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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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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.

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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