Growing Ochromonas malhamensis Above 35°C

GROWING Orhrommas ABOVE 35" C . 2 59

simple function including these parameters.

REFERENCES

1. Browning, J., Brittain, M . S. and Bergendahl, J . C. (1952). Synchronous and rhythmic reproduction of protozoa following inoculation. Texas Repts . Biol. Med., 10, 794-802.

2. Hughes, A. F . (1952). T h e Mitotic Cycle . London, But- terworth.

3 . Scherbaum, 0. (1956a). The application of a standard counting method in estimation of growth in normal and heat treated cultures of Tetvahymena pyriformis. Acta pathol. et nticrobiol. Scand., 37.

4. __ (1956b). Cell growth in normal and synchron- ously dividing mass cultures of Tetrahymena pyrijormis.

Exptl. Cell Res., 11, 464-476. 5. Scherbaum, 0. and Zeuthen, E. (1953). Induction of

synchronous cell division in mass cultures of Tetrahymena pyriformis. Expt l . Cell Res., 6, 221-227.

6. ___ (19%). Temperature-induced synchronous divisions in the ciliate protozoan Tetrahymena pyriformis growing in synthetic and proteose-peptone media. E x p t l . C~11 Res.,

7. Walker, P . M. B. (1954). The mitotic index and inter- phase processes. J . Ezptl . Biol., 31, 8-15. 8. Zeuthen, E. and Scherbaum, 0. (1954). Synchronous

divisions in mass cultures of the ciliate protozoan Tetrahymena pyriformis, as induced by temperature changes. Colston Papers, 7, 141 - 1 56.

SUppl. 3, 312-325.

J . PROTOZOOL., 4, 2.59-269 (1957)

Growing Ochromonas malhamensis Above 35" C."" S. H. H U T N E R , H E R M A N BAKER, t S. AARONSON,: H E L E N E A. N A T H A N ,

E U G E N E RODRIGUEZ,Q S A L L Y LOCKWOOD, M A R V I N S A N D E R S and R O B E R T A. P E T E R S E N

Haskins Laboratovips, .?Oj E . 43 St., New York 17, N . Y .

SUMMARY. Ochvontonas lnalhamensis (Pringsheim strain) can be grown above 3.5.5" C. ; below 35", the previous chemically defined medium supports dense growth. The BIZ and thiamine requirements rise steeply with temperature, and growth promotion by folk acid emerges; folk acid spares the enhanced B,? requirement. BI1 is spared also, perhaps wholly bypassed, by purines + pyrimidines + amino acids (below 35', exogenous purines, pyrimidines, and folk acid have little effect). Requirements also emerge for glycine (spared by serine), valine and isoleucine (their ratio is critical; leucine and threonine assist in maintaining a good balance), and, a t very slightly higher temperatures, phenylalanine, tryptophan, cystine, and lysine. Requirements for Mg, Fe, Zn, and M n appear to rise steeply with temperature ; metal toxicities have to be circumvented carefully. The proportion of histidine i- arginine to carbohydrate has to be increased, and a Krebs-cycle component such as succinic acid becomes stimulatory. At 36.3-36.7", a further supplement of crude natural materials such as an autoclaved suspension of Ochromonas cells is needed. Relevance of these findings to fever stress in vertebrates. Eeneral mitochondria1 function, and repair of radiation damaae, is discussed

FTER A simple medium was developed(27; cf. A Table I ) estimation of BI2 in body fluids by means of Orhromonas 1na2hamensis (ATCC 11532) began a t Mt. Sinai Hospital. The assay worked: growth was rapid and dense, linearly proportional to the Bl r content of natural materials as judged from parallel assays with Euglena gracilis, Lactobacillus leichmannii( 2 ) , and from clinical findings, and drift was negligible when natural materials were assayed : so we assumed the basal medium to be reasonably complete. Then Ford( 16) supplemented the basal medium with Tween 80 (a polyoxyethylene sorbitol

oleate), acid-hydrolyzed casein, choline, inositol. p- aminobenzoic acid, cystine, and tryptophan, for, "the organism proved susceptible to non-specific growth stimulants present in certain crude extracts." This discrepancy could arise from a difference in temperature of incubation: when we put cultures nearer the lamps, the incubation temperature rose and a richer medium was needed.

Common practice in assaying BIZ with the rat or chick is to administer thyro-active materials to increase the Blz requirement several fold and thus di- minish the experimental error introduced by intestinal synthesis and contamination of the basal ration with B I Z ( 29) . Since thyro-active materials raise body temperature and generally increase metabolism, we wanted to know whether the extra nutritional requirements of Ochromonas grown at elevated temperatures paralleled the effects of high temperatures in metazoa- specifically, the chick, mouse. rat, and man.

Aided by grants from the Rockefeller Foundation, the Loomis Institute, the American Cancer Society, and the American Cyanamid Co.

f Portions of this paper are from a dissertation presented by H. Baker in partial fulfillment of the requirements for the Ph.D. in Biology a t New York University; present address: DeDt. Chemistrv. M t . Sinai HosDital, Piew York 29, I-. Y.

f Dept. Biology, Queens College, N. Y. 6 Present address: Dept. Bacteriology, School of Hygiene

and Public Health, The Johns Hopkins Univ., Baltimore 5 , Md we find that OCh?'OmMm has a steeply enhanced

260 GROWING Ochromonas ABOVE 35” C.

requirement for B1 above a critical temperature (35’). The Ochromonas requirement for B12 seems identical in pattern with that of birds and mammals (8,17): is this identity fortuitous or does i t signal a special kinship between chrysomonads and metazoa -do such biochemical resemblances extend to the pattern of temperature factors, among other potential phylogenetic markers? Is the thyroid-enhancement of the BIZ requirement of mammals really similar? How, then. to interpret the ‘thermal” B12 requirement of Ochromonas? Some approaches are cited in the “Dis- cussion” section.

Several metabolites in addition to BIZ were needed as temperature factors and more are being identified with each small (0 1‘) increment in temperature. Temperature factors for “permeable” protozoa such as Ochromonas may thL s reveal hitherto hidden metabolic systems, and are discussed as an introduction to temperature studies with other protists and metazoa. The methods for studying them are novel in some details.

METHODS

Incubators and culture vessels. Water-jacketed incubators (National Appliance Co.) were necessary. Temperature variation with shelves almost fuIIy Ioaded was +_ 0.1’ C., provided space was left near the thermoregulator probe and a free air passage was left between shelves. Kimble thermometers (no. 43602, -0.1 to + 51” C.) were satisfactory; they agreed with a Bureau of Stanc’ards thermometer.

In work with thermophilic bacilli ( 1 ) , evaporation of culture media caused drifts in temperature and concentration of media. This difficulty was surmounted for Ochromonas work a’; follows: Experimental media were distributed in 5-ail. amounts in “10-ml. micro- Fernbach” flasks (Kiable no. 36502). Each flask had an aluminum cap ( 4 . S. Aloe no. 78300B), short- ened by us to hang without resting on the flask side- wall else, after autoclavmg, trapped water was sucked into the flask on cooling. The flasks were set in Pyrex trays, autoclaved , cooled to room temperature in the autoclave, inocul,ited, and a layer about 1 cm. deep of “tray solution’ (26) was poured around the flasks as a water bath to equalize temperature, lessen evaporation, and kill fungi. A duplicate tray was then used to cover the tray holding the flasks, and the joint between trays sealed with “freezer” tape (“masking” and Cellop lane tapes stuck poorly).

Culture conditions. Experimental cultures were incubated in the dark for the sake of precise temperature control; there was no difference except color between light or dark cultures. At first, experimental

TABLE I. Inoculation broth. Wt. for 100 ml. of final rriedium.

(NH,) IH citrate 0.12 g. L-Histidine HCl-H,O 0.05 g. CaCO, 0.015 g. 1,-Argiiiine HCl 0.05 g.

0.04 g. MgCO, (basic) 0.05 g. KH,HCO,

Sucrose 1.6 g. Biotin 0.4 &. DL-Methionine 0.06 g . B,, 1.0 rr i tZe .

L-Glutamic acid 0.3 g. “Metals 45A” 1.0 mg. KH,PO, 0.03 g. Thiamine HCl 0.2 mg.

- r - 0 .~

The p H is 4.8-5.2; adjustment is unnecessary. “Metals 45A” differs from “Metals 45”(26) in having Mo 0.005 m y . % instead of 0.035 mg. The basic MgCO, has the des- igiintioii 4MgCO, - Mg(OH), - 4H,O (Mallinckrodt).

cultures were inoculated from cultures grown in light a t room temperature (24-31”) in “inoculation broth” (Trypticase 0.2 g., yeast autolysate 0.2 g., “1:20” liver 5 mg., cane sugar 1.0 g., glycerol 0.5 g., water to 100 ml.) in screw-capped tubes. I t was superseded by a chemically-defined medium like the one from which the present study departed(27) (Table I ) , containing a limiting concentration of B12 (1.0 mpg. %). For inoculum, a drop of a weU-grown culture was sus- pended in 20 ml. of distilled water or the basal medium; each flask received one drop of the suspension, which contained 30-100 motiIe flagellates. Cultures were incubated for 7-14 days; growth was usually slower a t the critical temperatures. Growth was meas- ured as optical density with a Welch Densichron specially equipped with a cuvette-holder and a red- sensitive phototube. The IEensichron directly measures densities to 3.9-a time-saving.

Stored nonsterile culture media and solutions used for compounding media were treated with - 0.5% of a volatile preservative: chlorobenzene; 1,2-dichlorethane; and 1-chlorobutane (n-butyl chlor- ide) (1:1:2 v/v). Media were autoclaved a t 118- 122” for 35 min. This severe autoclaving removed the high-boiling chlorobenzene and killed the unusu- ally resistant spore-formers harbored in the laboratory.

To prepare basal media easy to store, uniform from experiment to experiment, yet flexible, dry mixes were employed; the latest one is detailed in Table VI.

Experimental design. Setting incubation temperatures higher enforced changes in the proportions of nutrients. An “accordion” design enabled us to dilute or concentrate the entire basal medium in testing supplements. This speeded decision on whether lack of growth at an elevated temperature was due to a defi- ciency or imbalance, and so supplements were tested more adequately (Table 111). This sort of experiment had to be done a t each temperature increment; growth promotion by crude natural materials was often the correction of an imbalance rather than the satisfaction of a need for additional factors.

Sterilization.

Dry-mix technique.

GROWING Ochromonas ABOVE 35” C. 261

RESULTS

Znocula. In early experiments, large inocula some- times permitted growth and small inocula such as described in “Methods” did not. Large inocula might be advantageous if growth a t supra-optimal temperatures depended on mutation: the larger the population, the larger the chance of a favorable mutation. Growth in those experiments was not random-it was reproducible; the decisive feature proved to be not the number of viable cells (although, of course, the fewer the viable cells, the longer time for growth to appear) but total cells, dead and alive. As Ochromonas is vigorously phagotrophic, dead bodies as well as solu- bles introduced with the inoculum were almost cer- tainly utilized; moreover, some flagellates in the inocula in flasks a t the higher temperatures might die, and be cannibalized by the survivors, thus sup- plying temperature factors. Addition of autoclaved culture fluid + cells from a dense growth of Orhrom- onus, cultivated a t ordinary temperature in the simple chemically-defined medium, allowed growth from small inocula (cf. Table 111). This “chrysomonad suspension” has again and again been an excellent source of temperature factors. Growth a t 36.3-36.7’ requires unidentified factors in natural materials; autoclaved chrysomonad suspension remains a good source. The small inocula now used allow ample time for temperature equilibration before growth is significant. Be- cause chrysomonad bodies supply temperature factors, a temporary lowering of the incubation temperature might permit otherwise impossible growth.

Casein hydrolysate effects. The early experiments led us to suppose that a t elevated temperatures acid- hydrolyzed casein promoted growth because of multiple factors-which seemed to harmonize with Ford’s recommendations. Amino acids in combination or higher concentrations of metal ions such as Ca and Fe--abundant in casein hydrolysates-supported good growth, yet combinations of amino acids and metal ions supported only poor growth. Low concentrations of intact or enzymatically digested casein sharply increased growth, which opened the seductive prospect of identifying new growth factors. The explanation was largely, as noted, one of nutrient balances. In many experiments, supplements of glycine, valine, and isoleucine permitted growth in the presence of high BIZ (or low BI2 + folic acid) + thiamine. As the temperature was raised, additional amino acids were required, eg., phenylalanine, tryptophan, cystine, and lysine, for good (though not maximal) growth.

The basal medium of the last experiment, with its supplements, epitomizes the main features of the present complicated position (Table VI) .

The experiments chosen for tabulation illustrate

TABLE 11. Enhanced B, and thiamine requirements. I n this and later experiments growth was expressed as opti-

cal density.

Coilcentration in final medium 260 Cl34O>, CL38097

Thiamine 0.1 mg. % ( 1) So B,, 0 0 0 i ( 2 ) 3) B,, ” 0:I30lpg;,% 0.003 0.58 0.76 0

1.15 1.47 0 ” 0.01 ” 1.55 1.93 0 ” 0.03 ” 1.76 2.28 0 ” 0.1 ” 1.78 2.46 0 ” 0.3 ” 2.09 2.42 0 ” 1.0 ” 2.22 2.51 0

( 4) ( 5) ( 6) ( 7 ) ( 8 )

” 3.0 ” 2.14 2.71 0.20 ” 10.0 ” 2.55 2.96 0.13

i 9) (10)

(11) No thiamine 1.90 2.74 0 (12) Thiamine HC1 0.001 mg. % 2.13 2.76 0 -

0.004 ” 2.10 2.86 0 0.02 ” 2.11 2.70 0 0.1 ” 2.04 2.66 0 0.3 ” 2.08 2.62 0 1.0 ” 2.17 2.62 0.82

(13) :: (14) ,, (15) ,, (16) ,) (17)

The basal mediuin was similar to that in Table I. ilt this stage large inocula were used.

In the light of later experiments, “34’)’ probably was ef- fectivcly about 35.2-36.0”, and ‘(38” ” about 38.0-36.8”.

The basal medium in this early stage was supplemented with DL-valine, glycine, and DL-lysine, and a large inocu- luni was used. Inability to demonstrate the thiamine re- quirenieiit in this experiment at 26” and 34” is attributable to carryover from the large inoculum. The concentration of thiamiue HCl permitting half -maximal growth a t ordiiiary tmiperatores is 0.2 pg. %.

some of the main advances: ( a ) Enhanced requirements for BIZ and thiamine

(Table 11). ( b ) The “accordion” design in the dissection of the

active components in chrysomonad suspension or other crude sources of temperature factors (Table 111). The use of “complete supplement” illustrates the means employed to differentiate between known and unknown factors. Experiments like this one identified most of the presently recognized temperature factors. In this experiment it will be noted that something in the basal medium appeared to be liniit- ing, and that additional factors were supplied both in the chrysomonad suspension and in the complete supplement.

( c ) Enhanced metal requirements. The additional active materials identified as an immediate follow-up of the experiment of Table I11 proved to be, predominantly, the metals shown in Table IV, along with threonine and extra isoleucine.

( d ) B12-folic interactions; sparing effects, mainly with purines and pyrimidines (Table V ) .

( e ) Chrysomonad suspension as a source of temperature factors for the last temperature range (36.7- 37.0’) accessible with the present basal medium

262 GROWING Ochromonas ABOVE 35" C.

TABLE 111. Dissection c4f clirysonioiiail-suspensioii factors by :iii ' accordion " expcrimcnt. The basal medium (similar to T:ible I) was supplemented tvitli valine, lysine, glycine, swine, isoleucine, tryptophan, cystinc, fo lk acid, folinic acid, thy-

mine, high B,, (cyanocobalainiii 2.0 pg. %), and adenosine.

35.9" 36.3" 36.7" C.S. No. 9 C.S. No. 9 C.S. s o . 9

4.0 m1./100 4.0 m1./100 4.0 m1./100 Medium C (basal iiiediuiiij _ _ _ ____.____-

( 1) Idediuni I 0.2X 0.4X 0.8X ( 31

( 2) "

0 0 0

( 4) " 1.2x 0 ( 5) " 1.6X 0

A. Chrysominail suspeiisioii 2.5 nil. / lo0 added to basal niecliuni

____ ( 6) Medium IA 0.2X 0.23

0.4X 0.12 0.8X 0.12 1.2x 0.1 7 .a 0.8

( 'i) ;: ( ,, ( 9, 3 ,

(10)

C.S. = ccnipletc snpplrineiit (see imtes)

11. (basal nwdiuui + cliryso monad suspension 2.0 r n l . / l i ) O )

(16) Mediurri I1 0.2X (17) " 0.4X

(19j " 1.2x (20) " l.6X

A. Complete t,upplenient No. 9 (see notes helou) 4.0 m1./100

(21) Medium ITA 0.2X (22) 0.4X (23) " 0.8X (24) " 1.2x (25) " 1.6X

Medium I1 + ' ' B "*

(26) Medium IIB 0.2X ~~ - -

(27) " 0.4X (28) " 0.8X (29) " 1.2X (30) " 1.GX

0.82 0.71 1.34 1.35 2.25

0 0 0 0 0

0 0 0 0 0

35.9" 86.3" Vit. S o . 12 Vit. No. 12

1.0 m1./100 + 1.0 m1./100 + amiiio acids No. 6 nniiiio acids No. 6

1 .O ni1./100 1.0 n11./100

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

36.7" Vit. Xo. 12

1.0 ni1./100 + amino acids No. 6

1.0 m1./100 - _ _ _ - _ _ _ ~

0.11 0.35 0 0 0 0 0.18 0 0 0 0 0 2.35 2.42 0 0.56 0 0 1.24 ".ti2 0 0 0 0 0.2G 1.41 0 0 0 0

35.9" 36.3" 36.7"

1.19 1.18 0 0 (I 0 1.71 1.78 0 0.18 0 0 2.26 2.46 (J.4i 2.42 0 0

0.99 2.68 0 1.09 0 0 2.67 2.66 1.09 2.52 0 0.28

35.9" 36.3" 36.7"

1.65 1.28 0 0 0 0 2.03 2.15 0 0 0 0 1.42 1.68 0 0.46 0 0 2.82 2.76 -.-li 2.58 0 1.04 3.11 2.81 1.50 2.03 0.56 1.83

.> '>

* "B" = Purine mix 0.2 1111./100; pyriniiiline mix 0.2 1111./100; amiiio acids X o . 6 1.0 m1./100 Also, a1k.-liyd. yeast iiucleic acid 0.01%, :uid ticid-liyd. DNA (see notcs below).

0.002%. . _ _ _ ~ ______

" C o i n p k t r a i r p p k n i ~ n t ."To. 9 " is iiitriidecl :is a conveniently eoiiipwliwsivc mixture of known factors; composition i n final medium a t full strengili (irhrii used a t the level of 5.0 n11./100 nil.) :

Gelatin liyrlrc~lysnte' 0.4 g. Alkali-li~~llolyz(.d yeast iiucleic acid? 0.02 g.

DL-Methionii e 5.0 mg. Vitninins S o . 11' 1.0 rn1./100 Stablr growth factors m.iy be iiitroduced with the Iiydrolysatcs; tiley seein to Iw more :ihuiiilant in casein liydrolysxte

' Prepared by Rutoclaviiig gelatin with 5% (w/v) H,SO, f J r 8 hr. a t 118-121", neutralizing while hot to pH 2.8-3.2 with

'Prepared by suspending 6.0 g. yeast iiucleic a c i d in 120 1111. H,O, adding SaOH to pII 9.0, and steaming 1 hr. Prepared by suspending (i.0 g. DNA (from hcrritig) in 120 IUI. RIO, adding H,SO, to pH 1.5-2.5, and steaming 2 Iirs. 1.0 nil. contains:

L-Tryptophai I 5.0 nig. Acid-liyclrolyzcd DSAS 0.01 g.

as iiitlicated by unliublislled work with other protists, ~ . g . , Liutrrrlln //1o110(iHtOyrll(,.s and SfrPptococcrts tlielmophilus.

barium Iipdrosidc, :iiid filtering off tlic RaSO,.

Tlii:iinine FIC'I 0.1 mg. Pjritloxnl * HC'1 0.02 mg. B,, 0.1 pg. Sirotiiiic acid (1.1 mg. liiositol 1.0 mg. Bic.titi 0.5 pg.

Pyridox:iiiiiiie.'LH(:I C .02 mg. p-Hydroxybeiizoir acid 0.01 mg. Folic acitl 0.004 tug.

C:i p i i t o t l i c ~ i i a t c (1.1 mg. Putrrsciiie Il,4-1li:11iiiiio- 0.04 lug. Iipoic acid (tll-tliioctic acid) 0.004 iiig.

('lioliiic H, citrxtc 1.5 nig. butane) * 2 HCl Ctrovoriutn fnctor (1,ruko- 0.2 pg. S a rihoflaviii PO, (,.()I nig. p-Aminobcnzoic acid 0.01 nig. vorin, the ('a * 51I,O salt)

Tlir tliioctie ac id and I,eukovorh \rere gifts of the Alrieric 111 C'y:iti:iii>icl ('oin1):\11y.

GROWING Ochromonas ABOVE 35” C . 263

(Table VIII) : amino acid interactions (Tables VI and VI I ) .

DISCUSSION

B,,-folk relations. “Folic acid” is used here in a general sense to denote all pteridines conjugated with p-aminobenzoic acid and one or more glutamic acid residues. “Folic acid” in our experiments was pteroyl- glutamic acid.

The “ordinary”-the base-line or low-temperature, thermally-insensitive BIZ requirement-of Ochromonas seems to differ from the thermally enhanced-“ther- ma1”-requirement in that the ordinary requirement cannot be bypassed, while the thermal portion of the total BI2 requirement is spared by methionine (but not bypassed); methionine in turn is spared (but not bypassed) by cystine. We found all but one of several commercial samples of “C.P.” L-glutamic acid to be significantly contaminated with cystine, hence our experiments with glutamic acid without cystine have limited quantitative value.

The nature of the thermal B12 requirement may be quite different from the ordinary requirement. Inabil- ity to completely bypass the ordinary B12 requirement of Ochromonas and Euglena is a reminder that an essential function of B I y is unknown(f7). If natural materials contain an unknown B,?-bypass factor, it must occur throughout nature much as does B12 itself, e.g., it must be absent, as is BIZ, from yeast and higher plants, (as indicated by assays with Euglena, Ochro- inonas, rat, and chick). Chemical identification, not biological activity, tells whether B I Z activity in natural materials is attributable to BIZ or to a B,?-bypass factor. With no clear discrepancies between Ochro- wzonas and metazoan assays. there is little practical incentive to identify chemically the BIZ activity in non-commercial crudes. The same applies to the fail- ure to bypass the B,? requirement of Euglena, but here possible discrepancies with metazoan activity are overshadowed by Euglena’s response to members of

Piir i i ic. m t i . 1.0 nil. contains (mg. ) : Adcninc 0.5 Hypoxantliine 0.5 ddenosine 1.0 Gnanine HCl 0.5 Yeast adenglic acid 1.2 Gumosine 1.0

I ’ i ~ r ~ ~ i i z d ~ n ~ m u . 1.0 nil. contains (nig.) :

TARJIE IV. Metals as temperature factors. The basal medium was almost the same as tha t i n Table V I except

for the metal concentrations being lower.

34” 35.5” 35.9”

f 1) Nedium 0.4X i 2) ” 0.8X

f 4) ” 1.6X ( 3 ) ” 1.2x

( 5 j *’ 2 . 0 ~

( 6) Fc 0.1 nig.% ( 7) ” 0.25 ”

( 8) ” 0.4 ”

( 9) MII 0.01 mg. % (10) ” 0.025 ”

(11) ” 0.04 ”

(12) Ca 1.0 mg.% (13) ” 3.0 ”

(14) ” 10.0 ”

0.1 ”

(18) Zn 0.01 rng. ’70 (19 ) ” 0.02 ”

(20) ” 0.05 ”

(21 ) “ l i i s ” 0.0170 HE1)TA 0.01%

1.79 2.47 2.50 0.68 0

2.50 2.50 0 2.35 2.23 2.36 2.40 2.60 2.58 2.54 2.52 2.52 2.58 2.50 2.59

2.50

1.28 2.47 0 0 0 0 0 0 0 2.54 0 2.56 2.38 2.42 2.62 2.52 2.50 2.61 2.48 2.61

2.54

0.04 0 0 0 0 0 0 0

0 0 0

0 0 1.52 0 0 0.04

0 0.13 2.56

2.64

Tlic tenil)er;rtiire-iiiduertl imbalance in the basal nicclium is shown in the ‘ ‘accordion” portion of the experiment (media 1 through 5). The likelihood of imbalance is further borne out by the growth-promoting effects of the eliclating agents “Bis” [N,N’-bis(2-hydrosyethyl)glycine~ arid HE- IITA (liydroxyethyl ethple~iedia.niiiie triaectie ncid) (medium 21 j ; by reducing the effective concentrations of otherwise toxic chelwtahle metals, the chelators permitted growth. In this espcriinent, Fe and Mi1 appeared to upset the metal balance. A high concentration of Ca (medium 14) gave the siinie growth response as did much lower concentrations of Z n ; further experiments were needed to decide vhether 211 acted by displacing Ca from a metal complex by ni:iss :ic- tion, or whether bot,li Zn and Ca were temperature factors. l in te r eq’erimerits made it likely that Zn was the primary reyuirtwicnt ; i t was also more effective th:in Cn or Mg in correcting the Fe and Mn imbalances. Tlic resnlts of t.hese cxperinients are embodied in the basal niediuni in Table VI.

“Bis” and HEDTA were used because their chelntes witli hfg, Ca, and Fe are more soluble than tlic XDTA or citrat.e complexes, and it was desirable to have in the basal nicdiuni an excess of cliclating capacity in case the metal reqiiircnients continued t o rise steeply witli tcniperature. Choice of IL low concentration of Zn rather than a high con- ceiitr:ition of Ca + Mg had the furt,licr advantage of lcav- ing a 1:irgt.r reserve of solubilixing rapacity.

Guaiiylic acid 1.2 Xanthinc 0.5 I nosiiie 1.0 Xantliosine 1 .O

Thymine 0.5 Thyniidinc 1.0 Uracil 0.5 Uridine 1.0 Orotic acid 0.4

I)I,-Alnninc 0.04 LGlutamic acid 0.1 DL-Methionine 0.006 DL-Tlireonine 0.01 L-Arginine (free base) 0.03 L-Histidine (free base) 0.02 DL-Phenylalanine 0.004 DL-Tryptophan 0.005

Amino acids S o . 6-At “ fu l l” concentration (g./100 ml. in the final medium) Jrhen used a t the levcl of 5.0 m1./100 nil.

DL-Aspartic ncid 0.05 DL-Isoleucine 0.005 L-Proline 0.004 L-Tyrosine 0.004 Glycinc 0.05 DL-Lysi11e * HC1 0.045 1)L-Serine 0.01 UL-Valine 0.005

Wliilr usc of L-amino acids made by resolution of synthetic amino acids might seem to be more natural than use of the r~iccniic products, resolution of these coninicrcial products is done cnzgniatically, and it is not yet established that the en- zyme prepparations do not introduce the very growth factor emtarninations that the m e of synthetic amino acids was in- tcwlcd to avoid. We have therefore continued t o use racemic mixtures in experinicnts designed to rcveal growth factors.

Tlir ehrysonion:id suspension had an optical density of 3.1.

264 GROWING Ochromonas ABOVE 35’ C.

TABLE V. B,,-folic interactions; sparing effects by deoxyguanosine and thymine. The basal medium was B,,-free ; otherwise it was rubstantially the same as tha t in the experiments represented by Tables 111 and IV.

A B C D E Deoxy-

guanosine B + thymine C + folic acid Folic acid No Add 1.0 mg. % 1.0 mg. VO 0.04 mg. 70 0.04 mg. 70

~

( 1 ) No R,, (2) B,, 0.0001 pg,.,”/”

(4) ” 0.005 ”

(5) ” 0.01 ”

( 7 ) 1.0 (8) ” 5.0

(3) ” 0.00(15

(6) :: 0.1

(1) No B,,

( 3 ) 0.0005 (2) 0.0001 pg;,%

(7) :; 1.0 ”

(4) ;; 0.002 1’

(5) 0.01 ”

(6) ” 0.1

(8) 5.0

(1) No Em ( 2 ) 0.000lpg;,%

(5) 1; 0.01 ”

0.000 5

(6) 0.1 (7) ” 1.0 (8) ” 5.0 ”

0.26 0.26 0.30 0.56 1.00 1.90 2.67 2.56

0.15 0.20 0.35 0.46 0.92 0.96 1.00 (1.94

0 0 0 0 0.08 0 0 0

0.42 0.42 0.52 0.72 1.10 1.90 2.56 2.64

0.25 0.26 0.42 0.26 1.40 2.42 1.91 1.29

0 0 0 0 0 0 0 0.07

35.5” 0.50 0.52 0.66 0.94 1.32 2.34 2.62 2.68

35.9” 0.30 0.38 0.54 0.80 1.50 2.47 2.04 1.74

36.3 0 0 0 0 0.12 0 1.98 0.54

0.45 0.66 0.75 0.94 1.51 2.14 2.63 2.66

0.40 0.43 0.66 1.00 1.44 2.52 2.70 2.71

0 0 0 0 0.52 0.72 0.08 0.20

0 0.10 0.12 0.34 0.62 1.88 1.84 0

0 0 0.15 0 0.25 0.12 0 0

0 0 0 0 0 0 0 0

The main conclusion from several follow-up experiments was tha t the puriues and pyrimidines had the broad specificity to be expected if they were acting simultaneously as bypass factors for f o l k acid and Bl,. Considerable latitude was permitted. Thus when thymine was replaced by thymidine, dcoxyguanosine could be replaced, at least partly, by free purines and purine ribosides such as guanosine or adenosine. It is likely tha t at slightly higher temperatures there are additional lesions in purine and pyrim, dine metabolism, since there was sparing of purine and pyrimidine deoxynucleosides by free purines and pyrimidines. The basal medium in Table VI embodies some of these results. Wheu media a re improved in respect to metals, amino acids, and known vitamins besides B,, and fo lk acid, it will obviously be desirable to go over this ground. Folic acid, whvn supplied alone, upsets a balance whose nature is uot yet clear. The high blanks are attributable to use of the old non-synthetic inoculation broth because a vigorous culturc in chemically defiued medium was not available.

the BIZ family (pseudo EI2’s) which are inactive for metazoa.

To decide, then, whelher one or several lesions underlies the thermal Blr and folk requirements, it is necessary to examine the folic requirement and the sparing of the thermal Blz requirement by folic acid; the lesion responsible for the ordinary B l a requirement is yet obscure. This examination may cast some light on the biogenesis of Crithidia factor, which behaves in Crithidia fasciculata as if folic acid were a precursor (3 5 ) . To see whether the folic requirement reflects a crippled synthesis of folk acid, we are now attempting to measure the folic content of Ochromonas as a function of temperature; if a folic requirement appears a t the temperatur? where the folic content of the cells falls to zero, the crippled-synthesis hypo- thesis would be supported. Present microbiological assays for folic acid are, unfortunately, of dubious validity when applied to intact organisms. Another approach is to poison Ockromonas with sulfonamides

or anti-folics, thereby inducing directly or indirectly a need for exogenous folic acid. Would B12 then act as a bypassing or at least a sparing factor for this antimetabolite-induced kind of folic requirement as it does for the thermal kind? Also do the folic and Crithidia-factor contents of Ochromonas run parallel with variations in temperature?

For some protists, B12 is a sparing factor for folic acid(30). I t is plausible that folic acid, in its familiar role as a car- rier of I-carbon units, figures in the synthesis of BIZ; indeed, it is suggested that in the synthesis of the benzimidazole part of BIZ, the ring is closed by incor- poration of a 1-carbon unit into a precursor such as o-phenylenediamine ( 19), similar to the insertion of the I-carbon unit which closes the purine ring. Re- ciprocally, BIZ may be essential for the de novo for- mation of the just-postulated 1-carbon unit entering folk acid. Perhaps folic acid triggers its own synthesis, as in the example of starch and glycogen. In

The folic acid-B12 link is obscure.

GROWING Ochromonas ABOVE 35” C. 265

portance of impaired folic function in the appearance of responses to &‘cine, serine, leucine, and lysine, nor have the responses to phenylalanine, tryptophan,

Weight f o r Weight for and cystine been studied in detail. The thermal interactions between leucine, valine, isoleucine, and thre- 100 nil. final

onine (Table VII) noted resemble observations on bacteria( 11) and rats(3).

HEDTA ( ” j 0.02 g. 8.0 g. Amino acid imbalances unexpectedly appeared at KH,PO, 0.025 g. 10.0 g. intermediate temperatures (e.g., 34”) ; the complexity

of this situation may be inferred from the fact that CaCO:, 0.015 g. 6.0 g. L-Glutamic acid1 0.2 g. 80.0 g . MgCO, (basic) 0.1 g. 40.0 g. media such as those used in Table VII supported Siicrose 1.0 g. 400.og. dense growth a t lower and higher temperatures but L-Argiiiinc FICl 0.05 g. 20.0 g. not at intermediate temperatures. This situation DTJ-Methionine 0.05 g. 20.0 g. seems most readily induced by threonine, homoserine,

and a-aminobutyrate, which are closely related meta- YH,HCO, 0.04 g. 16.0 g. Adenosiiic 1.0 nig. 0.4 g. Thymidine 1.0 nig. 0.4g. bolically. The results in Table V I I were consistent Thymine 2.0 mg. 0.8 g. with several preceding and later experiments.

rate \vith penta- In the light of later experiments, the effectiveness of erythritol chrysomonad suspension at 36.7” k 0.2 (e.g., as in

Table VIII) seems to depend on multiple known fac- Thiamine HCP 2.0 mg. 0.8 g. Biotin 5.0 fig. 0.2 g. of 1: 100 triturate Folic acidS 10.0 pg. 0.4g. ” ” 1 9 tors involving an altered balance of amino acids and Folinic acid3 10.0 fig. 0.4 g. ” ” vitamins already present in the basal medium.

Mg, Fe, Mn, and Zn in cliiiin). combination, along with the aforementioned amino Ilry mix 1.64 g . TXystine 2.0 nlg. acids and vitamins, duplicated the effectiveness of the MgSO,, 7H,O 0.06 g. Td-Lysine HC1 0.04 g. Ca 1.0 mg. DL-Phenylalnlline 8.0 mg. chrysomonad-suspension supplement a t the lower tem- F e 0.1 mg. L-Tryptophan 4.0 mg. peratures (35.4-36.1 ”) which had been the critical

ones in early work (Table IV). Reproducibility 211 0.1 mg. L- Valinc 5.0 nig. Mn 0.02 mg. L-Isoleucine 4.0 mg. ‘ ‘ Metals 45A ’ ’ 0.2 mg. Pyromellitic acid 0.1 g. hinged on full availability of iron; the procedure for

The additional nirtals besides those in the dry-mix portion preparing a stable stock solution of Fe (Table VI), of t,he basal medium represent tcnipcrature factors. Pyre- adopted from studies on the so]ubilization of ca and mellitic acid served t o lower the pH to the desired value (4.8-5.2). The isoleucine (California Foundation) was allo- Be(s9) , made it to prepare fresh solutions free. F e : 1.0 ml. of the stock solution contained Fc (as of Fe frequently or to prepare the solutions with high FeSO, * (NH,), SO, - 6H,O) 1.0 mg. + sulfosalicylic acid 2.0 mg. ; the solution assumes a deep port-wine color. etais is concentrations of acid. 468” when used at ‘‘full strength” (2.0 mg.yo) yields tlie The concentrations of histidine and arginine are fo11OWillg concentrations (me.%) in the final medium: Fc 0.2, Zn 0.1, Mn 0.005, Cu 0.008, Co 0.01, B 0.01, Mo 0.005, and v 0.001. cinic acid has been favorable. The results do not now

The cornposition of “vitamins X o . 1 2 ” and “complete lend themselves to a simple presentation and inter- supplement” (“C.S.”) No. 9 is given in the notes to Table pretation and it best to postpone a detailed 111. “Ami?zo acids No. 6 ” when used a t t.lie level in the Tahlc, yields the following concentrations (%) i n the final treatment.

It is tempting to equate nicdium as shown in Table 111. It is convenient to keep this as a dry mix. The stock sola-

tion is made up a t 20X final concentration, pH 4.8-6. Use of temperature factors with agents that help maintain arginine and histidine as free bases appreciably reduces the mitochondria1 integrity (criterion, the persistence of

oxidative phosphorylation), e.g., Mg(32), Mn( 14,28), anlourit of alkali needed t o adjust the pH. Cystine (or cys- trine) is :ilways added separately to final media; i t is convenient t o store cystine as a 1% solution (prepared by dis- and fatty acids(37). HOW Blz protects mitochondria SolVillg the cystine in a Small volunle Of concentrated KOH, is uncertain; some suppose that protection by B,? then diluting to 1%).

against thyrotoxicosis in the rat results from a partial all, the Blz-fo1ic system takes on the aspect of a self- restoration of the uncoupling of oxidative phosphoryla- regulating-a positive feedback-device, easily upset tion caused by thyroxine( 3 1 ) . Ungley’s omission of by temperature shifts. Investigations of pernicious BIZ as a temperature or stress factor in his critical anemia have emphasized, but done little to elucidate, review of BIZ in therapy(41) attests to the primitive the folic-Blz link. state of knowledge about this aspect of BIZ.

Amino acids. Folic acid participates in the synthe- Comparative biochemistry of temperature factors; sis of serine and glycine, methionine, histidine, leucine, fever. The issue of whether metazoa and Ochromonas and perhaps lysine. We have not assessed the im- need the same temperature factors may hang on anal-

TABLE VI. Final basal medium for growth at 36.7”.

Dry-mix portion of t h e basal medium. The ingredients are calculated for 40 liters of final medium.

40 liters of medium final medium

d ‘Ris, , (see TableIV, 0.02 g. 8.0 g. noles)

L-Histidinr I-ICl. H,O 0.1 g. 40.0 g.

H, 2 2.0 pg. 0.8 g. of 1:1,000 tritu-

- complete 1):isal meilium (aniounts/100 nil. of final me- Metals and substrates.

higher than in the original medium, also a t times SUC-

Mitochondria and stress.

266 GROWING Ochromonas ABOVE 35" C.

TdBLE VII. Amino acid internetioils. Basal medium a3 in Table VI.

-30-31"- ,----34"---, 7 3 5 . 5 ' - 7 3 5 . 9 " - - 7 7 3 6 . 3 " -

conc., n1g. '/4

f 1) KO additions

( 2) L-Isolrueiw 2.0 10.0

(14) 20.0

(( 15 ) I)l-a-amiiiobnt~rie 2.0

10.0 (16, ( 1 7 ) 20.0 (1 X ) T,-T,curiiie 1.0

1)L-Tlirconiiie 1.0 ( 19 ) T~-Lc~uciiie 4.0

I)T,-Thrronine 4.0

acid

2.5 2.4 2.5

2.5 2.4 2.4 2.5 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.5 2.4 2.5 2.4 2.4 2.4 2.5 2.4 2.5 2.5 2.4 2.4 2.4 2.4 2.4 2.4 2.5 2.4 2.4 2.5 2.4 2.5 2.4 2.4 2.4 2.4 2.4

2.4 2.4 2.4

2.4 2.4 2.4 2.4 2.4 2.4

2.4 2.4 2.4

2.4 2.4 2.4

2.4 2.3 2.4 2.3 2.4 2.3 2.4 2.4 2.4 2.4 2.4 2.4 0 0 0 0 0 0 0 0 0 0 0 0 2.8 0 0 0

0 1.1

0 1.7 0 0.42

0 2.1

0 2.3

yses of the special supplements permitting the growth of rats fed diets supplemented with thyro-active sub- stsnces. Superficially there is yet little convergence with Ochromonas temperature f ac txs aside from BIZ and perhaps thiamine. The basal diet for these animal studies contained 347% casein and water-soluble vitamins a t 10 X the level required by the normal rat( 13). The protective factors in liver were replace- able in part by unsaturated fatty acids, steroids (cholesterol or bile acids), and extra casein(36). This divergence from Ochromonas may be less than now appears: the Ochromonas factors identified to date were chosen from among water-soluble, thermostable supplements: the rat diets were not so restricted. In- teractions of Ochromonas temperature factors with fat-soluble or thermolabile factors, or a need for such factors, must be considered in attempts to grow Ochromonas a t still higher temperatures: chrysomonad suspension and the like may not be indefinitely re- placeable by salts and water-soluble, thermostable compounds. The stimulation noted by Ford with Tween 80 and inositol may hint at a convergence with the rat results. Another provocative animal result not yet paralleled with Ochromonas is the activity of bromide in protecting mice and chicks fed thyro- active materials( 6,25).

Thyroid and dinitrophenol fevers still offer no clear guide to Ochromonas temperature factors-and vice zwrsa. One wonders, nevertheless, about parallels to

2.4 0.82 2.02 2.4 1.6 2.5 2.4 0.22 0.12 0.86 2.4 1.7 1.8 2.5 2.0 2.5 2.4 0 0.86 2.10 2.4 1.3 2.3 2.5 2.0 2.4 2.5 0.18 1.3 2.2 2.4 1.7 2.5 2.5 2.0 2.4 2.4 0.78 1.9 2.4 2.4 1.4 2.4 2.5 1.8 2.4 2.6 0.22 1.2 2.3 2.4 1.4 1.9 2.4 1.9 2.5 2.5 0.38 1.4 2.3 0 CJ 0.44 0.14 0 0 0 0 0 0 0.12 0 0.22 0.68 0 0.1 1.5 0 0 0 0.33 0 0.44 1.5 0 0.1 0.22 0 0 0 0.86 0.14 0.44 1.4 0 0 0.4 0 0 0

1.2 0 0.12 1.0 0 0 0.14 0 0 0 1.5 0 0.16 1.6 0 0.52 0.30 0 0 0 1.3 0 0.08 0.84 0 0.1 0 0 0 0 1.8 0 0.06 0 0 0 0.18 0 0 0

1.9 0.36 1.4 1.4 0.14 0.32 2.3 0.52 2.0 2.4

2.2 0.08 1.2 1.6 - 0 1.5 1.4 2.2 2.5 2.4 0.74 1.3 1.1 0 0.5 2.5 1.0 2.3 2.4

2.5 0.14 1.3 1.3 0.14 0.68 2.5 1.1 2.5 2.6

2.4 0.2 1.4 2.2 0.36 1.7 2.6 1.2 2.5 2.6

other forms of temperature stress or fever in metazoa. Where tissue destruction is severe, i.e., in extensive burns, fever is usual(7); one might re-phrase this to "where mitochondria1 damage is extensive", as suggested by recent studies( 15 ) .

The literature on supportive measures for metazoa in fever holds few concrete suggestions for Ochromonas temperature studies. A constant in post-traumatic fever is negative nitrogen balance ; the countermeas- ures are unclear. Thus Cuthbertson (9) : " . . . despite substantial increase in the rate of increase in the intake of a diet rich in proteins and calories by patients with moderate or serious injuries, a negative nitrogen balance existed at the height of the catabolic period." A sampling of textbooks of mammalian physiology reveals a remarkable vagueness about supportive measures: e.g., Best and Taylor(4) cite work recom- mending a high caloric diet with a liberal intake of protein; Houssay( 24) warns, on the other hand, that if sufficient carbohydrate is not ingested, ketosis results. ( A sidelight on the damage inflicted by the malarial parasite is Houssay's statement that heat production in malaria may reach 5 X the normal out- put-in general, metabolism rises 13% for every ' Centigrade above normal.) Other texts are even vaguer; facts from experiment are few(38).

As with folic acid, and its possible thermally excessive diversion into Crithidia factor, do the enhanced amino acid requirements in Ochromonas reflect blocks

GROWING Ochromonas ABOVE 35" C. 267

w c

300t0 " c ?

j o o o o

in synthesis or, instead, excessive diversion of amino acids into other metabolites? Information is available for tryptophan in man: Dalgliesh( 10) notes hydroxy- kynurenine excretion in many patients in various dis- ease states; the common factor, he supposes, is the increased rate of breakdown of body proteins in fever. Accordingly, Ochromonas culture fluids should be ex- amined for breakdown products of tryptophan, and tryptophan-sparing by nicotinic acid should be looked into.

The thermal thiamine requirement in Ochromonas adds interest to the report that rats and mice(33) and chicks (34) subjected to high environmental temperatures have an enhanced thiamine requirement. Little effort seems to have been made to repeat this work: in another laboratory, temperature did not affect the thiamine requirement of rats(22). In view of the multiple nutrients required to prevent thyrotoxicosis with its accompanying fever in rats, it is not surprising that it is not simple to demonstrate thiamine to be a temperature factor for metazoa.

The steep rise in metal requirements with temperature offers the hope that inorganic requirements will be uncovered beyond what is permitted at lower temperatures by existing technology. The magnitude of the thermal BIZ requirement implies that cobalt as an essential element could well have been identified first as an index of the activity of an Ochromonas temperature factor, i e . , BI2 .

Radiation injury. Thermal derangement in Ochro- monas of the folic-B,? system, even without antece- dent derangements in amino acid metabolism, might induce the protistan equivalent of a negative nitrogen balance; it would also halt DNA and RNA synthesis. The constant, sensitive sequel of radiation damage. whatever the organism, is arrest of DNA synthesis ( 2 1 ) , in vertebrates-at least in warm-blooded ones -fever is another sequel. Clues to Ochromonas temperature factors from radiation research are scant. Inasmuch that radiation energy, for the most part, is eventually degraded to heat, the invariant element in radiation damage, as distinguished from the random events surrounding the absorption of high-energy radiation, might be thermal. Radiation injury might be exhibited in part through the appearance of temperature factors. There are resemblances between thermal and ultraviolet damage to Didinium( 20).

One may speculate that highly resonating molecules such as folk acid and BI? (or their presumably less- stable coenzyme forms) may be intrinsically vulner- able to low-quantum energy radiation as well as to thermal damage. Factors for hematopoietic regeneration after irradiation are assumed to exist, but identification seems remote ( 5 ) .

For survival of Esrherichia roli after X- and y-irra-

268 GROWING Ochromonas AmvE 35" C .

diation, glutamic acid, uracil, guanine, plus several amino acids, were clearly favorable; there was some evidence that inorganic nutrients played a part, but in the preliminary report by Stapleton et aZ. (40) , a clear distinction was not drawn between the concentration of inorganic nutrients needed for ordinary growth and the concentrations needed for restorative effects. In a continuation of this line of experimen- tation, guanine + uracil + glutamic-aspartic acid + phenylalanine-tyrosine + tryptophan restored growth and DNA production ( 12 ) . Krebs-cycle components have been claimed to restore the growth and DNA production of ultraviolet-inactivated bacteria ( 2 3) . Similar studies of protozoa seem absent from the literature. The voluminous, predominantly biophysical literature on temperature injury has hardly illumi- nated mechanisms of injury(42).

Concluding remarks. Comparative studies with other protists, notably those requiring B1? or folk acid, might answer some of the questions asked here. Euglena gracilis, for example, offers interesting possi- bilities. Since the z strain and others of this type are permeable to a wide variety of nutrients, and their Bla pattern diverges radically from that of Ochro- monas, its temperature factors, if they include BIZ,

should tell more about why B12 is prone to be a temperature factor. Added theoretical interest attends this kind of Euglena because of its susceptibility to irreversible bleaching by heat.

In interpreting these comparative studies, the entire pattern of a requirement must be taken into account. Certain soil bacteria, for example, seem to respond to precisely the same members of the BIZ family which are active for Ochromonas and vertebrates, but these bacteria differ in that their Blr requirement is not spared by methionhe( 18).

I t may be that inocula grown at elevated temperatures will support elevated temperature growth where room-temperature inocula will not. Such adaptation experiments will be more attractive when we no longer have leads to additional factors whFh can be identified with inocula grown a t room temperature.

The problem of temperature factors for Ochro- monas, as we have become aware of cognate problems ranging from thermophilic bacilli to fever in mammals, looms immensely greater than at the outset of this study-and Ford's medium ( 16) still challenges us. The whole enterprise has a t least been valuable for casting a slender shaft of light into vast expanses of ignorance.

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Nutrition of a Hemoflagellate (Leishmania tarentolae) Having an Interchangeable Requirement for Choline or Pyridoxal.

WILLIAM TRACER Rockefeller Institute for Medical Research, New York 21, New York

SUMMARY. Leptomonads of Leishmania tarentolae were grown continuously in a defined medium containing: inorganic salts, glucose, hemin, 1 7 amino acids, purines and pyrimidines, and a mixture of vitamins of the B group. In this medium the population of organisms reached about 20 to 50 million per ml. after 1 week at 27" C. Only slightly better growth occurred in a partially defined medium containing bovine plasma fraction V. In earlier experiments, however, omission of the plasma fraction resulted in decreased growth, and under these circumstances cholesterol or lecithin had growth-stimulating effects. In later experiments in the fully-defined medium no effect of these lipids could be found. The leptomonads were shown to require a t least the following substances: inorganic salts; a source of purines and pyrimidines; tryptophan and the nine other amino acids essential for the growth of rats, glutamic acid, tyrosine, proline, serine, one or more of the group alanine, glycine and aspartic acid ; folic acid, biotin, pantothenic acid, nicotinamide, riboflavin, thiamine, and either pyridoxine plus choline or pyridoxal or pyridoxamine. Choline at 2 X 1OP M gave optimal growth in the presence of pyridoxine a t 1 X 1 O P M. I n a medium with a suboptimal concentration of choline (0.4 X 10.' M) the leptomonads grew through nine transfers but they were mostly somewhat rounded and aflagellete.

HE hemoflagellates provide an excellent example of an evolutionary series of parasitic protozoa,

from relatively simple forms restricted to the alimen- tary tract of insects to more complex forms having alternate developmental cycles in a vertebrate host and an invertebrate vector. The complexity of the nutritional requirements of these organisms seems to parallel in a general way the complexity of their life 'cycle. A hemoflagellate of insects has been cultured in a synthetic medium(4,S). The invertebrate stages of most hemoflagellates can be grown readily in media containing blood, and these stages of a few species

have recently been cultivated in a medium of partially known composition(2,3). The stages found in the vertebrate host, such as the blood stream forms of the African trypanosomes and the intracellular leish- manias of kala-azar, have not been reproduced in culture( 16,17) with the exception of Trypanosoma mega of the frog(l5). A study of the nutritional requirements of a hemoflagellate which might be considered intermediate between those restricted to invertebrates and those parasitizing warm-blooded vertebrates seemed of interest, and the parasite of lizards Leish- mania tarentolae was used for this purpose.

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