The parasitic nematode, Neoaplectana glaseri, in axenic culture. II. Initial results with defined media

EXPERIMENTAL

PARASITOLOGY

The

12, 25-32

Parasitic Axenic

(1962)

Nematode, Culture. II. Defined George

U.S.

Public

Health

Neouplectuna

Initial Media

Results

with

J. Jackson

Service Research New York,

(Submitted

in

glaseri,

Fellow, The New York

for publication,

16 June

Rockefeller

Institute,

1961)

Growth is compared in various axenic, liquid media for Neoaplectana glaseri (Steiner, 1929), in nature a nematode parasite primarily of insect grubs. The best liquid cultures contained a Stoll extract of pregnant rabbit liver which supported population increments up to 22~ during a 3-week period. A chemically defined medium containing 18 amino acids, 1 fatty acid, urea, Krebs cycle intermediates, purines, pyrimidines, vitamins of the B group and glucose as well as inorganic constituents supported the development of third stage larvae into adults and low (2X to 3X) but consistent levels of reproduction. Defined and partially defined media did not give good results if the liquid level in culture tubes exceeded 1.5 cm, implying the importance of a gas phase. Temperatures of about 21” C were optimal for growth and reproduction.

The axenic culture (Baker and Ferguson, 1942) of many bacterial species,both parasitic and free-living, has been routine procedure for decades-since the era of Robert Koch. Culture techniques for protozoa have not come as easily, particularly for those parasitic specieslike trypanosomes and malaria which have complicated life cycles consisting of distinct morphological stages, each with its own special requirements. The failure to culture parasitic worms is even more remarkable. Although partial or complete maturation outside the host, in the absence of demonstrable contaminants, has been obtained for several species of platyhelminths and nematodes, the members of only one parasitic nematode genus have been cultured through consecutive life cycles (Glaser, 1940a; Glaser, McCoy, and Girth, 1942). Consequently, our knowledge of the animal parasites has been severely restricted. Advances, as in certain aspects of bacterial genetics, physiology, chemotherapy, etc., may be expected in parasitology only when larger numbers of animal parasites can be grown as routinely as bacteria under axenic conditions in the laboratory. 25

The present work was undertaken to study factors, both favorable and unfavorable, in the development and reproduction of Neoaplectana glaseri, the one parasitic nematode which has been grown axenically and also continuously throughout many successive generations (Glaser, 1940b; Stoll, 1953a, 1959). The effects of anthelminthics and antiserum in Stoll’s type of axenic, liquid culture were described in the first paper of this series (Jackson, 1961). Attempts to define the nutrition of axenic N. glaseri are described here. MATERIALS

AND

METHODS

Source of Worms Glaser isolated N. glaseri for axenic culture several times; the strain used in these experiments was isolated from a beetle grub in 1944 and has since been grown in Glaser’s and, later, Stall’s laboratories on agar slants with a pierce of sterile animal tissue (Stoll, 1953b). In Stall’s laboratory the slants are routinely made in large (22 X 100 mm), cotton plugged tubes with 2% agar containing dextrose and a 1% peptone-beef heart infusion broth as supplement to a piece of

26

JACKSON

fresh rabbit kidney placed at the bottom of the slant. The surface of the slant is wetted by water of condensation from the suspension of inoculated worms. Large, almost homogeneouspopulations of third stage larvae can be harvested from the inside walls of the tubes after 3 or 4 weeks. These are washed and stored in shallow, distilled water at 5” C for subsequent inoculation into new cultures. Keeping the storage flasks at room temperature for 24 or 48 hours prior to inoculating experimental cultures promotes ecdysis of the worms and, in the present experiments, was also used to allow elimination of some gut contents. Axe&c

of Liquid

Preparation

Media

The handling of media from natural sources is described by Stoll (1953b, 1959). The procedures used for partially and completely defined media were adapted from Trager’s (1957) work with leishmania. Tests used for detecting contaminating microorganismswere described in paper I of this series. Moreover, the defined and partially defined media themselves serve as additional controls. Incubation

of

Cultures

In experiments other than those with the temperature variable, all cultures were incubated in the dark at about 2 1’ C. Liquid cultures were on a shaking machine giving 100 strokes per minute of about 1.5 cm traverse. In the temperature experiments, cultures were not kept on the shaking machine.

Ax&c

TABLE I N. glaseri in Variozrs

St011 raw liver extract (pregnant rabbit) St011 raw liver (non-prqnant Trager Meat 1%

“S”

infusion peptone

broth

Defined

medium

dehydrated

Trager

medium

‘3” -

Trager

medium

“C”

199 XCTC

R R

with

serum

Beetle

++++ +++

Rabbit

Japanese

R

++.

N. glaseri

Commercial

+++++

extract rabbit)

medium

Media

grub

R

+R

ox liver bpf

+

\

digest’[

+

r D D D

extract

S S

109

S

a “Panmede” (Paines & Byrne Ltd., Greenford, England). The following abbreviations are used: R (development of third stage larvae into reproductively fertile adults and development of newborn larvae to at least third stage in turn, thus completing at minimum one life cycle) ; r (development of third stage larvae into reproductively fertile adults but little or no development of newborn larvae) ; D (development of third stage larvae into adults but no reproduction) ; S (survival of third stage larvae but little or no development) ; + (relative amount of reproduction).

alone; but with inclusion of l/lOth RLE: these increments in the present experiments were up to 22X during the same period. In an 8.3% solution of a commercial, dehydrated ox liver digest, the inoculated third stage larvae developed into fertile adults, but the majority of newborn worms did not complete RESULTS the life cycle by developing into third stage The growth of N. glasevi in various liquid larvae in turn. In rabbit serum, there was media has been compared and is summarized development into adults but no reproduction. in Table I. The best liquid cultures con- In a beetle grub extract prepared by Stoll tained Stoll’s raw liver extract (RLE). This (1959), third stage larvae survived but did material supported larger populations when not develop into adults. the source of the liver was a pregnant rabbit It was discovered that development and rather than a not pregnant one (Stoll, 19.54, reproduction of the worm occurred in 1961). In practice RLE was diluted 1:9 Trager’s (1957) partially defined medium with an Arnoldized peptone-meat infusion “S,” which had originally been devised for broth. Starting with a standard inoculum of the leptomonad stage of Leishmania taren25 third stage larvae, population increments tolae. Total populations of N. glaseri per ml per ml of culture medium of 2x to 4x could were approximately half as dense after 3 be grown during a 3-week period in the broth weeks as in Stall’s medium made with RLE

CHEMICALLY

DEFINED

from pregnant rabbits. The composition of the partially defined medium is shown in Table II. If bovine plasma fraction V (bpf V) was omitted, third stage N. glaseri larvae developed into adults but there was no reTABLE II Trager Medium

Cytidylic Adenine,

acid guanine,

0.02 0.08 0.20 0.30 0.50 0.80 0.30 0.05

xanthine

(each)

0.17

Hemin

2.50

KH,PO, Na,HPO, NaCl

50. 125. 200.

Glucose

500.

PORTION

2: Not

Bovine

plasma

Casein

hydrolyzate

Q “Parenamine” York).

defined

fraction and

chemically V tryptophana

(Winthrop-Stearns

27

GLASERI

Trager

TABLE III Medium

PORTION 1: Chemically folic acid increased from PORTION

2: Now

“C”

defined as in 0.08 to 0.16)

chemically

200. 1125. Inc.,

New

production. Among the adults there were also more stunted individuals. Amounts of 0.1 ml to 0.15 ml of buffered, unconcentrated tissue culture medium “ 199” (Morgan, Morton, and Parker, 1950) adequately replaced bpf V. The amino acid content of Trager’s (1957) completely defined L. tarentolae medium “C” is based on the composition of a lactalbumin hydrolyzate (Table III). In this medium N. gZaseri larvae developed into adults but did not reproduce. Addition to “C” of tissue culture media 199 or NCTC 109 (McQuilkin, Evans, and Earl, 1957) did not stimulate fecundity. There was survival, but no development or reproduction in the tissue culture media alone. Only when “C” was supplemented with 199 or NCTC 109 and certain amino acid concentrations were increased (Table IV, portion 2) did N. glaseri

coso,

. 7H,O

cuso, MnS04 FeS04 ZnSOJ

* 4H,O * 7H,O * 7H,O

CaCl, M&O,

* 7H,O

DL-Alanine L-Arginine HCI DL-Aspartic acid L-Glutamic acid Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine HCl nL-Methionine DL-Phenylalanine L-Proline DL-Serine m-Threonine m-Tryptophan L-Tyrosine DL-Valine Amino

acids

(total)

‘5”

(with

defined

0.002 0.01 0.02 0.10 0.20 0.50

W% ml

100

(each) uracil,

N.

Metal ion mg/lOO ml WV

Cl, inositol

FOR

“s”

PORTION 1: Chemically defined Biotin Folic acid Pyridoxal, pyridoxamine, pyridoxine. riboflavin, thiamine (each) p-Aminobenzoic acid Nicotinamide Ca pantothenate Choline

MEDIA

Mg/loo ml 0.011

0.044 0.05 0.40 1 .oo 2.20 2.6 100.00 70. 30. 120. 190. 10. 15. 60. 150. 125. 30. 40. 50. 40. 50. 20. 40. 50. 1090.

develop and reproduce in a defined environment. In these particular mixtures reproduction was consistent, but generation time was longer and yields were considerably lower than in medium containing RLE or in the partially defined medium. The composition of the simplest, most satisfactory defined medium so far devised for N. glaseri is given in Table IV. Generation time of the first generation was 6-7 days and did not differ significantly from some of the undefined and partially defined media. But, at best, there were population increments of only 2x to 3X. After the F1 generation had developed into third stage larvae, further development and reproduction was minimal unless fresh medium was added. The effects of temperature on Stoll cul-

28

JACKSON

TABLE Keoaplectana

Defined 1: removed)

As

PORTION

hemin

PORTION

the

2 :

following

in

Trager

IV glaseri

Medium

medium

As in Trager medium concentration changes) :

Mg/lOO

ml increased

r-Arginine HCI L-Histidine L-Isoleucine L-Leucine L-Lysine HCl nn-Methionine DL-Phenyialanine nL-Threonine m-Tryptophan m-Valine

“C”

(with

“C”

(with

from------+

to

3: Additional

PORTIOK

components Mg/lOO

Butyric

acid

ml

16.

Citric acid Fumaric acid m-Lactic acid r,-Malic acid Pyruvic acid Succinic acid

38. 11. 18. 13. 16. 11.

Taurine Urea

150. 0.02

Effect

TABLE on N. glaseri

T” C

Survival’r

Freeze-thaw 5 21 25 30 35 37 40 (I Days

DISCUSSION

156. 52. 208. 312. 312. 260. 100. 260. 80. 260.

30. 15. 60. 150. 125. 30. 40. 50. 20. 50.

survival

V in Stall Growth

-

-

>30 >30 >3O 30 24 10 7

+ + + + e -

of infective

At and about 21’ C there was optimal reproduction in axenic cultures. Above 25” C, there was a falling off of reproduction, development and, lastly, survival. Controls included media incubated at the indicated temperatures prior to inoculation with worms.

Medium Reproduction +++ ++ + &

larvae.

tures of IV. glaseri is shown in Table V. The worm did not withstand freezing; even the third stage larvae were killed when rapidly frozen and thawed. At 5” C there was survival of third stage larvae but no development. Third stage larvae stored in water and refrigerated at this temperature for periods as long as two years have been successfully subcultured (Stall, personal communication).

In nature, the worm parasitizes various stages in the life cycle of the so-called Japanesebeetle, Popillia japonica, primarily the grub stage which feeds in the soil during spring and autumn, ingesting the infective worm larvae with its meal (Glaser and Fox, 1930; Glaser, 1932). Occasionally infections can persist through pupation into adult beetles, or the adults become infected in early summer on emerging from the pupa in the soil. During the summer, adult beetles feed on plants where there is little opportunity to gain fresh contact with the parasite -which is very sensitive to drying and does not survive on or above the soil surface (Glaser and Farrell, 1935). In later summer adult females can become infected again, when they return to the soil to bury their eggs. The first evidence of this infective cycle was reported by Glaser and Fox (1930): at the Tavistock Golf Course near Haddonfield, New Jersey-‘L14 dead, flaccid and ocherous brown, fully grown Japanese beetle grubs” were found. The worms discovered on dissection of the 14 dead grubs were able to infect healthy grubs, developing, reproducing and spreading from the gut throughout the host, killing it by consuming the tissues. Steiner (1929), on receiving specimensfrom Glaser, designated the nematode a new genus and species,Neoaplectana glaseri. This parasite’s life cycle is in many respectsthat of a typical nematode with four larval stages, each separated by a molt. Adults are dioecious and ovoviviparous. The third larval stage retains the second stage cuticle temporarily as a sheath covering its new cuticle. When the beetle grub dies these ensheathed larvae survive in the soil for a long time and on being eaten are infective for new beetle grubs. Glaser’sfirst cultures of n’eoaplectana were

CHEMICALLY

DEFINED

not axenic (Glaser, 1931; McCoy and Glaser, 1936). The medium consisted of a dextrose-meat infusion agar with live yeast. The yeast grew as a mat on the agar surface, suppressing but not eliminating other microorganisms, serving as live food for the worms and maintaining moisture. With a generation time of 6-7 days and transfers to fresh medium every second week, several continuous generations of Neoaplectana were cultured. This was the first time that the entire life cycle of a parasitic nematode had been grown outside the host. However, after a number of subcultures, fecundity declined (Glaser, 1940a). At different times in different series, a generation appeared whose adult males looked normal but whose females, well developed otherwise, had undeveloped ovaries. This condition could be prevented by passing the culture through live beetle grubs, or sprinkling the surface of cultures with powdered beetle grub, bovine ovary or orchic tissues. At the time, the effect of the sex gland powders could not be attributed to any known hormones. To explain generation time variation in the loss of fecundity in different cultures, Glaser postulated the existence of physiologically different strains of Neoaplectana. Another interpretation is possible. It seems unlikely that inherent deficiencies of a medium should show up after as many as 32 worm generations-which was, occasionally, when loss of fecundity appeared. Perhaps the strain differences which explain Glaser’s results are those of the contaminants and not of the worms. Once axenic cultures had been established (Glaser, 1940b ; Glaser, McCoy, and Girth, 1942), not only was there no further mention of drastic fecundity losses in subcultures, but strain differences as to population size also disappeared and the total counts were higher. Comparing the early work with axenic culture work, it appears, at least for parasitic members of the genus Neoaplectana, that association with other microorganisms is not beneficial outside the host and that the population potential is more fully realized in an axenic environment.

MEDIA

FOR

N.

GLASERI

29

Glaser grew Neoaplectana continuously in three types of axenic culture systems. 1. A meat infusion agar slant with a piece of sterile animal tissue at the bottom, wetted with saline. In Stoll’s laboratory, kidney is the tissue choice since its capsule effectively protects it from contamination during autopsy and, after chilling, capsule and kidney are readily separated. Mammalian or avian liver, ovary, testis, as well as embryo tissues and, less adequately, spleen and muscle can be substituted for kidney but are more difficult to manipulate sterilely (Glaser, 1940b; Stoll, 1953b). The meat infusion component of the agar is not a necessary supplement for liver or kidney tissue, but in the present experiments spleen alone supported only small population increments as did dextrose-peptone-meat infusion agar without addition of the fresh animal tissue. 2. In a second type of axenic culture, Glaser eliminated the solid agar surface and grew the worms on rabbit kidney in 3-4 mm of O.SF sodium chloride. 3. In the third type, a semisolid agar containing autoclaved, ground meat was used to culture another parasitic species, N. chresima (Glaser et al., 1942). A difficult step in all of these cultures was obtaining, without injury to the worms, inocula which had been freed of contaminants. A method for doing this, developed by Glaser and Stall (1940), involves filtration through gauze and several 2% sodium hypochlorite washes to sterilize the surface of the worms, interspersed with periods of storage in sterile water to allow elimination of bacteria from the gut. Stall (1948) developed a liquid medium containing raw liver extract (RLE). This supports excellent growth of axenic N. glaseri (Stoll, 1951, 1953b, 1954, 1956, 1959, 1961). The extract, prepared in the cold, is acidified and Seitz filtered. At first it is a clear, pale yellow liquid but after several days storage at 4-5” C a flocculent precipitate forms and, on further standing, this aggregates into a solid pellet which can, however, be resuspended. The activity of RLE for supporting N. glaseri growth and reproduction is heat labile and, to a great extent, associated with the precipitate rather than the super-

30

JACKSON

natant. Indeed, extracts are less active prior to the formation of the precipitate. In practice heat-labile RLE is diluted with 9 parts of heat-stable meat infusion broth, 5% dextrose is added with an inoculum of about 25 third stage larvae per tube or flask. As in the case of slants, optimal yields of several thousand worms are obtained at the end of 3 weeks in the dark at about 2lo C. Shaking the liquid cultures results in greater populations by 50%. During the first generation in the best cultures, one female worm has approximately 200 offspring. The kidney-agar slant and the liquid liver medium offer different advantages for laboratory work. In slant cultures the new infective larvae leave the bottom of the tube and migrate up the glass walls. By going to the inside walls with a bacteriological loop one can remove almost homogenouspopulations of these third stage larvae for subculture. No such separation from other stages in the life cycle occurs in liquid media. However, the advantage here is that a uniform distribution of test substancesis possible, especially when the cultures are shaken. By inoculating from a slant into fluid media, by way of washes and storage in distilled water, one can combine the assets of both systems. One further aspect of the cultures should be mentioned. Considering the lability of cultured mammalian cells and certain protozoa, the extent to which N. glaseri has retained its wild-type character is remarkable. No morphological changes are apparent and Stoll has shown that the worm was not only infective but still pathogenic for the Japanese beetle grub after 7 years, representing 180-195 cultured generations (Stoll, 1953a) ; infectivity was shown again in the 17th year representing a total of about 216 cultured generations (Stall, personal communication). Also, after 15 years of culture, Pramer and Stoll (1959) showed that N. glaseri induced trap formation in predaceous soil fungi, associating this effect with a fraction of the worm excretions called Nemin. (When purified samplesof Nemin are available, it will be interesting to test for another effect: its antigenicity for vertebrates.)

One can not work with known chemicals which have unfavorable effects on a parasitic nematode (Jackson, 1961) without asking the reverse question-which chemicals favor growth and reproduction? Long before the present experiments, Stoll in 1948 had incubated third stage 12’. glaseri with synthetic media basedon then current studies of Tetrahymena and Drosophila, but there was no development (Stoll, 1959). An analysis of RLE was being planned in Stoll’s laboratory when it was discovered that N. glaseri developed and reproduced in the partially defined medium “S” for Leishmania tarentolae leptomonads. Hindsight rationalization tells that this result is not surprising. In nature both parasites are found in cold blooded species. The one host of the worm and one of the hosts of the protozoan is an insect. The first defined media in which N. glaseri completed a reproductive cycle had many constituents becauseof the inclusion of 199 or NCTC 109. These mixtures have a great variety of defined components, between 60 and 70. There are amino acids, purines, pyrimidines, vitamins, growth factors, cofactors, cholesterol, oleic acid, ATP, etc., as well as inorganic compounds-but at very low concentrations compared to most components in Trager’s defined medium. Consequently, two paths of investigation presented themselves. One was to look for the substanceswhich would stimulate yields to levels found in good, undefined media. In fact, a variety of compounds has been tested. Suggestionsas to quality and balance were culled from defined media for other types of organisms (White, 1946; Dougherty et al., 1959) from analyses such as Tallan, Moore and Stein’s of liver amino acids (1954); and from catalogues of assorted chemicals. Even so, the magic formula can be overlooked. The other path was to analyze the promising but imperfect medium one has, eliminating unnecessarycomponentsand adjusting concentrations to optimal levels. Enthusiasm for this undertaking is dampened when one considers all the combinations of 70 odd componentseven at only a few of the possible concentration levels.

CHEMICALLY

DEFINED

In the simplest, most satisfactory medium devised for N. glaseri so far, the value of all components at the particular concentrations listed in Table IV has not been determined. But a few statements about the composition can be made. Other factors being the same, the amino acids group-arginine, histidine, isoleucine, leucine and lysine-is needed at about the relatively high concentration used. This requirement may reflect the parasite’s adaption to the host. Insects are known to have especially high amino acid fluid levels (Buck, 1953; Wyatt, Loughheed, and Wyatt, 1956). Also, vitamins of the B group are required at and about their relatively high concentrations. Purines and pyrimidines are beneficial, although the approximate optimal concentrations have not been determined. A tacit assumption of this discussion has been that a nutritionally well balanced medium is the crucial condition for optimal axenic cultures of Neoaplectana. However, the influence of other factors can be shown. Defined and partially defined media do not give good results if the liquid level in a culture tube is much above 1.5 cm. Stoll’s medium gives good results at two and three times this depth, although shaking the tube is important. This perhaps implies the importance of a gas phase. The effect of changing temperature is shown in Table V. What conclusions can be drawn from the physical, chemical and biological variables to which N. glaseri has been subjected? If one thinks of the life cycle in relation to temperatures, anthelminthics, nutrients-it appears that the development of reproductively functional adults is a critical step. The other steps in the life cycle-from first stage larvae through morphological adultsalthough also sensitive to adverse conditions, can occur in an environment which temporarily or permanently prevents reproduction. It is proper to mention two experimental cautions. When one speaks of “axenic” and “defined,” one meansideal states. This should be remembered in respect to all cultureswhether of plant or animal tissues or intact organisms. Obviously the cultures are only as axenic as sterility tests are sensitive; and

MEDIA

FOR

N.

31

GLASERI

the media as defined as glassware is clean or chemicals pure. Within the limits of feasible methods both states were presumably of a high order in the present experiments. ACKNOWLEDGMENTS

As in the previous Dr. Norman R. Stall

paper, I am indebted to for the hospitality of his laboratory at the Rockefeller Institute. The technical assistance of Jane P. Elam is gratefully acknowledged. The initial samples of the partially defined medium “S” were supplied through the kindness of Dr. William Trager. REFERENCES

J. A., AND FERGUSON, M. S. 1942. Growth of platyfish (Platypoecilus maculatus) free from bacteria and other microorganisms. Proceedings of the Society for Experimental Biology and Medicine 51, 116-119. BUCK, J. B. 1953. Physical properties and chemical composition of insect blood. In Roeder, K. D., ed., Insect Physiology, New York, John Wiley & Sons, Inc., p. 147-190. DOUGHERTY, E. C., HANSEN, E. L., NICHOLAS, W. L., MOLLETT, J. A., AND YARWOOD, E. A. 1959. Axenic cultivation of Caenorhabditis briggsae (Nematoda:Rhabditidae) with unsupplemented and supplemented chemically defined media. Annals of the New York Academy of Sciences 77, 176-217. GLASER, R. W. 1931. The cultivation of a nematode parasite of an insect. Science 73, 614.615. GLASER, R. W. 1932. Studies on NeoaplectanG glaseri, a nematode parasite of the Japanese beetle (Popillia japonica Newm.). New Jersey, State of, Department of Agriculture, Bureau of Plant Industry, Circular 211, l-34. GLASER, R. W. 1940a. Continued culture of a nematode parasitic in the Japanese beetle. Journal of Experimental Zoology 84, 1-12. GLASER, R. W. 1940b. The bacteria-free culture of a nematode parasite. Proceedings of the Society for Experimental Biology and Medicine 43, 512-514. GLASER, R. W., AXTD FARRELL, C. C. 1935. Field experiments with the Japanese beetle and its nematode parasite. Journal of the New York Entomological Society 43, 345-371. GLASER, R. W., AND Fox, H. 1930. A nematode parasite of the Japanese beetle (Popillia japonica Newm.). Science 71, 16-17. BAKER,

GLASER,

R.

W.,

MCCOY,

E.

E.,

AND

GIRTH,

H.

B.

1942. The biology and culture of Neoaplectana chresima, a new nematode parasitic in insects. Journal of Parasitology 23, 123-126.

32

JACKSON

R. W., AND STOLL, N. R. 1940. Exsheathing and sterilizing infective nematode larvae. Journal of Parasitology 26, 87-94. JACKSON, G. J. 1961. The parasitic nematode, Neoaplectana glaseri, in axenic culture, I. Effects of antibodies and anthelminthics. Experimental Parasitology 11, 241-247. MCCOY, E. E., AND GLASER, R. W. 1936. Nematode culture for Japanese beetle control. New Jersey, State of, Department of Agriculture, Bureau of Plant Industry, Circular 265, I-10. MCQUILKIN, W. T., EVANS, V. J., AND EARLE, W. R. 1957. The adaptation of additional lines of NCTC clone 929 (strain L) cells to chemically defined protein-free medium NCTC 109. Journal of the National Cancer Institute 19, 885-907. MORGAN, J. F., MORTOS, H. J., AND PARKER, R. C. 1950. Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proceedings of the Society for Experimental Biology and Medicine 73, 1-8. %‘R.&MER, D., AND STOLL, N. R. 195’9. Nemin: a morphogenic substance causing trap formation by predaceous fungi. Science 129, 966.967. STEINER, G. 1929. Neoaplectana glaseri, n. g., n. sp. (Oxyuridae) a new nemic parasite of the Japanese beetle (Popillia japonica Newm.) . Journal of the Washington Academy of Sciences 19, 436-440. STOLL, N. R. 1948. Axenic cultures of Neoaplectana glaseri Steiner in fluid media. Journal of Parasitology 34 (6, suppl.), 12. STOLL, N. R. 1951. Axenic Neoaplectana glaseri in fluid cultures. Second report. Journal of Parasitology 37, (5, sect. Z), 18. STOLL, N. R. 1953a. Continued infectivity for Japanese beetle grubs of Neoaplectana glaseri (Nematoda) after seven years axenic culture. GLASER,

In Dayal, J., and Singh, K. S., eds., Thapar Commemoration Volume, Lucknow, University of Lucknow, p. 259-268. STOLL, N. R. 1953b. Axenic cultivation of the parasitic nematode, Neoaplectana glaseri, in a fluid medium containing raw liver extract. Journal of Parasitology 39, 422-444. STOLL, N. R. 1954. Further progress in the axenic cultivation in fluid media of Neoaplectana glaseri, a parasitic nematode. dnatomical Record 120, 745-746. STOLL, N. R. 1956. Axenic cultivation of the parasitic nematode, Neoaplectana glaseri Steiner, 1929, in fluid media. Proceedings of the International Congress of Zoology. 14th Congress, Copenhagen, 1953 p. 382. STOLL, N. R. 1959. Conditions favoring the axenic culture of Neoaplectana glaseri, a nematode parasite of certain insect grubs. Annals of the New York Academy of Sciences 7’7, 126-136. STOLL, N. R. 1961. Favored RLE for axenic culture of Neoaplectana glaseri. Journal of Helminthology R. T. Leiper supplement 169-174. TALLAN, H. H., MOORE, S., AND STEIN, W. H. 1954. Studies on the free amino acids and related compounds in the tissues of the cat. Journal of Biological Chemistry 211, 927-939. TRACER, W. 1957. Nutrition of a hemoflagellate (Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. Journal of Protozoology 4, 269-276. WHITE, P. R. 1946. Cultivation of animal tissues in vitro in nutrients of precisely known constitution. Growth U(3), 231-289. WYATT, G. R., LOUGHHEED, T. C., AED WYATT, S. S. 1956. The chemistry of insect hemolymph. Organic components of the hemolymph of the silkworm, Bombyx mori, and two other species. Journal of General Physiology 39, 853-868.