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