wax. (Note: this identical method was being used in the mid-forties at the time the author joined the working group at the Beltsville, Maryland USDA Plant Industry Research Station).

 

de Toledo Artegas, 1937 -- Memorias Instituto Butantan (1935-6) 10: 65-75.

Nematodes and other helminths were first treated in acetic acid and then thoroughly impregnated with creosote.  The animals were then mounted in a mastic-creosote fluid medium.

 

van der Vegte, 1959 -- Nematologica 4: 356-357.

A method for fixing and mounting nematodes in one process is reported.  In essence, gum arabic and pure glycerine were added to the fixative F.A.A.in the following proportions -- Gum arabic 50g, distilled water 100cc, 40% formalin 40cc, pure glycerine 40cc, 96% alcohol 50cc and glacial acetic acid 3cc. The pH of the mixture was 3.5 and it was summarily named ARFAGAL.

 

 

MOUNTS, WHOLE --MNW

 

Arant & Knapp, 1932 -- Science 75: 495-496.

A simple method for rearing and mounting hookworm larvae is described. Larvae were reared in a mixture of  2 parts charcoal and 1 part feces. Juveniles were isolated through the use of two general methods and were mounted using alscohol as a killing and hardening agent. Larval structures were more distinct when haematoxylin was used rather than Congo red, Orange G or alum cochineal

 

Jairajpuri & Rahmani, 1979 -- Indian Journal of Nematology 8 (yr 1978): 177.

An ideal sealing medium for nematode slides which can be substituted for 'Glyceel' was found in a commercial product named "PUTTY", manufactured  by the Royal Paints and Varnish Company of New Delhi, India.  The material contains amyl acetate which is used as a diluent for thinning the "putty".  The substance was found to be ideal for the purpose of sealing nematode mounts.

 

Lippens & Grootaert, 1973 -- Nematologica 19: 562-563.

A routine method for mounting nemtods in resin with high refractive index is proposed using ERL, an epoxy resin with low viscosity.  Some of the advantages of the method are claimed to be:no significant alteration of dimensions of the nematodes, no sealing problems or

"swimming", and higher refractive index without losing detail. The disadvantage is the high cost of the chemicals involved.

 

Pijanowski, Cleveland & Georgi, 1972 -- Cornell Veterinarian 62: 333-336.

A glycol methylacrylate embedding technic for nematode whole mounts is reported in which

the chemical was found capable of permeating intact specimens. The clarity of detail and uniform transparency achieved, even in large specimens, led the authors to believe that the material may actually excel conventional clearing and mounting media.

 

Rubin, 1951 -- Stain Technology 26:257-260.

A procedure is proposed in which the nematodes are cleared and permanently mounted in a mixture of polyvinyl alcohol, phenol and lactic acid. Larvae and eggs can be trasferred directly from formalin, water, alcohol or glycerine alcohol.  Use of the foregoing may result in excessive clearing with smaller nematodes.

 

Smith, 1966 -- Nematologica 12:177.

Dimethyl hydantoin formaldehyde resin is proposed as a mounting medium for nematodes. It is proposed that living nematodes can be narcotised in propylene phenoxitol and then fixed in T.A.F. overnight. They are then mounted in dimethyl hydantoin formaldehyde resin. Clearing occurs in just a few hours.

 

MOVEMENT & MIGRATION -- MOV                                                      

 

Buckley, 1940 -- Journal of Helminthology  18: 178-182.

 The vertical migrations of certain bursate nematodes were investigated by a simple method. A glass slide was marked out into 10 compartments. Infective larvae from horse or sheep feces was smeared onto a special area of the slide. Fine sand was sprinkled evenly over the slide and a variety of procedures were followed eventually resulting in data concerning vertical migration of various helminths at different temperatures.

 

Chen & Rich, 1963 -- Phytopathology 53: 348.

In studying the attraction of Pratylenchus penetrans  by plant roots, the authors induced the movement of the nematodes through special U-tubes which were first partially filled with a modified Hoagland's and Knop's medium containing 0.5% agar.  White clover seedlings were grown on one side and the nematodes were introduced into the other side.of each of the tubes.

 

Crofton, 1954 --- Journal of Helminthology 28: 35-52.

A study was made of the migration of larvae of strongyloid nematodes on a glass slide, their positions being plotted in relation to an eyepiece scale. The movement of larvae along channels in a special series of slides and over surfaces in a water film which was gradually reduced by drying also was observed.

 

Edmunds & Mai, 1967 --- Phytopathology 57: 468-471.

While investigating the effect of  Fusarium oxysporum on movement of Pratylenchus penetrans toward alfalfa roots the authors found that P. penetrans was attracted to carbon dioxide and to gases coming from the germinating alfalfa seedlings. More carbon dioxide was released from infected than from noninfected alfalfa roots.

 

Evans, 1969 -- Nematologica 15: 433-435.

The author refers to other work related to nematode movement and presents a modification of an apparatus suggested by Wallace, 1958. Two diagrams are presented which illustrate the suggested modifications.

 

Klingler, 1963 -- Nematologica 9: 185-199.

Experiments on agar sought to determine whether aggregations of nematodes near carbon dioxide were because of undirected (orthokinetic) or directed movements. It was found that Ditylenchs dipsaci was capable of a directed orientation reaction (klinotaxis).. Directed movements were observed with artificial as well as biological sources of carbon dioxide.

 

Peters, 1953 --- Journal of Helminthology 27: 107-112.

An investigation was made of vertical migration of the golden nematode Heterodera rostochiensis by growing a potato plant in a sectionialized wooden box of soil. Cysts were introduced in thin layers at known depths.

 

Pfaeltzer, 1935 -- Archief voor Koffiecultuur in Indonesie 9: 29-38.

In order to demonstrate the passages made by nematodes in their movement through roots, air is displaced from the roots in warm water.  The roots are then soaked in India ink for 30 min. followed by treatment with acidified water for 2 hrs which coagulates the ink.

 

Tarjan, 1971 -- Proceedings of the Soil and Crop Science Society of Florida 31:253-255.

The movement of three citrus-pathogenic nematodes through two Florida Soils over a was studied.  The progress of Radopholus similis, Tylenchulus semipenetrans and Pratylenchus coffeae through a light-textured "Lakewood sand" and a heavier-textured Parkwood sand contained in 1 x 1 x 4 ft.long boxes was studied over a two-year observational period

 

Tarshis, 1958 --- Proceedings of the Helminthological Society of Washington 25: 99-106.

For a field study of the lateral migration of nematodes parasitizing cattle, the author used eight concentric galvanized sheet metal rings spaced 2 inches apart as a measuring apparatus and  guide to the collection of forage clippings about the central point of inoculation.

 

Van Gundy & Stolzy, 1963 --- Nematologica 9: 605-612

The relationship of oxygen diffusion rates to life processes of  Hemicycliophora            arenaria

involved the use of the platinum-microelectrode technique at the interface between the solution containing the nematodes and the air in the pore spaces. The paper suggests the use of oxygen diffusion as a reliable measure for the availibility of oxygen to nematodes and on the effects of oxygen deficiencies on nematode biology.

 

Wallace, 1955 --- Annals of Applied Biology 43: 477-484.

The author presents a technique for the purposes of measuring nematode motility in sand. This was part of a study of the influence of soil moisture on larval emergence from cysts of Heterodera schactii.

 

Wallace, 1958 --- Annals of Applied Biology 46: 74-85.

A variety of simple techniques were used by the author to investigate the effects of pore size and moisture content on migration of nematode juveniles.

 

Wallace, 1961 -- Nematologica 6: 222-236.

For tests investigating the orientation of Ditylenchus dipsaci to physical stimuli, a  temperature gradient was sent up in a polyethylene tube, 15cm long and 6mm internal diameter, which contained water-saturated sand of particle diameter 250-500 microns.

It was found that light, gravity and water percolation do not affect the orientation of the nematode, although passive directional movement may occur with the last two stimuli.

 

           ----- N -----

 

NEMATICIDE APPLICATION -- NAP                                                        

 

d'Herde & van den Brande, 1959 --Mededelingen van de Landbouwhogeschool en de Opzoekingsstations van de Staat te Gent,  Belgium.24:637-644. (in Flemish)

A new applicator for soil fumigants allows a correct distribution of very low to normal doses of fumigants at the desired depth of soil. The mechanism by which rate of flow is controlled is of simple construction. Flow rate is not altered due to speed modification  Two prototypes are illustrated.

 

Good & Christie, 1956 -- Plant Disease Reporter 40: 987-988.

A water pressure injector which can reach all parts of a plant's root system is described and illustrated. It is constructed from cheap readily-available materials, and is composed of a reservoir tank of 3-1/2 quart capacity which is attached to the water line. It is fitted with garden hose fittings and has a plug which can be removed for inroduction of nematocide into the tank.

 

Ohr, Sims, Grech, Becker & McGriffen, Jr.,1996 -- Plant Disease 80: 731-735.

Methyl iodide was tested as a replacement for methyl bromide as a soil fumigant. It is a better methylating  agent than methyl bromide and is rapidly destroyed by UV light and therefore unlikely to be involved in stratospheric ozone depletion. When compared at equivalent molar rates,  methyl iodide was equal to or better than methyl bromide in controlling the tested soil-borne pathogens and weeds.

 

Tarjan, 1959 -- Plant Disease Reporter 43: 451-458.

A novel pressure-injection apparatus for introduction of  liquids into trees was used to inject 84 formulations of 54 chemicals into 89 Valencia Orange Trees , Citrus sinensis. Some of the materials showed promise as nematocides, but the extent of control varied with fluctuations in nematode populations during the sampling period.

 

Taylor & O'Bannon, 1968 -- Plant Disease Reporter 52: 218-222.

The authors conducted experiments with an apparatus for subsurface application of nematocides to nursery plants in containers. The applicator applied subsurface drenches of nematocides mixed with water for control of nematodes attacking plants in nursery containers. Root-knot, spiral and burrowing nematodes were controlled in the various tests.

 

Thorne, 1951 -- Proc. Helminthological Society of Washington 18: 18-24.

Experiments were conducted to determine the diffusion pattern of certain soil fumigants when applied with chisel-type commercial machines or as single injections with hand applicators. The method thus resulting gave a definite reading on the killing range of chemicals as they are applied in the field.

 

 

NEMATICIDE, LAB ASSAY -- NLA                                                             

 

Bishop, 1958 -- Nematologica 3: 143-148.

A technique was designed to test the nematicidal value of antibiotics against the root-knot nematode  Meloidogyne incognita acrita. The author tested application of the the antibiotics to the nematodes, penetration into the nematodes, and effect on reproduction. hey found that only a 2% solution of undecylenic acid was lethal to the nematodes.

 

Fenwick, 1951 -- Journal of Helminthology 25: 166-172.

The author studied varying effects of a nematocide against cysts of the golden nematode. He used two methods, in pot tests, to estimate the kill because on the inherent complications in determining kill versus hatchability.  The data obtained was subjected to a statistical probit analysis.

 

Goodey, 1945 -- Journal of Helminthology 21: 45-59.

Small seed samples infected with Ditylenchus dipsaci  were exposed to known quantities of methyl bromide sealed in glass ampules from which the chemical was released. Larger scale tests and inclosed in closed jars ccurred using tim canister boxes. A larger scale test involved using tin canister boxes in which as much as 8 lbs. of seed could be treated.

 

Johnson & Lear, 1962 -- Plant Disease Reporter 46: 742-743.

A method for assaying nematocidal activity of experimental chemicals suggests mixing root-knot nematode-infested soil and experimental chemicals to be a safe and efficient method of dispersing candidate materials throughout a soil mass.After storage the nematocidal activity of a chemical is evaluated by growing test plants in the treated soil.

 

Klotz, De Wolfe & Baines, 1959 -- Plant Disease Reporter 43: 1174-1175.

Lucite cylinders were constructed with ports at at different levels for testing the effectiveness of soil fumigants.  Plants are capable of being grown in the ports.  The cylinders are stood on end in racks and filled with soil. The candidate chemicals are added at the top.

 

Loos & Stessel, 1958 -- Plant Disease Reporter 42: 1187-1190.

Two methods were studied for investigating certain chemicals as contact nematocides. .One of these methods uses an "aeration-agitation" device which is described and which is compared to a previously developed contact test method.

 

.Peters, 1952 -- Annals of Applied Biology 39: 447-456.

The author tested various nematocides against Heterodera rostochiensis and proposed a pot test for evaluating the effects of nematocides injected into soil contained in 8-liter glazed pots.

 

Schuurmans Stekhoven & Mawson, 1954 -- Meded Landbouw.Opz.Gent 19: 373-376.

Two methods were proposed for testing nematocides in which nematodes in water were exposed to the action of chemicals suspended in an inverted cloth conical-shaped container, the apex of which just touched the dish with the nematodes. In addition they were exposed to crystals of the chemical which adhered to an inverted coverglass floating in the dish.

 

Staniland & Stone, 1953 -- Journal of Helminthology 27: 41-74.

The authors, in attempting to determine if solubilized chemicals could penetrate into cysts, immersed them into the solubilized chemicals in which a small quantity of blue dye was dissolved. Cysts were removed at varying internals, washed and placed in clean water.  By then putting pressure on the cysts, a stream of die coming out insured that the dye had penetrated the cysts.

 

Taylor, Feldmesser & Feder, 1957 -- Plant Disease Reporter 41: 527-530.

A new technique for preliminary screening of nematocides is described where  mixed populations of  Rhabditis sp. and Panagrellus sp are used as the test organisms. These are cultured on cellulose sponges soaked in potato broth. Test containers are glass vials of 5.1ml capacity into which 1000ppm of the test material has been inserted.. After 1-2ml of nematode suspension has been added to each vial, the vials are 2/3ds filled with clean dry sand.

 

NEMATICIDE, SPECIAL METHODS --NSM                                              

 

Bijloo, 1966 --Nematologica 11: 643-644.

The author introduces the"Pisum" test, a simple method for the screening of substances on their therapeutic nematocidal activity. By means of a special syringe, 0.02 ml of a suspension of Ditylenchus dipsaci containg about 50 nematodes is placed between the cotelydons on the plumule of pea plants, Pisum sativum. Two weeks afterwards, the plants are inspected and the degree of infestation estimated.

 

Bishop, 1958 -- Nematologica 3: 143-148.

Designed solely for laboratory testing, this method first exposes the nematodes to the test materials.  The nematodes are then washed by the aid of centrification after which they are tested for host penetration by being added to plants growing in vermiculite in test tubes.

 

Feder, 1954 -- Phytopathology 44: 428-430.

Two methods were described for testing the action of chemical vapors against Ditylenchus or Aphelenchoides spp. within plant tissues. Infected material was placed either in a watch glass in Petri dishes or it was supported on a loose cotton wool plug above the chemical in a test tube.  

 

Goodey, 1945 -- Journal of Helminthology 21: 45-59.

A fumigation chamber constructed from a section of sewer pipe sunk into a concrete base was capable of  conducting fumigation tests on as much as 30-lb. lots of seed samples infected with Ditylenchus dipsaci.

 

Harrison, 1959 -- Phytopathology 49: 540.

A technique was devised for the detection of lethal concentrations of nematocides in soil at various internals after injection. Heterodera rostochiensis eggs and juveniles were placed in thin glass bubbles, encased in nylon bags, and buried in soil so that their long glass necks protruded above the soil surface. Pressure applied to the neck broke the bubble at the other end and the nematodes were exposed to the vapors still present in the soil.

 

Ichikawa, Gilpatrick & McBeth, 1955 -- Phytopathology 54: 576-578.

Following earlier suggestions made by Thorne, 1951 for determining  diffusion patterns, a special trowel was used to cut two-inch cubes of soil.  Each of the cubes was processed for nematodes or placed in a small pot with sterile soil and planted with an indicator plant which was later examined for nematodes. The location of each cube with respect to the injection site was recorded.

 

Jensen, Caveness & Mulvey, 1954 -- Plant Disease Reporter 38: 680.

A 14-inch vertical column of soil was obtained using a soil tube with a 1-inch diameter.

A metal scraper cut 2-inch sections from the core. Two composite samples from each side of the injection site were combined to give about 50g of soil.. Samples were put in 2-inch pots in which 5 Hairy indigo seeds were placed as indicator plants for the test nematode, Pratylenchus penetrans.

 

Schuldt, Burchfield & Bluestone, 1957 -- Phytopathology 47: 534.

Quantitastive determinations were made on the stability and the movement of an experimental nematocide by a colorimetric procedure that is dependant on a reaction with pyridine and alkali.

 

Staniland, 1959 -- Technical Bulletin Ministry Agriculture 7: 41-147.

In working with contact nematocides, thae author found that a waxed paper cylinder marked in depths of one inch and filled with various soils was useful in studying the penetration of fluids through soil and into cysts. After cutting with care around each of the one inch depth lines with scissors, each layer could be separated with a wire, manipulated on to a flat surface, and then examined.

 

Tarjan, 1955 -- Proceedings Helminthological Society Washington 22: 33-37.

Eight test organisms, namely Panagrellus redivivus, Diplogaster sp., Pungentus pungens, Xiphinema americanum, Meloidogyne incognita, Dolichodorus heterocephalus, Ditylenchus sp., & Rotylenchus erythrinae were compared as to reaction to 1000ppm preparations of various wetting agents and emulsifiers. The test sought to investiate the durabilty of the nematode test organisms against the mentioned materials which are used

in pesticide investigations.

 

Taylor, Feldmesser & Feder, 1957 -- Phytopathology 41: 527-530.

The authors describe a new technique for the preliminary screening of nematocides. They suggest that preliminary tests should use small amounts of the material under test and be reproducible within reasonable limits. A testing method is described and illustrated in which chemicals can demonstrate toxicity in solid, liquid, or vapor phases. As little as 100mg of chemical is sufficient for several replications.

 

 

NEMATOPHAGOUS FUNGI -- NUN                                                          

 

Aschner & Kohn, 1958 -- Journal of Animal Science 19: 182-189.

The authors described a medium for culturing the nematode-trapping fungus,

Harposporium anguillulidae.  They observed and described the method by which this fungus traps nematodes.

 

Balan, Krizkova, Nemec & Kolozsvary, 1976 -- Nematologica 22: 306-311.

A quantitative method for the detection of nematode-attracting substances and proof of production of three different attractants by the fungus Monacrosporium rutgeriensis was devised by the authors who were with the Department of Biological Properties of Low Molecular Weight Substances, Institute of Molecular Biology, Janska, Czechoslavakia. The described method, in connection with thin-layer chromatography, indicates that the fungus produces three different compounds which attract the nematode Panagrellus redivivus.

 

Crump & Kerry, 1981 -- Nematologica 27: 330-339.

A quantitative method for extracting resting spores of  two nematode-parasitic fungi, Nematophthora gynophila and Verticillium chlamidosporium, from soil is described. The soil sample is wet-sieved and the spores separated by centrifugation on a high specific gravity solution of magnesium sulphate. The spore suspension is counted directly under a compound microscope.

 

Gorlenko, 1956 -- Nematologica 1: 147-150

Methods are presented for the culture of  nematophagous fungi in flasks.  The production of a dry powder inoculum of the fungi which retains its viability for a year more or less is also presented.

 

Johnson, Curl, Bond and Fribourg, 1959 -- Methods for studying soil microflora-Plant relationships. Burgess, Minn,, 178pp.

This work, as the title implies, is a comprehensive collection dealing with technques.for the isolation and culturing on artificial media the nematophagous fungi.

 

Mankau & Clark, 1959 -- Plant Disease Reporter 43: 968-969.

Routine procedures are presented for working with soil samples and fragments of roots for isolating nematophagous fungi.

 

Nicolay & Sikora, 1989 -- Revue de Nematologie 9: 97-102.

Two techniques were developed for determination of the activity of egg parasitic fungi in field soils. They are based on the examination of eggs newly formed either in observation chambers or in a soilfraction. Experiments demonstrated that the rate of parasitism of newly produced  Heterodera schachtii eggs was not correlated with the nematode density, but increased with increasing fungus density.

 

 Tarjan, 1960 -- Nature 185: 779-780.

For determining the potential usefulness of various nematode-predacious Hyphomycetes, it was found that the nematode Panagrellus redivivus was an excellent victim to test the utility of the fungal traps. It was found that the sacrificial nematodes could be produced in an oatmeal- cation exchage resis medium, and could be maintained for years at 2-5c refrigeration after becoming established on test-tube agar slants.

 

Tolmsoff, 1959 -- Phytopathology 49: 113-114.

Nematode-trapping fungi  were readily isolated from Oregon soils on milk agar containing 2g powdered milk and 20g agar per liter of water. When 1g or less.of soil was scattered over the surface of the medium,  Arthrobotrys appeared within 10 days only if nematodes were also presnt on the plate.

 

---- O -----

 

OBSERVATION -- OBS

 

Bedding, 1967 -- Nematologica 13: 643-644.

A simple technique for displaying the surface structure of nematodes is depositing metallic silver in the striae and lateral fields of nematodes so that those features can be examined using transmitted light. The method is fully described and depicted.

 

Bloomberg, 1963 -- Plant Disease Reporter 47: 455-458.

Acrylic molding beads gave the best combination of translucence, lack of optical distortion, chemical inertness, and ability to support normal growth in a search for a translucent rooting medium for the observation of Douglas Fir seedling roots and organisms surrounding the roots.

 

Bolla, Shaheen and Winter, 1982 --  Journal of Nematology 14: 431.

Phytotoxin production by Bursaphelenchus xylophilus in pine wilt.was shown by obtaining a lipid extract from nematode-infected Scotch Pine, Pinus sylvestus . The acquired extract produced symptoms in all  species of pine tested except P. jeffreyi. These symptoms were comparable to those symptoms produced by B. xyloplilus.

 

Chapman & Eason, 1969 -- Journal of Nematology 1: 279-280.

A technique for studying penetration of roots of plants by endoparasitic nematodes was reported. It consists of incubating nematodes and seedlings between two 50mm discs of

"Miracloth" in a 50mm petri dish. After incubation, water is added, the top layer of the cloth is removed, and the seedlings are fixed, stained, cleared and mounted for observation.

 

Chen, Kiernan & Mai -- Phytopathology 55: 490-491.

A technique for studying the feeding of Trichodorus christiei on isolated root cells of corn is reported. Single aseptic root cells were obtained from the emerging radicle of a corn seedling.and maintained in isotonic solution. Isolated  root cells were added to a drop of concentrated nematode suspension was placed in the well of a slide filled with 0.8% agar. The slides were then inerted and observations made through the cover glass at magnifications up to 1500x.

 

Cobb, 1906 -- Hawaiian Sugar Planters Association Bulletin 5: 178-179.

The author briefly described and illustrated the construction of molded metal micro-observation chambers which provide for aeration and moisture.

 

Cobb, 1918 -- Estimating the nema population of the soil, Agricultural Technology Circular, Bureau Plant Industry, United States Departmentof Agriculture, pp 1-48.

Microscope eyepieces with a cross-hair diaphram  are described using a spiders web fastened on a metal diaphram about 1/50th of an inch thick. The metal is ruled into squares with a convenient tool. Additional details are presented for completing for the finished diaphram.

 

Dean, 1929 -- Phytopathology 19: 407-412.

Two types of root-observation boxes are described  and illustrated by drawings.The smaller of the boxes is modeled after  the "root cages" first described by J. H. Comstock.  The second larger box is a "swinging" observation box. Drawings and illustrations of the boxes are given.

 

den Ouden, 1958 -- Tijdschrift over Plantenziekten 64: 269-272.

A method for growing plants in thin layers of agar is described. Enough agar is introduced into polyethylene bags under sterile conditions to form a thin, even layer containing numerous air bubbles. Sterile germinated seeds  are inserted through a cut in the bag which is then resealed . Cuts are subsequently made for the shoots to emerge. Sterile nematodes are introduced as is water, from time to time. The method  can be used for the observation of nematodes and other root paprasites while attacking growing roots.

 

Dickinson, 1959 -- Nematologica 4:60-66.

The behavior of larvae of  Heterodera schachtii on nitrocellulose membranes was studied so as to find out how an eelworm penetrates the outer cell wall of the root apex. Repeated penetration of the membrane by the nematode stylet was observed Passage right thru the membranes by the nematodes also was observed.

 

Doncaster, 1964 -- Nematologica 10: 306-312.

Sealed observation chambers of two large cover slips separated by a variable number of gaskets have been used for nematodes in agar or water. Perfusion chambers have been used for specimens that need their culture medium replenished frequently.  A chamber for observing roots at high magnifications incorporates sand whose moisture level can be controlled.  This chamber can be perfused and entire seedlings accomodated within.

 

Doncaster, 1966 -- Nematologica 12: 645.

Modifications to the moisture-controlled observation chambers described by the author  and published in 1964 are proposed. Additional suggestions pertaining to construction of the

chamber, sealing and filling the chamber, and placing seedlings and nematodes within it are

proposed.

 

Doncaster & Clark, 1964 -- Nematologica 10: 136-140.

Observations were made on gut pH and absorption of methyl red and neutral red dyes in the intestinal walls of  Pelodera and Mesodiplogaster. Except for Ascaris lumbricoides and Strongylus edentatus, The nematodes tested showed acid gut reactons.  Methyl red and neutral red pH indicator dyes were absorbed selectively by different regions of the intestinal wall and by the two species.

 

Estey, 1970 -- Canadian Journal of Plant Science 50: 209-210.

Various modifications are proposed to extend the usefulness of root-observation cells originally described by the author. Difficulties in using the original cells are referred to

and the  modifications described. The base of the original cell was enlarged sufficiently to support a container of soil at each end of the observation portion of the enlarged unit.

The modifications proposed are shown in three accompanying illustrations.

 

Godfrey, 1929  -- Phytopath 19: 611-629.               

In inoculation studies with Tylenchus [cf  Pratylenchus] brachyurus on roots of pineapple plants, a novel root observation box (illustrated) was used allowing excellent availability of roots for observation,  examination with microscope, and manipulation. By unhooking and swinging back the upper cross-piece and lifting the glass front, the roots become exposed without and disturbance.

 

Goodey, 1932 -- Journal of Helminthology 10: 33-44.

The author was among the first to construct a root-observation box. with a glass front. Using  this, he made observations of root-knot nematode on seedlings in sand and also in sand in Petri dishes.

 

Khera & Zuckerman, 1963 -- Nematologica 9: 1-6.

Feeding habits and parasitism of some ectoparasitic nematodes were simultaneously observed in vitro. . Resistance to attack by nematodes was also assayed. They postulated that feedig in vitro  is indicative of the habit of the nematode under natural conditions.

 

Kunz & Klinger, 1976 -- Nematologica 22: 477-479.

A method for direct or microscopic observation and photography of nematode tracks during orientation behavior studies was developed because of the need for a system providing a view of the experiment either by naked eye, through a surgeon's loup, or using a stereomicroscope. The method is described and illustrated which fulfills the requirements and permits photography with or without a stereomicroscope..

 

Linford, 1937 -- Proceedings Helminthological Society Washington 4: 41-46.

Observations were made on nematodes in hard agar within Petri dishes by inverting the dishes on the stage of the microscope. Details were observed at 600x using a 40x achromatic water immersion objective having a working distance of 1.9mm.

 

Linford, 1940 --.Phytopathology 30: 348-349.

Miniature root-observation boxes were constructed and filled with either black or white sand, depending on the desired background color. These were used for experiments dealing with the root knot nematode.

 

Mankau, 1975 -- Journal of Nematology 7: 119-122.

A semiquantitative method for enumerating and observing parasites and predators of nematodes was developed. The method described, with two illustrations of needed components has distinct advantages over others for enumerating organisms which attack nematodes.

 

Minton, 1962 -- Plant Disease Reporter 46: 199.

A root observation box for studying infectionm and pathogenesis of roots by nematodes is described and figured. The inside dimensions of the inoculation box are 3/4 x 11 x 30 inches. The box is constructed of 1/2 plywood and is lines with 4 mil thick plastic film. The removable plywood front side is attached with 1-inch wood screws. The medium in which plants are grown is a finely ground vermiculite.

 

Peacock, 1959 -- Nematologica 4: 43-55.

A technique for studying the host-parasite relationships of the root-knot nematode, Meloidogyne incognita under controlled conditions is presented. Among the advantages of this technique it was found that immersion of egg-masses for 15 min. in a

0.5% solution of 'hibitane diacetate' gave a high degree of asepsis, later improved by a preliminary washing in 0.1% 'Cetaflon'.

 

Sutherland & Adams, 1965 -- Nematologica 10: 637-643.

The authors investigated the pathogenicity of Tylenchorhynchus claytoni on the roots of red pine seedlings. The nematode was found to be parasitic, but not pathogenic. Twelve other species of forest nursery seedlings also were found to be hosts for this nematode.

 

Trudgill, 1976 -- Nematologica 22: 371.

A simple observation cell for studying the feeding of plant parasitic nematodes describes

observation chambers so thin that specimens can be viewed with high-powered microscope

objectives. The cells were made on 2.5 or 5.0cm x 7.5cm glass slides using 'Parafilm' folded three times to support a glass coverslip 2.2cm x 5.0 cm.

 

          ----- P, Q, R -----

 

PHOTOGRAPHY -- PHO

 

Cobb, 1905 -- First Ann. Rept., Div. Path. & Phys., Hawaiian Sugar Pltrs. Association

A solar camera  used to facilitate the production of illustrations from negatives and from transparent objects is described and illustrated.

                                                                         

Doncaster, 1956 -- Nematologica 1: 51-55.

The author describes and provides an illustration of electric flash equipment used for photomicrographic studies. He suggests that another possible use for the electronic flash technique is the measurement of nematodes from adequate enlargements of the organisms and of the stage micrometer at the same magnification.

 

Ferris & Ferris, 1958 -- Plant Disease Reporter 42: 1192-1193.

Four illustrations of nematodes are presented of the results using a simple method for making rapid routine photgraphs of nematodes. The article presents necessary details regarding the suggested photographic setup.

 

Malcolm, Minter & Doncaster,  1978 -- Nematologica 24:167-174.

A low-light remote recording system used to study nematode behavior was capable of recording oscillatory phenomena. Fluctuating output from a photocell applied to a closed-circuit TV monitor screen is amplified and displayed on a pen-recorder chart.  The system described has several advantages over cinephotography.

 

McVey & Gerdemann, 1959 -- Plant Disease Reporter 43: 403.

A method of mounting roots to be photographed is proposed in which the roots in water are mounted between two panes of glass. The frame is made from sheet aluminum.  Air bubbles which become trapped between the glass plates may be expelled by raising and slowly lowering one side of the glass.

 

Pitcher, 1968 -- Nematologica 13: 547-557.

The host-parasite relations of  Trichodorus viruliferus on apple roots was studied under field conditions by means of direct observation, time-lapse cinephotography, and soil and/or root sampling.  It was found that the most striking feature was the massing of nematodes around the elongating zone of one type of young root.

 

Taylor & Esser, 1975 -- Proceedings Soil and Crop Science Society Florida 35: 93-95.

Photography of living plant parasitic nematodes is suggested using a method of mounting living nematodes and using an improvised exposure meter for photo-micography. Several examples of the photograhic technique are presented as well as a diagram of an improvised  microphotographic exposure meter.

 

PHYLOGENESIS -- PHY

 

Maggenti, 1970 -- Journal of Nematology 2:7-15

In a paper entitled "System analysis and nematode phylogeny", the author proposes to clarify the phylogenetic relationships of the Nematoda without discussing nematode fossils A model systems analysis is presented which indicates that further in-depth analysis of all systems would reveal a phylogeny consistent with the manifested development of contemporary representatives.

 

PRESERVATION -- PRE                  

                                                               

Caveness, 1969 -- Journal of Nematology  1: 96.

Two silicone rubber preparations, Dow Corning's Silastic and General Electric's Silicone Seal were evealuated as slide ringing materials during a period of 26 months. Most water-formalin mounts sealed with silicone rubber or zut dried in 2-6 months. Glyderine slines ringed with silicone rubber or zut showed no signs of evaporation after 26 months at  room temperature.

 

Cobb, 1918 -- Estimating the nema population of the soil, Agricultural Technology Circular, Bureau Plant Industry, United States Departmentof Agriculture, pp 1-48.

"The Differentiator" is illustrated and described. It is a glass instrument facilitating the very gradual transfer of a specimen from one fluid to another so as to avoid distortion due to osmotic effect. The apparatus controls the flow of liquid through it by means of a capillary orifice and by means of tilting the instrument thus altering the pressure.

 

Hwang & Sayre, 1969 -- Journal of Nematology 1: 292-293.

The fourth larval stage of  Ditylenchus dipsaci that aggregates into masses when drying was the only stage of the nematode successfully preserved at subzero temperatures.The results of the various tests conducted indicate that partial dehydration of the nematode is not a necessary prerequisite for the survival of the nematode populations at subzero temperatures.

 

Meyer, 1957 -- Transactions American Microscopical Society 76: 344-345.

The author proposed a spring device which would apply a uniform pressure to coverslips which cover relatively thick or curved specimens.

 

Petana, 1955 -- Journal of the Biological  Photographic Association 23: 65-68.

The author was able to preserve small animal parasitic helminths almost indefinitely in a mixture of isotonic saline solution 100 ml,  absolute alcohol 30 ml,  40% formalin 1.5 ml, and glacial acetic acid 1 ml.

 

Shaikh, 1956 --  British Medical Journal 2: 1545.

Eggs of helminths parasitic on animals, which can not be preserved satisfactorily in 3%  formaldehyde, retained their normal morphological features for 2 years in a 1:1 mixture of  glycerine and normal  saline.

 

Tarjan, 1967 -- Nematologica 13: 153-154.

An abbreviated lactophenol-glycerine procedure for preserving nematodes involves using four solutions for processing and terminating with the nematodes in pure glycerine.  A minimum of 35 minutes is needed to transfer the fixed nematodes to anhydrous glycerine. The method had been used on 53 genera of nematodes oer a period of three years.

 

Tarjan, 1973 -- Proceedings of the Helminthological Society of Washington 40: 296-297.  

Properly fixed specimens are immersed into a lactophenol-glycerine-water mixture containing cotton blue dye and kept at a temperature of 72C for 1 to 3 minutes.  The nematodes are then transferred to glycerine also at 72C for at least 5 minutes. There are no anatomical parts of the nematodes that are distorted or obscured any more by this method than by other preservation methods.

 

Zuckerman, 1963 -- Proceedings of the Helminthological Society of Washington 30: 65-66.

Preservation of plant parasitic nematodes by freezing presentds a method whereby the animals can be kept in fresh appearing condition for long periods of time

 

PRESSURE -- PRG                                                                                           

 

Barker & Sasser, 1959 -- Phytopathology 49: 664-670.

The use of pressure in conjunction with the cotton-blue lactophenol staining method for young seedlings was reported by the authors.   To better detect the presence of nematodes in the tissues, the plants were crushed between two glass slides.

 

Cobb, 1920 -- Transactions American Microscopical Society 39: 231-242.

A 'compressorium' for flattening chromosomes by applying pressure is fully described and illustrated.  Principal parts consist of two curved perforated steel springs affixed to a 3x1 inch microscope slide.

 

RADIOACTIVITY,  RADIATION, IRRADIATION -- RAD         

 

Dropkin & King, 1956 -- Experimental Parasitology 5: 469-480.

For studies on plant parasitic nematodes homogeneously labeled with radiophosphorus, a technique of homogenous labeling previously developed but which also allowed quantitative

determinations of total phosphorus present was used. Results are given in a number of drawings and charts.

 

Green & Webster, 1966-- Nematologica 11: 638-642

The effect of ultraviolet irradiation on the stem and bulb nematode, Ditylenchus dipsaci,

was investigated. Suspensions of 4th-stage larvae of the nematodewere exposed to radiation,

about 254 nm, at doses from 16 ergs/sq cm to 84,000 ergs/sq.cm. The nematodes failed to reproduce after doses of  100 ergs/sq cm, became immobilized after 10,000 ergs/sq.cm, and ruptured after 80,000 ergs/sq.cm.

                                        

Ishibashi, 1965 -- Nematologica 11: 361-369.

An increase in male adults  by gamma-ray irradiation of the root-knot nematode Meloidogyne incognita is reported. An irradiation of genital  primordia before sexual differentiation increased the percentage of small male adults with one testis. Irradiation retarded growth of nematodes in proportion to the dosage.

 

Roessner, 1971 -- Nematologica 17: 320-321.

The sterile culture of plant parasitic nematodes in a hermetically sealed glass container was described and illustrated. It was pointed out that similar culture systems have been described but are complicated and need a supply of sterile air and germ-free water requiring suitable filters and conmtinuous care and control. Experiments using Rotylenchus robustus with red clover as the host were in excellent condition after six months in the cultural chamber described.

 

Rohde & Jenkins, 1958 -- University of Maryland Bulletin A-97: 1-19.

Rhodamine B, a fluorescent vital stain, and the radioactive isotope P32 were

used in order to establish that an ectoparasitic nematode fed upon the roots of a certain plant.  Uptake of the materials from the plant by the nematodes was considered proof of feeding activity.

 

Sprau & Suess, 1962 -- Nematologica 7:  301-304.

In order to prove that Longidorus maximus fed on sugar beet roots,  sodium hydrogen phosphate labeled with radioactive phosphorus (P32) in solution was sprayed on the leaves of sugar beet growing in soil infested with the nematode. The radioactivity of nematodes extracted from near the roots of treated plants was greater than that of nematodes from untreated plants. It was thus concluded that the nematode fed on the roots.

 

Townshend, 1966 -- Nematologica 12: 100.

When increasingly greater dosages of gamma radiation were applied to a sandy loan containing cysts of Heterodera schactii, emergence of second-stage juveniles declined The dosages ranged arithmetically from 12,500 to 800,000 rads in seven treatments. Only juveniles from cysts that had developed on  table beet, Beta vulgarus, grown in irradiated infested soil that had been exposed to 50,000 raqds  or less developed to maturity.

 

REFERENCE  SYSTEMS -- REF      

 

Esser, 1974 -- FAO Plant Protection Bulletin 24: 1-6.

Two simplified data retrieval methods are given.  One method is a file consisting of four basic sections -- A. Subject, B. non-phytoparasitic nematodes, C. Phytoparasitic Nematodes, D. Botanical Name.  The second data retrieval method, Taxonomic Retrieval System (TRS), contains a list of  146 categories of nematology subjects into which data can be inserted.

 

Tarjan, 1963 -- Plant Disease Reporter 47: 859-862.

A bibliographic reference system is proposed for recording published nematological inforrmation using  a 2- and 3-holed punch card, namely the E-Z sort Bibliographic Research 4758 card. Only simple codes, including two external codes (for nematode genus and for subject) are needed.

 

Tarjan, 1967 -- Nematologica 13: 153-154.

A rapid preservation technique is proposed requiring 35 min. and using four solutions for transfering nematodes from fixative into glycerine. The method had been used for over three years involving 53 genera of nematodes. Detailes are presented on the preparation and use of the phenol-lactic acid-glycerine- water solutions needed.

 

 

RELAXING BEFORE FIXING -- REL          

 

Abdel-Malek, 1951 -- Journal of Parasitology 37: 321.

Menthol relaxation of helminths before fixation was proposed for a variety of small metazoans. Half of a g.of Menthol in powdered or crystalline form sprinkled on the surface of 100cc of water produced a saturated solution which caused relaxation of the contained parasites.The parasites are affected more readily if the temperature of the water is slightly

above room temperature                                                                                    .

                                                                                                                                      

 

RESISTANCE/SUSCEPTIBILITY, DETERMINATION -- RDO

           

Andersen, 1963 -- Nematologica 9: 527-530.

For determining resistance to cereal root eelworm, Heterodera avenae, in testing methods, porous clay land drains are the best method for growing plants. As supplementary methods, however, glass tubes and plastic bags may be used.

 

Dropkin, Helgeson & Upper, 1969 -- Journal of Nematology 1: 55-61.

In studying the hypersensitivity reaction of tomatoes resistant to Meloidogyne incognita,: reversal by cytokinins, the initiation of larval growth, induction of cell necrosis and gall formation in the host were measured as criteria of resistance or susceptibility to the nematodes. Exogenously supplied cytokinins shifted the response of resistant plants toward the susceptible reaction.

 

Janssen, van Silfhout,  & Hoogendoorn , 1999 -- Nematology 1: 31-36.

So as to avoid using an undesirable field population of mixed species for resistance screening against the root-knot nematode, a special protocol was developed producing species-specific populations from single females. In addition, clear differential reactions were obtained by inoculation of juvenile nematodes on roots of potato cultivars when "the Petri dish method" was evaluated.

 

Saichuk, Williams & Birchfield, 1976 -- Plant Disease Reporter 60: 868-870.

A technique for rapidly screening large numbers of soy bean lines for resistance to the Wartelle race of the root-knot nematode was needed. A number of tests were conducted and the results indicated that resistant, moderately resistant, and susceptible soybean seedlings were easily separated by root gall index at 25 days after planting, provided that good root development occurred.

 

Scheetz, 1966 -- Phytopathology 56: 586.

To demonstrate contrast between resistant and nonresistant roots, 30-day-old infected roots are quick frozen, sectioned in a cryostat, stained with Sudan IV, and photographed. Peripheral lining of lysigenomata is dark orange red in susceptible, and light orange red in resistant roots. 

 

Seinhorst, 1945 -- Tijdschrift over Plantenziekten 51: 39-52.

For purposes of testing plant reaction to Ditylenchus dipsaci, about 100 nematodes in a droplet of water are placed between the desired tissues.  For seedlings, a vertical incision is made in the coleoptile or, in older plants, in the leaf sheath.

 

Seinhorst, 1952 -- Tijdschrift over Plantenziekten 58: 103-108.

A new method for testing the susceptibility of rye plants to attack by the stem eelworm, Diylenchus dipsaci (Kuehn) Filipjev,  involves the growing of rye seed on filter paper. Infected rye, oats and mangolds served as the source of supply of the stem nematodes.

 

Shepherd, 1958 -- Nematologica 3: 127-135.

Three methods of producing an infestation of the beet eelworm, Heterodera schachtii on beet plants were investigated. The first was the addition of 2,000 hatched juveniles per pot of potting compost (20 nematodes /gm of soil). The second was the addition of 50 hand-picked cysts per pot of compost. The third was the dilution of infested field soil with compost to approximately the same infestation level.

 

RESPIRATION -- RES

 

Atkinson & Smith, 1973 -- Journal of Experimental Biology 59: 247-253.

A constant-temperature microrespirometer, based on a Clark-type oxygen electrode,

is described and illustrated. The apparatus originally was devised for use with small, individual nematodes weighing 50-380 ug body weight.  The experimental procedure for accurate and reliable measurements of oxygen consumption of the order of 0.02-0.2 cubic mm O2/h are given.

 

Bryant, Nicholas & Jantunen, 1967 -- Nematologica 13: 197-209.

In a study dealing with aspects of the respiratory system of Caenorhabditis briggsae,

a method is described for the production of large numbers of the nematode free from culture medium. Studies were conducted on the effects of oxygen, cyanide, carbon monoxide, and sonic disruption on the worms

 

        ----- S -----

 

SAMPLING -- SAM  

                                                                                     

Bor & Kuiper, 1966 -- Mededelingen Rijksfaculteit Landbouwwettenschappen Gent 31: 609-616.

In working with the genera Trichodorus and Longidorus, the authors suggest obtaining wider, rather that narrower, soil cores. They further suggest that samples shold be handled gently because rough handling decreases recovery of nematodes.                                                                                                                      

 

Cobb, 1918 -- Estimating the nema population of the soil, Agricultural Technology

Circular, Bureau Plant Industry, United States Departmentof Agriculture, pp 1-48.

1. A sampling tube for soil-census work is an open cylinder of thin metal with the rim at one end reinforced and the other end sharpened. The area of the internal cross section is one-millionth of an acre. A half-sized illustration is presented.

2. For purposes of mixing and subdividing the desired soil sample, a funnel with a jointed spout held up by a metal stand is depicted. A small sieve with an amount of soil is shaken over the funnel. The soil collects in conical fashion within a metal ring below the tip of the funnel. The collected conical amount of soil is subdivided by thin metal vertical blades passed through the apex of the pile.

3. An air-blast nozzle for roiling a mixture of soil and water contained within a cylinder is illustrated. At the end of a 4-5 foot pipe there is a spiral tube with numerous perforations

through which air coming in through the pipe escapes thus completely mixing the soil and water mixture.

 

Cooke, McKinney & Thomason, 1979 -- Journal of Nematology 11: 202-203.

A rapid method for sampling surface soil from many points situated on a relocatable pattern is reported. The soil sampling unit is mounted on the rear of a Honda All-terrain cycle. A photograph and labeled drawing of the unit are presented.

 

Esser, MacGowan & van Pelt, 1965 -- Plant Disease Reporter 49:  265-267.

Two new nematode sub-sampling tools are described and illustrated. One is named "The D.P.I. Field Subsampling Tool", and the other, "The D.P.I. Nursery Subsampling Tool "

A listing of 4 advantages of the named tools terminates the article.

 

Jutras & Tarjan, 1964 -- Soil & Crop Science Society of Florida Proceedings 24: 154-158.

A novel hydraulic soil auger-shaker unit was constructed for the collection of root samples from trees suspected of being parasitized by the burrowing nematode, Radopholus similis. The unit developed was designed for the collection of citrus roots, in a minimum of time, using a two-man crew. The auger assembly is lowered into the soil to collect the citrus root sample then again raised after which it shakes off the soil and root sample on to a mechanized shaker screen. The vibrating screen shakes off soil from the roots after which the feeder roots can be collected, stored and later analyzed.

 

Maas & Brinkman, 1980 -- Meded. Facult. Landbouww. Rijksuniv. Gent 45: 769-773.

In the  quest for a suitable detection method for the Longidoridae, the soil extraction technique was modified by using a wide-caliber auger and penetrating deeper than into just the topsoil.

 

McSorley & Parrado, 1983 -- Plant Disease 67: 182-184.

A bioassay sampling plan for Meloidogne incognita involved obtaining 200 okra samples from two large fields so as to simulate predetermined sampling plans which consisted of composited samples from each of two large fields. It was determined that instead of composite samples, use of 20 samples, each from a different location, resulted in a standard error to mean ratio of 49.6%.

 

Menon & Singh, 1963 -- Nematologica 9: 160-161.

A modified core-type soil sampler was designed which consists of a thin-walled steel tube

2.5cm in diameter and about 45cm long , split into two halves along its length. Construction details, drawings and photos accompany the article.

 

Peters, 1952 -- Annals of Applied Biology 39: 447-456.

In discussing the pilot test and methods under the broader heading of "Pot tests of nematocides against potato-root eelworm",  it is proposed that spacial variability in sampling is often necessary in research and may be attained by using very large pots.

 

Tarjan, 1950 -- Phytopathology 40: 1111-1124.

So as to obtain a reasonable estimate of infection of  Buxus sempervirens (boxwod) by meadow nematodes, Pratylenchus pratensis , a special sampling pattern was devised around the trunk of each plant. Two opposing root samples were dug from each of five concentric rings spaced 1/2 foot apart.

 

SCANNING  MICROSCOPY -- SEM

 

Baujard, 1978 -- Revue de Nematologie 1: 266-267.

A modified technique to prepare nematodes for Scanning Electreon Microscope observation is described. Details are presented on preparation of the specimens. Illustrations are presented showing the results of the technique.   The article is in French.

 

Clark & Stone, 1975 -- Nematologica 21: 256-266.

A simple method of preparing nematodes for scanning electron microscopy using Spurr's low- viscosity epoxy resin is reported. A complete description of the steps envolved is accompanied by 6 photos showing the results.

 

deGrisse, 1973 -- Mededelingen Rijksfaculteit Landbouwetenschappen 38: 1685-1695.

A method for preparing soft tissues for the Electron Scanning Microscope is described. The method is based on penetration of resin into the tissues, followed by rinsing the excess resin from the surface, after which polymerisation is achieved.

 

deGrisse, 1979 -- Journal of Nematology 11: 196-199.

A modification of the mini-sieve  and prepolymerized plate techniques for use in electron microscopy is described and amply illustrated. The article is concise, devoid of superfluidity, and without the usual introductory comparison of the proposed technique with similar techniques.used in the past.

 

Eisenback, 1986 -- Journal of Nematology 18: 479-487.

Using second-stage juveniles of Meloidogyne incognita prepared by several methods for scanning electron microscopy, it was found that sequential fixation at cold temperatures was superior to rapid fixation at room temperature. Also that glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone.  Critical point drying with carbon dioxide or Freon gave only slightly better results than air drying with Freon.

 

Ferris & Siegel, 1957 -- Nematologica 2: 16-18.

Conducting electron microscopy of the golden nematode cyst wall, the authors reported that an exo- and endocuticle were visible in electromicrographs of ultrathin sections of the cyst wall. More detail was observed in the endocuticle than previously reported from light microscope studies.

 

Green, 1967 -- Nematologica 13: 279-282.

The stereoscopic electron microscope at Cambridge, England facilitates detailed examination of the nematode cuticle. Those specimens least distorted were those impregnated with anhydrous glycerol or polyetylene glycol (400), the excess of which was drained from the surface before they were mounted and plated with gold/palladium in a vacuum chamber.

 

Green, Stone, Turner and Clark, 1975 -- Journal of Microscopy 103:89-99.

Tylenchid and Rhaditid nematodes were fixed and processed in several different ways for examination with the scanning electron microscope. The different processes evaluated were

drying from acetone, critical point drying, infiltration with glycerol and a conducting agent, and 

infiltration with an epoxy resin.  The last of these processes was the most promising.

 

Hiroyuki, Chen, & Maramarosch, 1967 -- Phytopathology 57: 646.

Dealing with electron microscopy of  Pratylenchus penetrans, the authors excised 2/10ths  of the total length of the cephalic region.  This was fixed in 3% glutaraldehyde in a 0.1m sodium cacodylate buffered solution, then postfixed in 0.1% osmium tetroxide in a 0.14M Veronalacetate buffered solution. After rinsing with 50% ethanol, the material was embedded in 3% agar and trimed to fit within a gelatin capsule.

 

McClure & Stowell, 1978 -- Journal of Nematology 10: 376-377.

A simple method of processing nematodes for electron microscopy is proposed. The small chamber suggested is figured,  while a photograph of the chamber with syringe attached is presented. An illustration of a nematode processed in the chamber is also included.

 

Sayre & Wergin, 1979 -- Scanning Electron Microscopy 3: 89-96.

The use of SEM to classify and evaluate the parasites and predators of pest nematodes is discussed.  If the activity of parasites and predators could be assessed, it may indicate their use as a form of biological control instead of chemicals.  The study used the scanning electrol microscope to evaluate that possibility.

 

Spurr, 1969 -- Journal of  Ultrastructure Research 26: 31-43.

A low-viscosity  epoxy embedding medium based on ERL-4206 is recommended for use in electron microscopy. The medium is easily and rapidly prepared and has been successfully used with a wide range of specimens, including endosperms with a high lipid content and tissues with hard lignified cell walls.

 

Stone & Green, 1971 -- Nematologica 17: 490-491.

A simple method of preparing nematodes for scanning electron microscopy involved transferring killed and fixed nematodes to 3 cm diam. glass cavity blocks each with approx. 0.2ml of distilled water. The blocks were placed in a dessicator with dry acetone and left for 24 hrs. Water in the blocks was slowly replaced  with acetone by vapor exchange.  Nematodes were placed on an adhesive stub surface, the acetone evaporates and the stubs then coated with gold, 200 angstrom units thick on a rotating turntable.

 

SECTIONING METHODS -- SMI                                                                  

 

Cairns, 1960 --  in Cairns Sasser & Jenkins, Chapt. 5, pp.33-84, N. Carolina Press.

The author refers to a handbook (anon., 1959) that can be purchased from the Sorvall Co. and which contains descriptions of preparation techniques and ultra-microtomy. A reference also is presented to an 84-page work by Richard, 1949 (American Optical Co) effective use and care of the microtome.

 

 Minderman, 1956 -- Plant and Soil 8: 42-48.

A technique for the study of organisms in situ in unaltered soil is described. Samples are cooled to -10C. A portion of the frozen sample is slowly immersed in  5%  glycerine solution. After infiltration and cooling, it is fixed in formalin and then treated with hydrofluoric acid  for the removal of sand grains. The sample is then immersed in gelatin solution a second time, and then affixed to a wooden block. It is then hardened in methylalcohol, clamped to a microtome,  and cut in sections 7.5-10 microns thick.

 

Newman, Borysko & Swerdlow, 1949 -- Science 110: 66-68.

Some new sectioning techniques are presented for  use with light and  electron microscopes.

 

Overgaard Nielson, 1948 --  Quarterly Journal of Microscopical Science 89: 437-438.

This Danish nematologist/zoologist presented a simplified method of orienting small nematodes prior to sectioning.

 

SECTIONING, NEMATODES -- SNE

 

Cole & Sykes, 1974 -- Stain Technology 49: 387-400.

Glycol methacrylate (GMA) in light microscopy presents a routine method for embedding and sectioning animal tissues. A procedure for the infiltration, polymerization and sectionng of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques

 

Deubert & Zuckerman, 1967 -- Nematologica 13: 343-346.

A technique  is described for the preparation of fresh frozen sections of free-living and plant parasitic nematodes with a cold microtome.  The described technique fulfills most requirements for histochemical work. The quality of the sections obtained is satisfactory.

                                                             

Tromba & Douvres, 1953 --  Proc. Helminthological Soc. Washington 20: 59.

Towards the goal of improving the method of sectioning nematodes for microscopic examination, the authors propose using a plastic slide containing grooves to aid in orientation of the worms.

                                                                         

 SECTIONING, SOIL --SSO                                                                          

 

Alexander & Jackson, 1955 -- in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pgs. 433-441.

A new method for studing organisms in soil was developed from standard geological methods and may be applicable to the study of nematodes.  It involves sampling the soil, pretreatments which may include fixing and staining, impregnation with a synthetic resin, and final preparation of sections by cutting, grinding and polishing.

 

Haarlov & Weis-Fogh, 1953 --  in Soil Zoology, ed D.K.M. Kevan, London, Butterworth,  pgs. 428-432.

A method is described in which agar is used as the embedding medium permitting serial sections to be made of  undesturbed wet soil.

 

Minderman, 1956 --  Nematologica 1: 216-226.

It was possible to cut sections of soil  to 10 microns in thickness after using gelatin as the

embedding substance and also dissolving the hard soil grains with fluoric acid.

 

Townshend, 1971 - Canadian Journal of Soil Science 51: 310-311.

A rotary spindle for the preparation of small uniform soil columns is illustrated for studying the behavior of plant nematodes. An inexpensive rotary spindle, a 1/6 h.p. electric motor, two pulleys, a rubber band, 18-gauge aluminum sheetin and 1.9cm plywood were all of the materials used.

 

 

SPECIAL TECHNIQUES -- SPT

 

Atkinson & Smith, 1973 -- Journal of Experimental Biology 59: 247-253.

A constant-temperature oxygen electrode microrespirometer based on a Clark-type oxygen electrode is described. The apparatus was designed for use with small individual nematodes weighing 60-380 micrograms body weight. An experimental procedure for accurate and reliable measurements of oxygen consumption is given.

 

Bedding, 1967 -- Nematologica 13: 643 - 644.

A simple technique for displaying the surface structure of nematodes deposits metallic silver in the striae and lateral fields of nematodes so that these structures can be examined in detail by using trasmitted light.

 

Bergeson, 1968 -- Phtopathology 58: 49-53.

Evaluation of factors contributing to the pathogenicity of Meloidogyne incognita was given       as well as  injury to tomato seedlings of different ages by the nematode. In addition, a split-root technique was used to isolate nutritional effects from toxin or auxin effects of infection.

 

Bijloo, 1966 -- Nematologica 11: 643- 644.

The "Pisum Test" is suggested as a new simple method for the screening of substances on their therapeutic nematocidal activity.. A suspension of Ditylenchus dipsaci in 1% "C.M.C".

in water containing about 2500 nematodes per ml is prepared. Using an Agla-micrometer syringe, 0.02 of the suspension, containg about 50 nematodes, is placed between the cotyledons on the plumule of the pea plants.  The plants are inspected two weeks later and degrees of infestation estimated.

 

Bollinger & Willett, 1978 -- Nematologica 24: 398-403.

A method for age synchrony of  Panagrellus redivivus in xenic culture is described. Synchronous cultures  are begun with 1-2 day old larvae and resynchronized at 7 days of age and every 3 days thereafter. Synchrony is obtained by separating parents from their progeny, and females from males, on sucrose gradients.

 

Bollinger & Willett, 1980 -- Nematologica 26: 491-493.

This publication describes a method that uses settling and filtration to obtain synchronized populations of adults of Caenorhaditis elegans var. Bristol in sufficient quantity for life history studies.

 

Cobb, 1914 -- U.S. Journal of Agricultural Research 11: 217-230.

For incubation of eggs, it was thought advisable to create favorable conditions for the eggs

and developing larvae. Material was placed in small glass brood samples and pasteurized at a temperature of 130-140F..The procedure was successful. A description and drawing of the brood capsule previously mentioned is given.

 

Cobb, 1920 -- Transactions American Microscopical Society 39: 231-242.

A method is proposed for systematically examinig and recording a large series of microscopical objects. The method consists in making a low magnification camera lucida drawing, or diagrammatic chart, of all the objects to be recorded. An example is presented of such a chart showing the exact position of nematodes, and other objects using a numbering system identifying the subjects to the side of the chart showing positions

 

Croll, 1967 -- Nematologica 13: 472.

A "Y" shaped trough is proposed for use in nematode behavioral studies. The trough is ground from a perspex block and has a "Y" channel with a flat bottom and sloping sides.

The trough is used to investigate the galvanotactic response of Panagrellus redivivus.

 

Crosse & Pitcher, 1952 (published 1953) -- Ann. Rpt. East Malling Res. Sta., pp 138-140.

A study was conducted of methods for freeing the nematode, Aphelenchoides ritzema-bosi,

from bacterial contamination using chemical sterilants.  Best results were obtained by immersion of the nematodes in low concentrations of various organic compunds for periods up to 90 hours.

 

Durie, 1959-- Journal of Helminthology 33: 189-196.

A new technique for the recovery of infective strongyle larvae from soil and pasture is described which is based on the difference in density of larvae and debris under the influence of an upward-flowing stream of water the flow rate of which is gradually decreased.. The results from trials gave an average recovery of 78% from soil and 74% from pasture.

 

Esser, 1974 -- Nematologica 19: 566-567.

The author proposes use of "ZUT" as a cover glass support for nematodes.This material replaces fingernail polish, which was advocated in an earlier publication He states that specimens are supported without body flattening.

 

Esser, 1986 -- Proceedings of the Helminthological Society of Washington 53: 254-256.

A water agar en face technique is described by which en face views of live or fixed nematodes are prepared using water agar. Such preparations are suitable for study, photography, or camera lucida drawings.

P

Ferris, Ferris & Callahan 1972 -- Tech. Rpt. 30, Purdue Univ. Water Resource Res. Cen.

Methods were developed by which nematode data on community structure from water research areas can be interpreted.for an increased understanding of ecological conditions.

Data for physio-chemical parameters also were collected. The study indicated that analysis of benthic nematode comunity structure can be a useful tool for evaluating disturbance to aquatic habitats.

 

Geraert, 1961 -- Nematologica 6: 258-259.

It is pointed out that nematodes may be flattened in prepared slides.  To decide whether a given nematode is flattened, the horizontal diameter is measured using a camera lucida, or with a caibrated micrometer eyepiece and compared with the vertical diameter, obtained by reading the calibrated micro-screw of the microscope.

 

Good, Sasser & Miller, 1963 -- Plant Disease Reporter 47: 159-163.

A suggested guide for reporting experiments on nematocidal chemicals is offered for providing meaningful comparisons and an understanding of the results obtained. The article  suggests  1) the information to be given for identification of chemicals, 2) methods of reporting dosage, 3) terminology for reporting methods of application, and 4) formulas for calculating chemical dosage.

 

Haight, Leushner, Noble & Pasternak, 1976 -- Nematologica 22:118-120.

The authors presented an account of sucrose gradient fractionation of Panagrellus silusiae into monotypic groups. They developed a method where large homogenous populations of worms at various stages of postembrionic development could be isolated sequentially and efficiently within a relatively short period of time. In summation, the procedure enables large mixed populations of the free-living nematode Panagrellus silusiae to be fractionated into homogenous groups that are based on length with a high overall recovery of the imput sample.

 

Hawn, 1969 -- Journal of Nematology 1: 290

A new technique was developed using twin-crowned alfalfa plants to deternine if

Ditylenchus dipsaci predisposes wilt-resistant alfalfa to infection by

Corynebacterium insidiosum.  The technique was designed to demonstrate the effect of different periods of nematode infection on the subsequent reaction  of wilt-resistant alfalfa to bacterial wilt.

 

Heip, 1974 -- Nematologica 20: 266-267.

A rapid method to evaluate nematode density is a modification of an earlier method reported by Jenkins, 1964.  To test the method, the number of nematodes appearing in the sample after each centrifugation of a given sample was counted. The conclusion reached presented the possibility that the first supernatant is a representative sample of the whole population

and would allow estimates of fauna composition and biomass as well as estimates of density.

 

Hirschmann, 1959 --  Proceedings  Helminthological. Society of. Washington 26: 73-90.

Mulvey's 1955 method for histological studies of nematodes was used except that the anterior end of the nematodes was severed instead of  the nematodes being squashed.

Nematodes were treated with 5% sodium hypochlorite to aid in a propsed study of cuticular layering. The specimens were immersed in the solution either alive or after being subjected to 65C for 10 min.

 

McClure & Robertson, 1974 -- Nematologica 19: 428-434.

A method of producing uniformly infected root segments is described. Previously washed , oven-dried vermiculite is moistened with distilled water and then packed into boxes lined with plastic wrap. Seedings are placed in the boxes with their root tips extending 3-4mm over the upper edge of a 2-cm wide strip of Miracloth  A second strip of the cloth is placed over the first covering the root tips. A suspension of 2nd-stage larvae is pippetted directly on to the upper strip. 25% of the larvae penetrate

the seedlings.

 

Mulvey, 1955 -- Canadian Journal of Zoology 33; 295-310.

A new technique for the handling of small nematodes was proposed.  A 2% agar solution is prepared and a small watch glass is partially filled with this solution. The living nematode is placed in the center of the warm agar and oriented as desired. After s  olidification, the agar is trimmed into a small rectangular block with the nematode in the center.

 

Myers, Chen & Balasubramian, 1971 --Journal of Nematology 3: 85.

Two methods for separating larvae of uniform length from mixed adults and larvae of  Pangrellus redivivus were proposed.  The first used nylon monofilament bolting cloth while the second used glass microbeads.

 

Peacock, 1959 -- Nematologica 4: 43-55.

In working with Meloidogyne incognita infecting Lycopersicon esculentum, the author noted that attraction of the juveniles to the plant was to a very closely defined area immediately behind the apical meristem.  To demonstrate the chemical nature of the attraction to the juveniles,  a technique prevously used to demonstrate toxic secretions of fungal hyphae in the soil was used.

 

Perry, 1976 -- Nematologica 22: 446-450.

Separation of stages of  Ditylenchus dipsaci and D. myceliophagus by body lengths was accomplished using the development of the genital primordia as the initial guide for identification.  This was done using the relationship between stage and body length.

 

Reid, 1952 --Journal of  Helminthology 26: 67-68 (appendix to Fenwick paper).

An improved method of cyst dissection suggests the use of one needle with cutting edge, instead of two needles. The cysts are spread along a line on a damp slide and each cyst can be bisected with a single cutting motion. Perplex slides are recommended for use with the needle

 

Rodriguez-Kabana, Hoveland & Haaland, 1977 -- Journal of Nematology 9: 323-326.

An evaluation was conducted of a seed-treatment method with acetone for delivering systemic nematocides with wheat and rye. The paper reports on the efficacy of oxamyl, carbofuran and phenamiphos in acetone solutions as seed treatment for controlling certain plant parasitic nematodes.

 

Roggen & Asselberg, 1971 -- Nematologica 17: 187-189.

The use of ratios in nematology is proposed for their use in the description of nematodes. In this highly technical article, an easy procedure is proposed which makes it possible to decidewhether a ratio is statistically acceptable or not.

 

Rubinstein & Owens, 1964 -- Contributions ofh Thompson Institute 22: 491-502.

The incorporaton of tritium-labeled thymidine and uridine into root galls of Meloidogyne incognita acrita on tomato, Lycopersicon esculentum, was studied using microautoradiographic techniques. It was shown that the developing syncytium is a region of intense ribo- and desoxyribonucleic acid biosynthesis.

 

Schaad & Walker, 1975 -- Journal of Nematology 7: 203-204.

The use of density-gradient centrifugation for the purificatiom of eggs of  Meloidigyne spp is reported.  Its use, in combination with filtration, might be a useful technique for getting

"clean"  eggs for biochemical analysis. The described method should be of value in determining the macromolecular composition of nematodes.

 

Taylor, Ferris & Ferris, 1973 -- Journal of Nematology 5: 68-69.

A technique for quantifying injury to seedling soybeans by Pratylenchus penetrans without sacrificing the plant is presented. Significant reductions in growth were apparent using the  parameters: of root weight,  tap root weight, weight of longest lateral root and total number of lateral roots. Since significant reductions in growth were apparent using all four parameters, sacrifice of the plant to obtain root weight data was not necessary.

 

 Townshend, 1971 -- Canadian Journal of Soil Science 51: 310-311.

A rotary spindle for the preparation of small uniform soil columns is illustrated for studying the behavior of plant nematodes. An inexpensive rotary spindle, a 1/6 h.p. electric motor, two pulleys, a rubber band, 18-gauge aluminum sheetin and 1.9cm plywood were all of the materials used. The article contains two illustrations of the proposed method.

 

van Cleave & Ross, 1947 -- Science 105: 318.

A method was successfully used which allowed the authors to reclaim large nematodes which had dried. Specimens were placed in 0.25 to 0.5% trisodium phosphate in distilled water.  The hydrated forms could be stained and remounted.

 

Viglierchio & Maggenti, 1965 -- Trans. American Microscopical Soc. 84:  284-293.

A programmable solvent delivery and removal system was designed for solvent exchange in specimens and tissues at ambient and above ambient temperatures.  The unit allows for the exchange of any series of solvents mutually soluble in sequence.

 

Warner & Hague, 1960 -- Nematologica 5: 71-72.

Another method for the construction of perspex cavity slides is presented. A piece of 1/2 inch perplex is placed in a mould in the oven, heated at 118C for 15 minutes, then tightened in a vice until the projecting perspex surface is fluch with the mould and left there until cold. Extensive drawings provide construction details.

 

Wong & Mai, 1972 -- Journal of Nematology 4: 237.

A technique for studying the influence of oxygen on the invasion of  Meloidogyne hapla in lettuce growing in soil was propose and fully described. The results demonstrated that invasion is adversely affected by oxygen levels below and above 21%.

 

Zullini, 1976 -- Nematologica mediterranea 4: 13-22.

Nematodes as indicators of river pollution are proposed as a biological pollution parameter.

In the analysis of fresh-water sediments, it was found that members of the Diplogasteroidea are more numeros in water with medium to high pollution.  In highly polluted waters, members of the Rhabditoidea are more numerous.  

 

STAINING,  ACID FUCHSIN -- SAF

 

Franklin, 1940 -- Journal of Helminthology 18: 63-84.

Nematode-infected roots are stained and cleared by boiling for a length of 2-3 minutes in lactophenol solution which contains acid  fuchsin dye.

 

Minderman, 1956 -- Nematolgica 1: 216-226.

Nematodes present in a sample of forest liter immersed in water .were shaken for 24 hours, bleached  with 6% hydrogen peroxide until the (leaves) were "yellowish-white),

and then stained with a hot solution  (60-70C) of acid fuchsin or cotton blue dye.

 

STAINING,  DEATH DETERMINATION  -- SDD

 

Boyd, 1941 -- Nature 148: 782.

The determination of death in the larvae of the potato root eelworm is covered. The author proposes a test where dead larvae can  easily be differentiated from the living by staining with a solution of 0.025g iodine in 100cc of 1% potassium iodide solution.

Use 5 drops of the solution in 2cc of a larval suspension.

 

Chaudhuri, Dick, Engelbrecht & Austin, 1966 -- Nematologica 12: 337- 342.

A technique is given for distinguishing live from dead free-living nematodes using the stain eosin-Y.The two test organisms used were Diplogasteroides sp. and Diplogasteritus nudicapitatus.Various methods for killing the nematodes using different approaches are given

 

Ogiga & Estey, 1975 -- Nematologica (1974) 20: 271-276.

So as to accurately determine nematode survival after use of nematocides or hot-water treatments, it is necessary to determine living from dead nematodes. . Meldola B and Nile Blue A generally were superior and more dependable than Phloxine B, New Blue R and somewhat better than Chrysoidin. The stains were effective on a given list of  eight nematode genera.

 

STAINING,  NEMATODES -- STM    

   

Alves & Bergeson, 1967 -- Plant Disease Reporter 51: 511.

It was found that placing stained roots in clear lactophenol and autoclaving for 10 miutes at 250F and 15 pounds pressure accomplished destaining that is as good as or even better than a 1-2 day period at room temperature. This allows the worker to stain, observe and count nematodes in roots within a 1/2 hour period.

 

Blair, Perry, Oparka, and Jones, 1999 -- Nematology 1: 103-111.

Live nematodes were treated with dimethyl sulfoxide for 10 min. @ 37C to assisit permeabilisation of the cuticle. They were then transferred to a medium containing 50 uM acridine orange and incubated for 2 hrs. at ca 20C. The nematodes were viewed with an MRC 1000 confocal laser scanning microscope to image  the fluoyrescent dye.

 

Chaudhuri, Dick, Engelbrecht & Austin, 1966 -- Nematologica 12: 337-342.

A staining technique was developed which employs the dye eosin-Y that stains dead nematodes while live nematodes remain unstained. The authors point out that the study of samples containing nematodes killed by less severe methods than they employed  may result in a systematic error greater than 3 %. Therefore the method proposed gives a conservative indication of the number of dead organisms present.

 

Ciorida & Johnson, 1950 -- Stain Technology 25: 114.

A rapid staining method using dye from Eye-Ease Hammerhill Filter paper during the fixation stage was proposed. The treated nematodes were gradually dehydrated and mounted in Yetwin's medium.

 

Cobb, 1917 .. Notes on Nemas in Contrib. to a Science of Nematology V, pp 117-128.

In an article on "Intra vitam color reactions in nemas", the author states that a great variety of coal-tar compounds, and other color compounds, can be fed to nematodes apparently without interfering materially with their normal metabolism. Small quantities of the dye in the medium containing the nematodes allowed the dye to act for days or weeks

 

Doetschman, 1944 -- Transactions of the American Microscopical Society 63: 175-178.

A simple method based on the Berlese technique was proposed for mounting, clearing and staining in acid fuchsin small insects to detect the nematodes.

 

Doliwa, 1956 -- Wissenschaftlichen Zeitschrift der Universitaet Rostock 5: 133-149.

The author used the stain 'chrysoidin' to distinguish between living and dead nematodes.

 

Doncaster & Clark, 1964 -- Nematologica 10: 136-140.

Observations were made on gut pH and absorption of methyl red and neutral red dyes in the intestinal walls of  Pelodera and Mesodiplogaster. Except for Ascaris lumbricoides and Strongylus edentatus, The nematodes tested showed acid gut reactons.  Methyl red and neutral red pH indicator dyes were absorbed selectively by different regions of the intestinal wall and by the two species.

 

Elsea, 1951 -- Proceedings of the Heminthological Society of Washington 18: 53-63.

While working on sectioned materials,  Heidenhains hematoxylin was used with iron alum mordant and fast green counterstain, iron alum-hematoxylin alone and Harris' hematoxylin with eosin counterstain.

 

Feldmesser & Taylor, 1953 -- Journal of Parasitology 39: 32.

A weak solution of aniline blue WS was a very useful stain for nematodes in water or for fixed specimens. It aided detection of amphid and phasmid openings, secretions from these openings, and it gradually penetrated the body openings. Keeping the specimens overnight in the dilute stain mixture had a beneficial accumulative effect in revealing fine surface structural details.

 

Fenner, 1962 --  Plant Disease Reporter  46: 383.

The author used the stain 'phloxine B' to distinguish between living and dead nematodes.

 

Franklin & Goodey, 1949 -- Journal of Helminthology 23: 175-178.

A cotton-blue lactophenol technique for mounting plant-parasitic nematodes is described.

 

Gemmell, 1939 -- Phytopatholgy 29: 287-288.

A staining procedure for demonstrating nematodes in plant tissue was proposed. The procedure uses a 2% solution of iodine in alcohol, decolorizes with alcohol, and finally clearing and mounting in clove oil.

 

Hasbrouck, 1959 -- Phytopathology 49; 523-524.

The author suggests using  gold chloride and picric acid iodine for in toto permanent slide mounts,  Nerve rings and associated cells and the gonads are differentially stained a bright red.  A fine black precipitate deposited on the cuticle aids in definition of cuticular details.

 

Haverkate, 1972 --  Nematologica 18: 414-418.

Specific staining of living plant nematodes with 4-dimethylaminobenzenediazocyanide, a neutral azo compound, specifically stained internal structures of living plant nematodes.

Various species of plant-parasitic nematodes reacted favorably the the staining technique,

but not all species used reacted favorably.

 

Karanastasi, Vellios, Roberts,MacFarlane & Brown, 2000 -- Nematology 2: 237-245.

Safranin-O stain was used to distinguish nematode specimens in resin blocks.Different parts of the nematode body were apparent and nematode ultrastructure was not affected.

 

Kirkpatrick & Mai, 1957 -- Phytopathology 47: 526.

A new staining technique for in situ observation of Pratylenchus penetrans and other parasitic nematodes is described. The abstract presented gives a complete description of the series of procedures to be followed.

 

McBeth, Taylor & Smith, 1941 -- Proc. Helminthological Society of Washington 8:26.                                                                                                                                                                A simplified method of staining nematodes in root tissue involves the use of Goodey's formula (Jour. Helminth. 15(3):137-144, 1937).  Roots are boiled in the specified stain for 1 min., washed in tap water, and then placed in clear lactophenol until clear from one to several days. Desired cleared root sections containing stained nematodes can be mounted directly in glycerine for the purpose of making permanent mounts. 

 

O'Brien, Gemmell, Prentice & Wylie, 1939 -- Journal of Helminthology 17: 41-50.

An in situ staining procedure was described using a 2% solution of iodine in alcohol, then decolorizing in alcohol, clearing and then mounting in clove oil.

 

Rodriguez-Kabana & King, 1976 -- Nematropica 6: 34-40.

Halogen-silver staining techniques for clarifying cuticular details in nematodes for light microscopy are presented. Nematodes are killed and fixed in formaldehyde solutions, rinsed in water, and immersed in 0.01N iodine solution. The specimens were then rinsed in water and then immersed in 0.2N silver nitrate. A bromination method was found superior to the iodine method for Hoplolaimus galeatus.  This consisted of immersion of the nematodes in potassium bromide solution, rinsing in water, then immersion  in silver nitrate for 3-4 min.

 

Shepherd, 1962 -- Nematologica 8: 201-208.

The stain "New Blue R" was found to color the body contents of members of the order Tylenchida from a pale 'mauve' to a deep purple. Living nematodes, however, resisted the treatment and remained unstained.

 

Stubbs & Live, 1936 --  Veterinary Ext. Quarterly, Univ. Pennsylvania 36: 11-15.

It was discovered that by allowing blood to clot, microfilariae passed into the blood serum in which they could be kept alive for several days and then stained as desired.

 

Taylor, Feldmesser & Fassuliotis, 1952 -- Plant Disease Reporter 36: 269.

An improvement in the method of searching for Heterodera cysts involved adding stains to the residue caught on sieves. Whereas not all stains will color the cysts, janus green, brilliant green, malachite green and gentian violet stains were found suitable

 

Whitlock, 1940 -- Journal of Parasitology 26: 45-47.

It was discovered that a few drops of concentrated iodine-potassium iodide solution added to washed worms in physiological solution stained the worms, debris from the gut, and the supernatant liquid a dark brown color. A solution of concentrrated sodium thiosulfate was then stirred in until the debris and fluid were decolorized. The nematodes then showed up prominently to aid in counting.

 

 

STAINING, OSMIUM TETROXIDE -- SOT

 

Bird, 1959 -- Nematologica 4: 31-42.

Buffered osmium tetroxide at 1% was found to be more suitable than the cotton blue/lactophenol technique for differential staining of nematodes in plant tissue. There was advantage to combining fixing and staining into one operation with the results allowing for electron or light microscope study.

 

Cole & Howard, 1958 -- Journal of Helminthology 32: 135-144.

Roots initally were fixed and then stained in Flemming's Solution in order to insure that the material contained nematodes.  Selected root pieces were embedded in wax, sectioned and then stained with iodine-gentian violet.

 

Godfrey, 1935 -- Phytopthology 25: 1026-1030.

Infected plant parts were first treated with hot 80% alcohol which was beneficial for subsequent clearing of tissues, then stained in hot Flemming's Solution. The roots were t washed, dehydrated with alcohol, cleared with clove oil and then mounted for examination

 

Pfaeltzer, 1935 -- Archief voor Koffiecultuur in Indonesie 9: 29-38.

Infected root pieces are immersed in a strong Flemming's solution and then in chromacetic acid for 24 hours, followed by a final treatment for two hours in aqua regia which prevents subsequent blackening of the roots.

 

Steiner, 1927 -- Journal of Parasitology 14: 71-72.

It was shown that osmic acid, a principal ingredient of Flemming's Solution, blackens the fat

molecules within nematodes. This enables the worms to be more readily detected against the unstained plant tissues.

 

Tarjan & Ford, 1957 -- Stain Technology  32: 171-174.

For purposes of clearly demonstrating nematodes in infected citrus roots, roots are immersed in a covered jar for 2 hr.at 52C in a solution composed of 16 parts of  distilled water, 10 parts of 10% acetic acid,  and 2 parts of  aqueous 2% osmium tetroxide. The stained roots are then washed in running water for an hour and then bleached in 10-30% hydrogen peroxide until the color of the roots lightens perceptibly.

 

 

STAINING , NEMATODE EGGS -- STS            

 

 Bittner, 1950 -- Jour. of Laboratory and Clinical Medicine 35: 121-122.

A technique was described making use of  aqueous solutions of fluorescein  which would render ova and other living organisms a bright fluorescent blue color.

 

Dickson & Struble, 1965 -- Phytopathology 55: 497

A sieving-staining technique for the extraction of egg masses of  Meloidogyne incognita

from soil is described whereby the soil is washed and the separated eggmasses collected.

These are then stained with a 2% Phloxine B for at least 5 minutes

 

Homeyer, 1953 -- Mitteil. Biologische Zentralanstalt, Berlin-Dahlen 75 : 232-235.

Use of acridine dyes and ultra-violet light were employed to produce fluorescent color differences between living and dead eggs and larvae.

 

Pusch, Senne & Beyer, 1950 -- Tieraerztliche Umschau 5: 54-55.

It was pointed out that detection of helminth eggs in fecal samples can be facilitated by staining floatation samples with yellow water-soluble eosin.  The eggs will show a green fluorescence which contrasts with the red-stained plant remains.

 

Rankl, 1947 -- Zentrallblatt Bakteriol., Parasitenk., Infectian. Hyg.152: 152-154.

The author discovered a fluorescence staining technique for easier detection of helminth eggs. An acid-alcohol pretreatment for eggs of  Trichurus and Ascaris after which berberine sulphate was used and the eggs could be detected by their secondary fluorescence was recommended

 

Saperno & Lawless, 1953 -- American Journal of Tropical Medicine 2: 613-619.

Tests of  MIF (merthiolate-iodine-formaldehyde) were reported as a wet fixative stain method for use in detecting protozoa and eggs in  fecal smears.

 

Southey,1986 -- Reference Book 402, Ministry of Agric., Fish., & Food, p 23.

To aid in counting nematode eggs, two drops of 0.35% acid fuchsin in 25% lactic acid is added to 25ml of a suspension of the eggs in water and boiled for one minute. The author suggests using a stereomicroscope with about 30X magnification to count the eggs.

 

 

STAINING,  NEMATODES IN PLANTS -- STP  

 

Byrd, Kirkpatrick & Barker, 1983 -- Journal of Nematology 15: 142-143.

An improved method for clearing and staining plant tissues for detection of nematodes is described. It uses a modified acid-fuchsin staining-destaining procedure using sodium hypochlorite (NaOCl) as a pre-staining treatment for nematode-infected plant tissues.

                                

Franklin, 1949 --- Journal of Helminthology 23: 91-93.

For the purpose of demonstrating nematodes contained in leaf tissue, a rapid acid fuchsin-lactophenol method was suggested. Following the staining, the material is dehydrated in iso-butyl alcohol and finsally mounted in Euparol.

 

Godfrey, 1929 -- Phytopathology 19: 611-629.

The technique presented essentially is a modified "Arzberger method".  Plant tissue containing nematodes is killed and kept in hot Flemming's solution for 30 minutes. It is then immersed in chrom acetic fixative for 12 to 24 hours followed by washing,dehydrating and then clearing in clove oil or something similar.

 

Godfrey, 1935 --  Phytopathology 25:  1026-1030.

An earlier technique (supra) was further modified so as to allow for the treatment of green plant parts as follows. 80% acetone in water is boiled and small pieces of nematode-containing plant parts are immersed and left in the cooling solution for 3-4 hrs.or until the chlorophyll is removed . The acetone is poured off,  the plant parts washed with water 3 or 4 times and then immersed in Flemmings Strong killing fluid. Once the nematodes are sufficiently colored, as observed under magnification, the parts are washed, dehydrated in in alcohol and cleared with clove oil.

.

Goodey, 1937 -- Journal of Helminthology 15: 137-144.

Lactophenol  with acid fuchsin or cottonblue stain is used to demonstrate the presence of stem nematodes in root tissue.  When plant material is thus treated in the hot staining solution, the contained nematodes stain a highly visable color while the surrounding plant tissue and soil particles remain unstained. For staining nematodes in situ in shoot structures, the stain "Scarlet R" was used.

 

Pfaeltzer, 1935 -- Archief voor Koffiecultuur in Indonesie 9: 29-38.

Infected roots were immersed in a strong Flemming's solution  and then treated for 24 hrs.in chromacetic acid with a final 2 hr. treatment in aqua regia to prevent subsequent blackening if the roots.

 

Tarjan & Ford, 1957 -- Stain Technology 52: 171-174.

Citrus roots infected with nematodes are immersed in a solution composed of distilled water, acetic acid and aqueous osmium tetroxide in a covered jar for two hours at 52C.

The stained roots are washed in running water for 1 hr and then bleached in 10 - 30% hydrogen peroxide at 32C for a few seconds until the color of the roots lightens perceptibly. The roots are then washed in water and dehydrated in 70, 95 and absolute ethanol held at 52C for 30 minutes at each concentration.

 

 STATISTICS -- STA

                                                                                     

Baeza-Aragon & Tarjan, 1983 -- Nematologica 28: 134-135.

Egg masses of  4 Meloidogyne species from Gardenia jasminoides cuttings were identified using Principal Components Analysis and Discriminant Analysis, both being multivariate statistical methods. When measurements selected by the  Principal Components analysis were combined to ratios highly significant in the "F" test, and analysed by Discriminant Analysis, separation of discrete species occurred.

 

Blackith & Blackith, 1976 -- Nematologica 22: 235-259.

In a multivariate study of the subfamilies Tylenchinae and Psilenchinae, 136 individual nematodes were assessed using 34 characters.  A principal coordinate analysis showed that the species formed a homogenous cluster and that in the genus Aglenchus  there was a tendency for the members of one genus to separate from those of other genera. There were some very high similarity coefficients both between species within a genus and between species from different genera.

 

Jones, 1955 -- Annals of Applied Biology 42: 372-381.

In an article entitled  "Quantitative methods in Nematology" the author feels that the subject is composed of 4 groups, namely: (1) Estimation of soil populations, (2) Assay techniques for the estimation of cyst nematodes, (3) Estimation of nematodes on or within plants, and

(4) Quantitative morphology. He feels that those entering this field of work will be greatly helped  by some knowledge of statistics and by mastery of the implications of the Poisson distribution, which plays more of a part in present nematological methods than in other

branches of biology. It is suggested to workers entering the field of nematological techniques to carefully study how time and effort could be saved without a loss in accuracy.

 

STERILIZATION -- STE       

 

Boswell, 1963 -- Phytopathology 53: 622.

A method for the separation and sterilization of  Pratylenchus brachyurus from mixed populations of nematodes is described. The nematode was immersed in and was tolerant to solutions of 0.25% "Clor-o-fen" for 45 min. or 0.5% for 20 minutes. These solutions effectively surface-sterilized  Pratylenchus brachyurus  but eliminated other free-living and parasitic nematodes.

 

Chantanao & Jensen, 1969 -- Journal of Nematology 1: 277-278.

Surface sterilization of  Pristionchus lheritieri was performed by immersion of the worms in different concentration of chlorine prepared from commercial sodium hypochlorite. It was found that all species of bacteria failed to survive 3 ppm of chlorine for 2 min, therfore a 20 ppm immersion for 20 min accomplished adequate surface sterilization.

 

Chen, 1964 -- Phytopathology 54:127.

A technique for the selection and disinfestation of individual nematodes first concentrates the nematodes by centrifugation. The suspension is poured into a petri dish and equal volumes of 2% agar and a disinfesting solution are mixed and added aseptically to the nematodes.

 

Fenwick, 1956 -- Nematologica 1: 331- 336.

Sterile, viable larvae of the potato root eelworm, Heterodera rostochiensis , were obtained by the sterilization of larvae, and of eggs. It was found that 8 hrs. of treatment with hydrogen peroxide  resulted in complete asepsis, with no loss of hatchability or invasive power.

 

Glaser & Stoll,1940 --Journal of Parasitology 26: 87-94.

A method is described which incorporates the use of Labarraque's or Milton solution for obtaining sterile, uninjured nematodes. This is used for the exsheathing or ecdysis of certain nematode larvae.

 

Goodman & Chen, 1967 -- Phytopathology 57: 1216-1220.

Two methods for the surface sterilization of  nematodes ( Trichodorus christiei ) are presented.  In one, the nematodes are suspended for 20 hrs. in 1-1/2% agar containing

100 ppm of Aretan (methoxyethyl mercury chloride).  In the second method the nematodes are allowed to migrate through absorbent cotton in a Melpar-Tiner storage trap filled with 100ppm of Aretan.

 

Green & Hornsey, 1983 -- Nematologica 29: 362-365.

Apparatus for collecting and surface-sterilizing nematodes and similar small aquatic organisms is described, and  illustrated.  The opening statement suggests that the equipment described can be bought or made with a drill and glass-working blow-lamp. Three drawings illustrate the necessary components. Several steriliation materials are listed of which the authors feel that 0.05% Aretan is preferred since residues are more easily eliminated and less phytotoxic.

 

Goodman & Chen, 1967 -- Phytopathology 57: 1216-1220.

Two methods for disinfestation of Trichodorus christei are proposed.  In one method, nematodes are suspended for 20 hrs. in 1.5% agar containg 100ppm of  methoxyethyl

mercury chloride (Aretan). In the second method, the nematodes migrate through absorbent cotton in a Melpar-Tiner storage trap filled with 100ppm Aretan.

;

Mueller, 1970 -- Nematologica 16: 154-155.

A method for the sterilization of  nematodes is presented and adequately illustrated in a short, concise article.  The description is written in German.

 

Peacock, 1959 -- Nematologica 4: 43-55.

Meloidogyne incognita egg masses were surface-sterilized by washing for 5 minutes in 0.1% Cetavlon (Cetyl trimethylammonium bromide); decanted and rinsed in distilled water.Then,

immersed in 0.5% Hibitane diacetate (Bis [chlorophenyldiguanido]-hexane-diacetate) for 15 minutes and then rinsed in sterile distilled water.

 

Roessner, 1971 -- Nematologica 17: 320-321.

A simple method for sterile culture of nematodes was proposed.  A glass cylinder of sufficient volume is filled with moist sandy soil which is pressed down and covered with a layer of light soil. The top of the cylinder is greased with silicon paste and a glass cover with a bore hole is affixed.

 

 STORAGE --STG

 

Bingefors & Bingefors, 1972 -- Journal pf the Swedish Seed Assn. 82: 320-329.

A "nematode bank" for production of inoculum of stem nematodes, Ditylenchus dipsaci was developed using available methods of rearing stem nematodes. The "nematode bank" is intended mainly to supply inoculum to institutes within Scandinavian countries, but nematodes for other purposes have also been sent to other countries, but on a smaller scale.

                                                                                      

Franklin, Clark and Course, 1971 -- Nematologica 17: 575-590.

It was found that soil samples, especially those obtained between October and April, will yield more juveniles if they are incubated for a week at 20C than if processed on the day the samples were obtained. Samples obtained during the  Autumn and kept at 0C for a week before incubation yield more larvae than incubated non-chilled samples.

 

Hwang, 1970 -- Nematologica 16: 305-308.

Free-living nematodes of the genera Aphelenchoides, Panagrellus, Turbatrix,and

Caenorhabditis were successfully frozen in heat-sealed glass ampoules using dimethyl sulfoxide as a protectant. After 6 months storage in the liquid nitrogen refrigerator (below160C) living nematodes were recovered after thawing.

 

MacGowan, 1986 -- Journal of Nematology 18: 419-420.

A new efficient technique for permanent nematode storage  places nematodes preserved in 2% formaldehyde into heat-sealed ampoules to eliminate problemsof slow evaporation or time-consuming dehydration for storage in glyceriine. Two figures accompany the article.

 

Whyte & Gowen, 1974 -- Nematropica 4: 27- 31.

The recovery of Radopholus similis from from roots incubated in polyethylene bags was greater from refrigerated samples (5-8C) than from samples stored at room temperature. Refrigeration had a depressing effect on recovery of  Helicotylenchus spp. from soil.

 

 

SURVIVAL -- SUR                                       

                                                 

Lees, 1951 -- Journal of Helminthology 25: 97-104.

Panagrellus redivivus was investigated as a possible facultative parasite in the mammalian body using the mouse as the host. It survived for one day in gastric juice from the mouse stomach but was quickly killed in pancreatic juice. The nematode could survive without oxygen for 24 hrs. When fed to mice it remained alive a short time in the stomach but was killed in the intestine. It invaded the vagina of the mouse remaining alive for 3 days during which time it caused some irritation to the mouse.

 

Simons, 1973 -- Meded.  Landbouwhogeschool Wageningen 73: 85pp.

In water-saturated soils, nematode populations were reduced to about 20% after 20 weeks. Results of experiments in unsaturated soil revealed that survival of ectoparasitic nematodes  in fallow soil at pF values between 0.5 and 4 is not directly affected by soil moisture quantity.

 

Slack, Riggs & Hamblen, 1972 -- Journal of Nematology 4: 263-276.

Soy bean-cyst nematode larvae, Heterodera glycines,  survived in water up to 630 days depending on incubation temperature. From 16 to 36C, survival was inversely correlated with temperature. Juveniles survived 7-19 months in flooded soil, 29-38 months in dry soil, and for 90 months in soil maintained at near its field capacity.

 

Solomon, Paperna & Glazer, 1999 -- Nematology 1:  61-64.

 The dessication tolerance traits of three strains of Steinernema feltiae were modified. A slow dehydration regime of pre-conditioning  at 97% relative humidity [RH]for 3 days at 23 degrees Centigrade induced a quiescent anhydrobiosis state in all strains, which enabled them to survive at lower humidities of 75% and 85% RH.

 

Specht & Walker, 1969 -- Journal of Nematology 1: 27-28.

Survival of  Pratylenchus penetrans under increased carbon dioxide was investigated in a chamber where nematodes were exposed to gaseous atmospheres for extended time. The results indicated that the nematode is capable of survivng extremely high concentrations of carbon dioxide for at least 2 weeks

 

 

          ----- T -----

 

TEMPERATURE  --TEM

                                                                                               

Lees, 1951 -- Journal of Helminthology 25: 97-104.

Panagrellus redivivus was investigated as a possible facultative parasite in the mammalian body using the mouse as the host. During accompanying investigations of   the nematode, it was found that it withstood temperatures of up to 37C.

 

Steele, 1967 -- Plant Disease Reporter 51: 171-173.

A constant temperature bath for pot-grown plants was constructed using a water heater, tank, lumber solenoid valves, screens and vermiculite for a cost of less than $200 US. Three photographs and a drawing of the electrical components are included.

 

THERMAL CONTROL -- THC   

                                                                   

Fassuliotis, 1962 -- Plant Disease Reporter 46: 61-62.

A diagram and photographs of an inexpensive temperature tank is presented.which can be used for experiments in which the soil temperature requires control within narrow limits.

                                                                           

THERMAL DEATH TEMPERATURE -- THD          

                                      

Bergeson, 1959 -- Nematologica 4: 344-354.

The influence of temperature on the survival of some species of  Meloidogyne in the absence of a host was studied. Temperatures of 32F and 40F were usually lethal to larvae in 7 and 14 days respectively. Temperatures between 80 and 90F reduced the rate of survival.  

 

TROPISMS & TAXIS --TRT 

 

Bird, 1959 -- Nematologica 4: 322-335.

The attractiveness of roots to the plant parasitic nematodes  Meloidogyne javanica and M. hapla was investigated.  More specifically, the physiological mechanisms associated with root growth which causes them to be attractive to the root-knot nematode was studied. The effects of pH, carbon dioxide, redox potential and oxygen were discussed

 

Cobb. 1917 -- Contributions to a Science of Nematology 5: 124-125.

It was discovered in a study of tides and beach sand, that the top layers of sand, between high and low tides and under ordinary circumstances,  afford the nematodes a habitat of considerable stability which interferes in no way with the activity of the nematodes. On open ocean beaches, however,  the force of the waves induce the nematodes atypically to have a tougher cuticle and roll up in a ball. Accordingly to observe the beach sand nematodes in their normal form, the top layers of sand should be sampled.

 

Croll, 1967 -- Nematologica 13: 17-22.

A study of the mechanism of orientation in nematodes was conducted using stimuli at different pulse rates. Accordingly, the galvanotaxis of Panagrellus redivivus and the phototaxis of Chromodorina viridis were investigated  A drawing of an oscilloscope which investigated

the influence of a pulsating current on Panagrellus galvanotaxis is presented. Phototaxis in

Chromodorina was accomplished using a transistorized stroboscope with a xenon discharge tube,  thus investigating the influence of a flashing light on the phototactic response of the nematode.

 

Edmunds, 1967 -- Nematologica 13: 174-175.

A method for studying the influence of hydrogen ion concentration on the aggregation of  Aphelenchus avenae is given. The system described offers the nematodes a choice of different pH values and the migration and accumulation of nematodes can be determined in time and space.

 

El-Sherif & Mai, 1969 -- Journal of Nematology 1: 43- 48.

Attraction of Ditylenchus dipsaci and Pratylenchus penetrans to a temperature gradient was investigated. The latter species oriented their heads toward a heat sourceand moved directly towards it from a 1-cm distance within 10 minutes.  When the heat was eliminated, the nematodes dispersed.

                                                                          

Peacock, 1958 -- Nematologica 4: 43-55.

To demonstrate the chemical nature of the root exudate which attracts the root-knot nematode, Meloidogyne incognita, a sterile piece of cellophane was placed on an agar film in a test tube. A sterile egg-mass was inserted between the glass and the cellopane in the agar and an excised root tip was placed on the top surface of the cellophane. Tubes  examined in 24 hours were found to have  large numbers of larvae clustered around the growing point  As the root tip grew, the larvae moved with it but penetrated only when the root tip had reached the edge of the cellophane piece.

 

Weischer, 1959 -- Nematologica 4: 172-186.

In an article written in German on Experimental Investigations on the Migration of Nematodes, the author reports on the conduct of 3 experiments. The hatching of larvae as influenced by the proximity of roots, the effect of root diffusates on larvae, and the apparent

showing of a rheotaxis by larvae of Heterodera but not by larvae of Ditylenchus.

 

 

TURBIDOMETRIC METHODS -- TUR

 

Blake, 1958 -- Proceedings Linnean Society of New South Wales 83: 241- 244.

A turbidometric method for estimating the number of nematode larvae in a suspension stabilized with 0.5% carboxymethyl-

cellulose is described.. The results rapidly obtained are amenable to statistical analysis, and for Anguina agrostis, the percentage error in the prediction from the standard curve was less than 5%.

 

        ----- U-Z -----

 

VIABILITY -- VIA

 

Bird, 1979 -- Journal of Nematology 11 103-105.

 A method was used which earlier was proposed for the presence of esterases which hydrolyse nonfluorescent fatty acid esters of fluorescein to yield fluorescein, which accumulates and is detectable by its fluorescence. The nematodes tested were larvae of Meloidogyne javanica and Caenorhabditis elegans. Lining nematodes displayed fluorescence in discrete particles in the gut, while dead specimens showed a general diffuse fluoresce throughout the body.

 

Chapman & Eason, 1974 -- Journal of Nematology  6: 55-56.  

Cysts of  Heterodera trifolii recovered from pots of  Kenland red clover were placed in 90-mm glass petri dishes and covered with molten, cooled 1% or 5% agar. Te dishes were placed in plastic freezer bags and stored in a refrigerator @ 10C. At various time intevals cysts were picked from the agar and placed with seedlings of clover  Cysts stored in 1% agar were viable after 62 days but not after 96 days. Cysts in 5% agar were viable after 546 days

                                                                                                   

Granek, 1976 -- Journal of Nematology 8: 91-92.

The author points out that it is difficult and time-consuming to determine if juveniles of  Heterodera rostochiensis juveniles are living or dead. He proposes the technique of applying an electric current through the  nematode using stainless steel electrodes set 1mm apart. At 15-20V and a maximum of 10amps, juveniles which were alive projected their stylets whereas dead nematodes did  not.

 

Jatala, 1969m-- Phytopathology 59: 1033.

A new staining technique for differentiating between living and dead nematodes using, at least, nematodes from 10 genera is proposed. The material used is potassium permanganate in aqueous solution. The time required for dead nematodes to pick up the stain was as short as 5 seconds.

 

VIRUS TRANSMISSION -- VIR

 

Bergeson, Athow, Laviolette & Thomasine, 1964 -- Phytopathology 54: 723-728.

Transmission, movement and vector relationships of tobacco ringspot virus were investigated. Xiphinema americanum transmitted the virus readily from infected cucumber or soybean to cucumber. Pin-pricking soybean roots immersed in inoculum resulted in more infection than rubbing the roots with inoculum.

 

                                   

 

                                                                                        

 

SECTION C -- SUBJECTS ALPHABETICALLY  ARRANGED

                        WITH RELATED ACRONYM

    

     ----- A to C -----

 

AERATION --AER

 

AGAR, CALLOUS TISSUE -- ACT

 

AGAR CULTURES -- AGA

 

AGAR, SPECIAL METHODS -- ASM

 

AGAR, SPECIAL PROCEDURES -- ASP

 

AG ING -- AGE

 

ANAESTHETIZING -- ANS

 

ANALYSIS -- ANA

 

AXENIZATION -- AXE

 

BIOASSAY -- BIO

 

BIOCHEMISTRY -- BCM

 

CHROMATOGRAPHIC TECHNIQUES -- CHR

 

CLASSIFICATION -- CLA

 

COLLECTION, BAERMAN FUNNEL -- COF

 

COLLECTION, MISCELLANEOUS -- COL

 

COLLECTION -- SIEVING, SUCTION, SIPHON -- COM

 

CONTROL,  CHEMICAL, USING GASES -- CONG

 

CONTROL. CHEMICAL, USING LIQUIDS -- CONL

 

CONTROL, CHEMICAL, MISCELLANEOUS -- CONM

 

CONTROL, CHEMICAL, USING SOLIDS -- CONS

 

CONTROL, NON-CHEMICAL -- CNC

 

COUNTING EQUIPMENT -- CNS

 

COUNTING METHODS -- CNM

 

CULTURE -- CUL

 

CYST PRODUCTION -- CSP

 

CYST RECOVERY -- CSR

 

CYTOLOGY -- CYT

 

     ----- D to E -----

 

DEHYDRATION  & CLEARING -- DIE

 

DETECTION --DIC

 

DIAGNOSIS -- DIA

 

DRAWING --DRA

 

ELECTRICITY, ELECTROPHORESIS -- ELC

 

EMBEDDING -- EMB

 

ENZYMATIC ASSAY -- ENZ

 

ESTIMATION OF CYST CONTENTS -- ESC

 

ESTIMATION OF INFESTATION -- ESI

 

ESTIMATION OF NEMATODES -- ESN

 

EXSHEATHMENT -- EXS

 

EXTRACTION, BAERMANN FUNNEL -- EXBF

 

EXTRACTION, CENTRIFICAL FLOATATION -- EXCF

 

EXTRACTION, CYST METHODS -- EXCM

 

EXTRACTION, EGGS -- EXEG

 

EXTRACTION, ELUTRIATORS -- EXEL

 

EXTRACTION, ERLENMEYER FLASK -- EXES

 

EXTRACTION , FILTRATION -- EXFT

 

EXTRACTION, FLOATATION -- EXFL

 

EXTRACTION, INCUBATION -- EXIN

 

EXTRACTION, MACERATION -- EXMC

 

EXTRACTION, MISCELLANEOUS -- EXMI

 

EXTRACTION, MISTIFIERS -- EXMS

 

EXTRACTION, SIEVING -- EXSV

 

EXTRACTION, SUCTION -- EXSU

 

     ----- F to L -----

 

FIXATIVES -- FIX

 

GROWTH & DEVELOPMENT -- GRD

 

HATCHING,  CHEMICAL  USAGE -- HMC

 

HATCHING, MISCELLANEOUS -- HMS

 

HATCHING,  PHYSICAL METHODS -- HMP

 

HATCHING, ROOT DIFFUSATES -- HMR

 

HISTOLOGY -- HST

 

IDENTIFICATION -- IDE

 

ILLUMINATION -- ILM

 

INFECTION, INDEXING INFECTION -- IND

 

INOCULATION,  FOLIAGE -- INOF

 

INOCULATION,  MISCELLANEOUS -- INOM

 

INOCULATION, ROOTS & TUBERS -- 1NOR

 

INOCULATION, USING ANIMALS --INOI

 

ISOLATION -- ISO

 

KILLING BEFORE FIXATION -- KIL

 

LIFE PROCESSES -- LIF

         

LYOPHILIZATION -- LYO

 

     ----- M -----

 

MANIPULATION -- MAN

 

MEASURING -- MEA

 

MICROPLOTS --- MCP

 

MICROSCOPY -- MIC

 

MICROTECHNIQUE -- MTE

 

MODELS -- MOD

 

MOISTURE -- MOI

 

MOLECULAR BIOLOGY -- MOL

 

MOUNTS, BODY PARTS -- MNV

 

MOUNTS, EN FACE -- MNF

 

MOUNTS, PERMANENT , MISCELLANEOUS -- MNP

 

MOUNTS, PERMANENT, MOUNTING FLUIDS -- MNR

 

MOUNTS, PERMANENT, SPECIAL SEALS -- MNS

 

MOUNTS, TEMPORARY -- MNT

 

MOUNTS, WHOLE -- MNW

 

MOVEMENT & MIGRATION -- MOV

 

    ----- N to P -----

 

NEMATICIDE, APPLICATION -- NAP

 

NEMATICIDE, LAB ASSAY --NLA

 

NEMATICIDE, SPECIAL METHODS -- NSM

 

NEMATOPHAGOUS FUNGI -- NUN

 

OBSERVATION -- OBS             

 

PHOTOGRAPHY -- PHO

 

PHYLOGENESIS -- PHY

 

PRESERVATION -- PRE

 

PRESSURE -- PRG

 

      ----- R to S -----

 

RADIOACTIVITY, RADIATION, & IRRADIATION -- RAD

 

REFERENCE SYSTEMS -- REF

 

RELAXING BEFORE FIXING -- REL

 

RESISTANCE/SUSCEPTIBILITY, DETERMINATION -- RDO

 

RESPIRATION -- RES

 

SAMPLING -- SAM

 

SCANNING MICROSCOPY -- SEM

 

SECTIONING METHODS -- SMI

 

SECTIONING, NEMATODES -- SNE

 

SECTIONING, SOIL -- SSO

 

SPECIAL TECHNIQUES -- SPT

 

STAINING, ACID FUCHSIN -- SAF

 

STAINING, DEATH DETERMINATION -- SDD

 

STAINING, NEMATODE EGGS -- STS

 

STAINING NEMATODES -- STM

 

STAINING, NEMATODES IN PLANTS -- STP

 

STAINING , OSMIUM TETROXIDE -- SOT

 

STATISTICS  -- STA

 

STERILIZATION -- STE

 

STORAGE -- STG

 

SURVIVAL -- SUR

 

      ----- T to Z -----

 

TEMPERATURE  -- TEM

 

THERMAL CONTROL -- THC

 

THERMAL DEATH TEMPERATURE -- THD

 

TROPISMS & TAXIS -- TRT

 

TURBIDOMETRIC METHODS -- TUR

 

VIABILITY -- VIA

 

VIRUS TRANSMISSION -- VIR

 

 

 

SECTION C' -- ACRONYMNS ALPHABETICALLY