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I I

T H E J O U R N A L O F

P R O T O Z O O L O G Y Volume 3 Number 3

A U G U S T , 1 9 5 6 I ‘

J. PROTOZOOL., 3, 101-112 (1956).

A Sugar-Containing Basal Medium for Vitamin B13-Assay with Euglena; Application to Body Fluids“

S. H. HUTNER, MICHAEL K. BACHt Haskins Laboratories, New York 17, New York

and G. I. M. ROSS Westminster Medical School, London, S . W . 1, England

~

SUMMARY. An improved assay of vitamin B I ~ is described. The e strain of Euglena gracilis, which grows more vigorously than the bacillaris strain previously used, is recommended. The pattern of Blz specificity of the two strains appears to be the same. A new medium containing sucrose, aspartic acid, glutamic acid, and glycine at pH 3.6 is well buffered and allows luxuriant growth. Vigorous utilization of sugar appears to depend on readily available nitrogen and a Krebs-cycle component; these requirements are well met by aspartic acid (or asparagine) and glutamic acid. The proposed procedure is especially suitable for the measurement of BIZ in blood serum because rise in pH and precipitation of serum proteins during incubation are minimized. Like bacillaris, the z strain allows the distinction between “combined” and “uncombined” Blz in serum. Serum may be an appropriate test material to tell whether such phagotrophs as Peranema can better utilize bound forms of vitamins than can the related osmotrophs. Patterns of BIZ requirements and occur- rence are discussed as phylogenetic markers.

HE EUGLENA assay for vitamin B12 finds use where extreme sensitivity must join with fairly

narrow specificity. I t is the most sensitive of the BIZ

assay methods( 1 2 ) , detecting as little as 0.25 ppg./ml. by the technique described here. I t is not completely specific for vitamin B12: i t responds to some “pseudo- Bls’s” e x . Factor A. but these have not been shown

recommendation of the Committee on Growth of the- National Research Council ; and the Westminster Hospital.

t Present address: Department of Biochemistry, University of Wisconsin, Madison.

these materials. Furthermore, assays with this method permit differentiation between combined and uncombined BIZ: heating at 100°C. liberates BIZ from com-

101

102 VITAMIN Bl2-Ass~y WITH EUGLENA

bination and allows the measurement of both combined and uncombined B12; only uncombined B12 is measured in unheated fluid (2 1 ) . Because the basal medium is as acid as p H 3.6, bacterial contamination is no problem though fungi may grow when preliminary heating of assay tubes is not done, as in the measurement of uncombined BIZ.

The point of departure in the present study was the glutamic-malic acid medium (pH 3.3-3.7). The development of this medium was discussed elsewhere (1 1 ). Unforeseen advantages were that BI2 is most stable in acidic solution, and bound B12 in such materials as serum and gastric juice is efficiently freed by steaming at acidic pH’s. Also, the initial p H was below the isoelectric point of the bulk of the serum proteins; thus dilutions of sera remained clear during heating of the assay batch. But as the glutamic and malic acids were consumed, the p H of the basal medium rose, and in assays of serum the fluid became turbid as the isoelectric points of the proteins were approached. With profuse growth the p H rose as high as 7 or 8 and there was gross precipitation of protein, making turbidity readings of Euglena growth impos- sible( 2 1 ) . Then, because the basal medium was over- simple, there was a tendency for “drift” in assay results-high concentrations of serum usually gave dis- proportionately greater growth than did low concentrations. Also, owing to slow growth of Euglena gracilis var. bacillaris in the simple basal medium, it was usually necessary to wait 6 or 7 days before reading the results.

These dissatisfactions prompted collaborative study of the Euglena assay. The American authors studied the general method, e.g. modifications of the basal medium, and the British author studied its applications to assays of body fluids.

The principal improvements over the previous Eu- glena methods descri’bed in the present paper are ( a ) development of an easily standardized basal medium in the form of a dry mix; ( b ) elimination of excessive alkalinization during growth which caused precipitation of serum proteins; (c) selection of the more vigorous z strain of Euglena gracilis.

Recent reviews cover B12 and microorganisms( 3,6, 13,23), B12 and hematology (in which the use of Euglena is discussed in detail) ( 2 S ) , physiology of Euglenidae (9) , and earlier Euglena methods for BIZ assay( 8) . Besides those specifically cited here, the other papers in reference(26) should be consulted.

P.4RT I . COMPOSITION OF BASAL MEDIUM A N D SELECTION OF T H E z STRAIN OF

Euglena gracilis The methods followed those previously described

(10). Experimental media were distributed in 10-ml.

amounts in glass-capped “ lO-ml.” Kimble borosilicate flasks or in 25-ml. Kimble or Pyrex flasks. Experi- mental cultures were kept under a bank of 4 “warm- white” fluorescent lights. The temperature varied between 22°C. and 29°C. As the flasks were kept on glass trays, culture media spilled and retained on the trays often supported the growth of molds, which occasionally spread to the cultures. These molds were later eliminated by wetting down the trays with fungicide solution containing cetyldimethylbenzylammonium chloride 0.5 mg.% + butyl 9-hydroxybenzoate 10 mg.%. This solution was conveniently dispensed from a 300-fold concentrated stock solution in ethylene glycol and diluted with water as needed. I n experiments in which BI2 contamination was not critical, glassware was satisfactorily cleaned by boiling with any of several commercial detergents and rinsing in tap water. Ordinary distilled water prepared in a tin-lined still was used for the studies in New York.

Stock Cultures. In recent experiments, cultures were maintained for months in 125 X 16 mm. screw cap tubes on Trypticase 0.2% + agar 0.25% or, for slants, agar 1.6%. Cultures persist for many months at 15- 18°C. Cultures ( z strain) a t 6°C. in the dark in the basal medium survived erratically; the addition of a mixture of amino acids or of an acid hydrolysate of casein (General Biochemicals, Inc.) allowed excellent survival. These experiments provide justification for supplementing conservation media with digests of casein. Until more is known of the requirements for growth at low temperature, it appears unwise to store cultures below 10°C.

Incubation. Experimental cultures generally re- quired from 5-10 days; there was no set period.

Measurement of growth. Growth was measured as optical density (O.D.) in a Welch Densichron with a red-sensitive photo-tube and specially designed cuv- ette holder; most readings were made with 1 cm.-deep Pyrex cells. The instrument directly measures optical densities up to 4.0, but because of Rayleigh scattering the cultures were diluted to O.D. 1.0 or lower. Such dilution is unnecessary with Ochromonas and bacteria.

Vitamin BIZ standard solutions for experimental media. Because BIZ assay experiments were done only intermittently, it was convenient to keep the standard stock of B12 (as cyanocobalamin) in the form of triturates with mannitol, pentaerythritol, or sucrose (BIZ is commercially available as a 1 : 1000 triturate in mannitol) . As cyanocobalamin is hygroscopic, absorbing up to 12% H20 a t SQ% humidity(l5), solid BIZ to be used as a standard should be kept in a desiccator or as a triturate so that the gain in weight of BI3 is negli- gible. I t is convenient to keep on hand triturates of varying strengths ( 1 : 10,000 etc.) . Dilutions of Byl in water are buffered at p H 4.5-5.0, the region of opti-

VITAMIN BII‘-Ass~y WITH EUGLENA 103

ma1 stability; citrate buffer is recommended( 15). It should be noted that this is not the procedure routinely used by the British author; see Part 2 of this paper.

Assembly of basal medium. The new basal medium is shown in Tables I and 11. The concentrations of ingredients are expressed in percentages (w/v in the final medium; the calculations for preparing enough dry mix for 10 liters of final medium are shown). The ingredients are ground together in a mortar or ball mill. The mix is not hygroscopic. For use, the desired amount of mix is made up in double-strength concentration with distilled water and warmed to speed solution. The pH usually does not require adjustment and is about pH 3 .6 . Some of the analytical results described later were based on media which differed in unimportant details such as minor differences in the levels of trace metals.

In selecting ingredients for the basal medium various carbohydrates, nitrogen sources, and additional stimulants were tried; the results are shown below. The bacillaris and t strains were used in these preliminary experiments.

Bujering of the medium. I n the earlier simple media such as those without amino acids, and with “sugar activation” effected by succinic acid + NH4, the pH often became very acid-as low as pH 2.0; this ap- 3.0t NEW BASAL MEDIUM

> b 2.5 v) z w 0

-I a 2 a t- 0

P

peared to be the lower limit for active growth. Media with asparagine tended to become alkaline, presumably through liberation of excess NH4. The pH of corresponding media made with aspartic acid remained almost constant. These pH shifts have not been ana- lyzed in detail; perhaps the acid produced from sugar balances the alkalinization induced by consumption of succinic, malic, glutamic, and aspartic acids.

With profuse growth in the old medium( 2 1 ) the pH rose as high as 7.0 or 8.0 (Fig. 1). This p H rise has been eliminated or much reduced in the new medium by following up the observation by Cramer and Myers (5) that E. gracilis var. bacillaris grew with glucose if 5% C 0 2 was bubbled through the culture, and that a low pH (4.5) was favorable to glucose utilization. In- stead of supplying C02 as such-an unwieldy procedure-it was hoped that sufficient C02 to activate sugar utilization might be formed from readily oxi- dizable substrates. Another possibility was that the function of CO2 was to maintain the levels of the C4 dicarboxylic acids of the Krebs cycle; by supplying these the C02 requirements might be bypassed. As these Ca acids promote growth in acidic media, both hypotheses suggested the same experiments.

In the new medium the pH rise is usually very small (Fig. 1 ) . With heavy prolonged growth, however, the

1 OLD BASAL MEDIUM

’ BACILLARIS T t

I I I I I I 10 25 50 125 2.5 S 10 25

VITAMIN B,, ppg./ml. Fiq. 1. Growth of z and barillaris strains of Euglma grarilis in old and new basal medium after 7 days’ incubation, show-

ing greatest growth of :: strain in the new medium.

104 VITAMIN B1,-AssAY WITH EUGLENA

pH may rise to 4 or 5 and this rise is greater in serum than in water with an equivalent amount of B12 (see later).

Carbohydrate. Glucose and sucrose promoted equally good growth in the presence of good N-sources. Sucrose was selected because of its greater stability, low cost, and lower osmotic pressure for a given amount of growth. In New York no BIZ was detected in ordinary sucrose of the “super-fine” type sold for domestic use; this was used in the medium. Such sucrose, as well as certain “chemically pure’, samples, has apprecia’ole biotin, but this does not affect the results; c.p. sucrose should be used for assays in the absence of information on the purity of domestic-grade sucrose. Fructose was utilized ; mannitol, glycerol, and gluconolactone were inert.

N-Sources and tricarboxylic acid cycle intermediates. In experiments under widely diverse conditions, diffi- cult to summarize in tabular style, it was found that smooth curves for glucose or sucrose utilization re- quired the presence of a high concentration of ammonium together with a Krebs-cycle component such as succinic acid. The most consistent growth was obtained by using an amino acid such a glutamic or aspartic acid which is readily swept into the metabolic pool. The components were tested in acid media with succinic acid alone, sucrose alone, and combinations of sucrose and succinic acid; (NH4)2S04 served as the positive control. The bacillaris and t strains yielded perfectly concordant results.

It was noted that in the glutamic acid-asparagine medium, addition of a mixture of amino acids made up to imi- tate the composition of a casein hydrolysate, supplemented with tryptophan, increased growth. This stimulation was duplicated by glycine (or serine) + NH4 (conveniently supplied as the succinate) . The further addition of malic acid + trace elements gave another increment in growth as did “complete supplement” (a mixture of vitamins, amino acids, and an alkaline hydrolysate of yeast nucleic acid similar to B mixture previously described (4). Deficiencies in the basal medium in any microbiological assay are likely to show up in assays as a “drift” upwards, i .e. by greater growth than is accountable by the content in natural materials of the compound being assayed. That the basal medium was now approaching adequacy was shown by the only slight growth stimulation by such Blz-free materials as yeast autolysate and Phytone (a papaic digest of soybean protein, Baltimore Biological Lab.). Another index of the adequacy of the basal medium was that it now supported the same rapid growth when used at double strength. Methionine in concentrations equal to or much greater than those entering as a contamination in glutamic acid did not

Additional stimulants and assay “drift”.

TABLE I. “Dry mix” basal medium for Bln assay.

Concentration (mg. %) in final medium

Wt. (g.) for 10 liters final medium

KH,POa MgSOa * 7Hz0 L-Glutamic acid CaCOs Sucrose DL-Aspartic acid DZ-Malic acid Glycine Ammonium succinate* Thiamine HClt “Metals 45”:

30 40

300 8

1500 200 100 2 50

60 0.06 2.2

3 .O 4.0

30.0 0.8

150.0 20.0 10.0 25.0

6 .O 0.6 0.22

* This appears to be no longer commercially available in the U. S. A.; it may be replaced by an equivalent amount of suc- chic acid 4- NH,HCOs.

t 0.6 g. of triturate containing 1 g. of thiamine HCI + 99 g. sucrose (“1: 100’’ triturate).

t See Table 11. The basal medium is made up double-strength by dissolving

the ingredients in distilled water by steaming for 15 minutes. Cool; check pH-it should be pH 3.6; if adjustment is necessary use NaOH or HzSOI. Filter. Bottle. Sterilize by autoclaving at 10 Ibs. for 15 minutes.

For assay of BD in sera of patients treated with sulfonamides, add p-aminobenzoic acid (PABA) 0.125 mg. 70 to the final medium. PABA does not alter the growth curve in the absence of sulfonamides.

spare BIZ or influence growth in the presence of excess BIZ.

The available strains of E. gracilis were now examined in the hope of finding one with a higher temperature optimum than the bacillaris or t strains and growing at least as vigorously in the new concentrated sugar-containing media. The higher temperature optimum was desired in the hope that growth would be faster a t higher temperatures while the cultures would be no more susceptible to irreversible heat damage. These experiments have been detailed elsewhere( 2). The strains, from the Cambridge Collec- tion of Algae and Protozoa, were mostly those isolated and studied earlier by the Pringsheims(20) ; strain’ t = Pringsheim’s strain 9. The z strain (Pringsheim’s no. 25) grew best in concentrated media. The z strain is available in the U. S. from the American Type Cul- ture Collection and the Culture Collection of Algae, Department of Botany, Indiana University, Blooming- ton. The stimulation by a “complete supplement”, even with a further supplement of Trypticase + oxbile, was later matched by modifying the medium to that shown in Tables I and 11.

While this paper was being written, Pringsheim published the results of similar experiments( 20) : the t strain used glucose vigorously as sole substrate, as did also his strains 7 and 13; the z and bacillaris strains grew poorly. Sucrose as such was not used.

The poor growth of the z strain in our experiments with very simple media (Table 111) , unexpected at the time, parallels Pringsheim’s findings. The concentra-

Choice of strain.

VITAMIN B,.-AssAY WITH EUGLENA 105

TABLE 11. “Metals 45” dry mix.

Concentration Gravi- Wt. for 1000 (mg.%)in final Compound metric liters of final

medium used factor medium (g.)

Fe 0.2 FeSOI * (NHI),SO~ * 6HsO 7.02 14.0 Zn 0.1 ZnS04-7Hs0 4.4 4.4 Mn 0.05 MnSOI-H,O 3.1 1.55 CU 0.008 CuSOI.SHs0 3.9 0.31 CO 0.01 CoSO*.7HsO 4.8 0.48 B 0.01 HaB03 5.6 0.57

*Mo 0.035 (NH,)oMo;Ozr * 4HzO 1.8 0.64 V 0.001 NaaVO, * 16Hp0 9.3 0.093

~~~ ~ ~~ .

Total 22.043 g.

* The value for Mo is almost certainly needlessly high ; in practice no inhibition was observed even at much higher levels.

Notes on the metal m i x . The ingredients are ground together and stored in a dry place. No direct evidence is a t hand that Euglewa requires boron, Mo, V, or Co beyond that in Bn; they are added for the sake of completeness in the light of data for other algae.

tion of trace metals (not shown) was decreased to about one quarter of the usual concentration to com- pensate for the omission of malic acid. Pyromellitic acid ( 1,2,4,5-benzenetetracarboxylic acid) served as an inert pH buffer. The classical Mainx strain was used in many of these experiments and grew poorly or not a t all in these concentrated media.

Temperature and apochlorosis. Pringsheim’s paper brings up to date the correlations in physiological characters among strains of E . gracilis. Strains of his Groups 2 and 3 grow a t 34”C., while the Group 1 strains grow poorly if a t all above 28°C. The Group 2 strains tend to bleach at 34°C.; Group 3 strains grow a t 34°C. and remain green. The 3 strains found by Pringsheini to utilize sugar belong to Group 2 . Our own results with all the available strains, grown either in the full-strength or half-strength standard medium, fully confirm Pringsheim’s conclusion: there is an un- fortunate correlation between proneness to permanent bleaching, utilization of sugar, and ability to grow at 34°C.; the z strain, like bacillaris belongs to Group 2. The Mains strain and others of its type (Group 1) grew in our half-strength basal medium. The inhibi-

tory component of the basal medium was not identified; it was not phosphate as there was no inhibition with 0.08% KH2POa. The Group 2 strains all grew without obvious inhibition in double-strength medium.

Our failure, like Pringsheim’s, to find a sugar-utilizing strain that would resist permanent bleaching when grown at 34°C. or higher, led us to re-attempt to develop culture media that would support growth above the usual limit. It was hoped that such experiments might also show how to prevent ( a ) chloroplasts from being lost in the dark (the chloroplasts reappear when dark cultures are illuminated) and ( b ) heat-induced irreversible apochlorosis. Achievement of the latter aim might help solve the problem of how streptomycin induces permanent loss of chloroplasts, as Group 2 strains appear to be the ones especially susceptible to streptomycin-induced bleaching. One of us (G.I.M.R.) has observed that heat-induced apochlorosis is not always permanent: in one experiment the z strain turned almost completely white a t about 34”C., and then on sutbculture a t a lower temperature it grew lux- uriantly green again with no sign of white cells. The z strain in the new medium appears to be more susceptible to heat-bleaching than the bacillaris strain in the old medium: a whole z batch turned white in the bath a t about 34-35OC., which meant it was completeIy useless for assay purposes while a parallel bacillaris batch in the old medium grew completely green and satisfactorily.

By analogy with Ochromonas malhamensis, where analysis of “temperature factors” is more advanced( 1) , the difficulty in obtaining growth a t the higher temperatures( 2) almost certainly resides in a combination of ( a ) distortions in metabolism that make necessary drastic changes in the basal medium, and ( b ) multi- ple blocks in synthesis that can be compensated for by appropriate supplementation of the medium. For routine assay purposes it is, then, clearly preferable to adhere to the use of basal medium of the new “standard” type and not to incubate cultures above 31°C.

TABLE 111. Growth in simple media. O.D. reached in 7 days in light at 25-28°C.

Additions to basal medium “Permeable” strains “Impermeable” strains

1 ) Kone 0.43 0.54 0.17 0.51 0.04 0.01 0.09 0.25 0.41 0.17 0.25 0.15 0.37 0.07 2 ) DL-Aspartic acid 1.0

3) DL-Asparaghe 0.1 0.89 1.09 0.78 0.72 0.08 0.22 0.12 4) Glucose 1.0 1.92 1.42 10.0 10.0 0.03 0.03 0.05 5 ) Sucrose 1.5 0.26 4.0 12.0 10.0 0.02 0. 0.04 6 ) Sucrose 1.5 + DL-Aspartic acid 0.1 1.2 1.54 18.0 20.0 0.06 0.04 0.04 7 ) Sucrose 1.5 + DL-Asparaginp 0.1 1.45 10.0 18.0 20.0 0.03 0.02 0.03

(”0 final conc.) z bac. v t b C d ~~~~ - __ . .- ~ -~ ~ .~ ~ .~ ~~ ~~~ ~~~~~~ ~~~~

-~ . ~. ~~ ~ ~ ~ . -. - .. . . - ~ ___ “Permeable” denotes strains using a wide variety of substrates. Basal medium (wt./100 ml. fina1 medium) #.

Ii HZPO, 0.02 6. I Pyromellitic acid 0.1 g. MgSOI * 7HzO 0.04 g. Thiamine HC1 0.05 mg. CaC03 0.005 g. B12 0.4 pg. NHIHCOI 0.1 g. pH 3.2-3.6

106 VITAMIN BI2-Ass~u WITH EUGLENA

Osmotic pressure. When either bacillaris or z is used for assaying BIZ in sea water, the increased 0s- motic pressure will inhibit growth unless the B12 concentration is high enough to allow the sea water to be diluted 10-fold or more. All the strains were tested for osmotic tolerance in suitable dilutions of the basal medium supplemented with NaCl or pentaerythritol (a physiologically inert non-electrolyte) . If allowance is made for the more vigorous growth of the Group 2 strains, all the 24 strains studied in the sequence of experiments had the same osmotic tolerance, except that the r strain (= Pringsheim’s strain 13) was consistently, if only slightly, more osmo- tolerant. The slope of B12 response curves of the z and r strains was unaffected by 0.25% NaCl; a t 0.5% inhibition was evident. A detailed study of the z , r, t, and bacillaris strains showed that inhibition appears between the following concentrations: NaCI, 0.1- 0.25% ; pentaerythritol, 0.5-0.75%; sea water, 5-10%, by volume. The osmotic tolerance of the r strain did not seem higher enough to recommend its substitution for the z strain in assaying sea water.

Specificity of Euglena strains for Biz. The change to the z strain raised the question of whether it had the same specificity as bacillaris. In experiments by K. Carsted, both strains responded to Factor A (contains 2-methyladenine instead of 5,6-dimethylbenzimidazole-gift of Dr. E. S. Holdsworth) ; pseudovitamin B12 (contains adenine instead of 5,6-dimethylbenzimidazole-gift of Dr. J. J. Pfiffner) ; and did not respond to Factor B (the isoporphyrin part of the B12 molecule without any nucleotide-gift of Dr. Holdsworth). All these results accord with earlier results of several laboratories with the bacillaris strain( 7 ) .

PART 2 . ASSAY OF V I T A M I N Biz I N B O D Y FLUIDS

MATERIALS AND METHODS The procedure used in assays with E. gracilis var.

bacillaris( 2 1) proved satisfactory with the z strain of E . gracilis in the new medium apart from a few minor modifications described below. Cotton-plugged 6 X 5/6 in. Pyrex test tubes have been used for assays and 6 x 3/4 in. tubes for maintenance of stock cultures. Aluminum caps are convenient if the tubes are incubated standing in racks but not if packed close together in bundles or compartments. Very clean glassware is essential for accurate assay, for even less than 1 x g. BI2 (1 ppg./ml.) can stimulate growth of Euglena. The tubes are washed well with soap and water but without detergent and are thoroughly rinsed in tap and then distilled water. They are twice filled with distilled water and autoclaved to drive any contamination on the glass into the water in the tubes.

After drying, they are plugged with cotton and sterilized by hot air at 160°C. for 1 hour; charring of the cotton must be avoided. Non-absorbent cotton contains insignificant amounts of BIZ. Bottles used for preparation of BIZ standard solutions and bottles or tubes used to store material for assay are similarly treated. Pipettes are soaked in water after use and rinsed thoroughly in an automatic pipette washer overnight. Then, after autoclaving in distilled water, they are rinsed in distilled water, dried, plugged, packed in tins and sterilized by hot air in the same way as the tubes. Glass-distilled water was used throughout.

Conduct of assays. Dilutions of test fluids are made in distilled water in test tubes. Volumes are made to 2 ml. with distilled water, and when the whole assay batch is ready, 2 ml. of double-strength basal medium are added to each tube.

The dilutions used will vary with the likely B12 content of the fluid assayed, With serum, final dilutions of 1 in 20, 1 in 40 and 1 in 80 are suitable for nearly all sera and each dilution is set up in two or three tubes. In choosing dilutions it should be re- membered that the physiological range of vitamin BIZ concentrations in the serum of man is from less than 16 ppg./ml. in subjects with B1z deficiency(l6) to 10,000 ppg./ml. in patients with chronic myelocytic leukemia ( 17) ; the mean normal concentration is about 360 ppg./ml. Higher dilutions may be necessary in the assay of serum and urine taken after therapy with vitamin BIZ. Tissues, especially liver, may contain high concentrations of vitamin B12-up to about 1.0 pg./g.-so that very high dilutions must be used.

To increase accuracy, the assay of each fluid should be repeated in a t least one other assay batch and provided results agree within the error of the method the mean value found may be accepted as the “B12” concentration.

While reasonable care should be taken to avoid contamination of test fluids, distilled water, and glassware with microorganisms, strict sterility is unnecessary in preparing the assay batch. Any organisms which are introduced are killed in tubes heated at 100°C. before inoculation (see below). Spores of aerobic spore-bearing bacilli able to resist this heating have not been found to germinate a t the acid pH of the culture medium. Tubes which are unheated, or heated a t no more than 56”C., however, have very occasionally become contaminated with molds.

Measurement of total, uncombined, and combined B12. Vitamin BIZ in test material can be measured either as “total” BIZ or as “uncombined” BIZ; the difference between these two values is considered to rep- resent the concentration of “combined” BIZ( 16).

To measure “total” BI2, tubes containing dilutions

VITAMIN B1?-Ass~y WITH EUGLENA 107

of test fluids in 2 ml. volume plus 2 ml. of double- strength basal medium are heated a t 100OC. for 15 minutes. This degree of heating at the acid pH of the medium (pH 3.6) appears to liberate a maximal amount of B12 from combination with protein in material such as serum. Such heating is conveniently made by placing the tubes in a water bath and allow- ing 15 minutes heating from the time the water begins to boil. This has given more reproducible results than free steaming in an autoclave where control of temperature may be less accurate.

To measure "uncombined" Blz, tubes are either unheated or are heated at not more than 56°C. for half an hour; this degree of heating releases little BIZ from Combination but may help to kill any fungi or vegeta- tive bacteria introduced during preparation of the assay batch.

Vitamin B I Z standard solutions. Owing to the greater growth of the z strain with a given amount of BIZ, slightly lower standard concentrations of BIZ are included than in assays using the previous medium. Standards are conveniently prepared in bottles in 100 ml. volumes, by dilutions in distilled water of an accurate commercially-supplied 20 pg. B12/ml. solution -for example Cytamen (Glaxo Laboratories Ltd.). These stock solutions are kept a t 4°C. and used for not more than 3 weeks. The standards routinely in- clude 10 different strengths of BIZ, covering the range 0.25 to 50 ppg./ml., and also control tubes with no BIZ.

Stock cultures. Stock cultures are preserved in 6 x j/, in. tubes containing 10 ml. of single-strength basal medium plus 0.25% agar, 0.2% Tryptone (an enzymic digest of casein; 0x0 Ltd.) and 50 ppg. B12/ml. Such cultures after a few days' incubation in the water bath can be left a t room temperature for several months without death of the culture. With this proportion of agar, cultures can be mixed and pipetted with ease. In 0.125% strength the agar fails to stay as a gel throughout the medium and settles in the lower half of the tube.

Cultures f o r inoculation. Cultures for inoculation of assay batches are maintained in similar tubes in 10 ml. volumes. Each tube contains 10 ml. of single- strength basal medium plus 0.2% Tryptone and 50 ppg. BI2/ml. The Tryptone itself in this strength contains an equivalent of 20 ppg. Bl/ml. giving a total of 70 ppg. B12/ml. in the culture. Such cultures for inoculation are set up in each assay batch and after incubation for 6 to 8 days are used to prepare the inoculum. The Euglena cells in the 10 ml. of culture are centrifuged in a sterile screw-capped container. After removal of all the supernatant fluid they are resuspended in 20 ml. sterile single-strength medium and are shaken to wash. After centrifugation the supernatant is again removed and the cells resuspended

evenly in a further 10 ml. single-strength basal medium. This dense washed inoculum with an optical density of about 3 is satisfactory for routine assays. One drop is added to each tube. The value of washing the cells used as inoculum was shown by Qstergaard Kristensen (19).

Incubation. A 5 - or 6-day period has been found convenient, but frequently obvious differentiation of growth can be seen within 3-4 days so that provisional readings may then be made. Incubation is a t a temperature between 25 and 30°C., preferably about 28" C., and the tubes are brightly and uniformly illuminated. A convenient way is to incubate tubes arranged a t random in a Perspex (methyl methacrylate) water tank (e.g. 2 ft. 6 in. x 1 ft. 3 in. x 7 in.) illuminated from below by two 3-ft. 40-watt fluorescent (warm white) strip lights; 'by extending beyond the ends of the bath they provide a uniform brightness over the whole length of the bottom of the tank. Cells grown above 33°C. may become devoid of chlorophyll. At temperatures about 40°C. the alga is quickly killed. Cells incubated in inadequate light grow more slowly and fail to develop a full complement of chlorophyll.

The amount of growth in each tube is measured in a photoelectric colorimeter with a red filter; the turbidity is recorded as optical density (O.D.) X 100. A 10 mm.-deep colorimeter cell able to hold the 4-ml. volume in each tube (reduced to about 3.8 ml. by evaporation during heating and incubation) is used. When the turbidity exceeds O.D. 1.0, readings may be made in a 5 mm.- deep cell or, if very turbid, in a 2.5 mm.-deep cell; such readings are then adjusted to be comparable with the readings in the 10 mm.-deep cell by reference to a conversion graph for the appropriate 5-mm. or 2.5- mm. cell. Alternatively and more accurately, all cultures in an assay batch may be dtiluted equally with distilled water so that the densest give a final O.D. of less than 1.0 when read in the 10 mm.-deep cell. I t must be noted that the addition of distilled water to cultures containing serum may lead to increased turbidity due to precipitation of proteins. This is less troublesome with the new medium (Tables I & 11) than with the old medium(21) and may be avoided to some extent by weakly acidifying the distilled water for dilution.

After removal of caps or cotton plugs all tubes are shaken very vigorously to break up clumps of algae. One drop of a dilute caprylic alcohol-water (about 1 in 6) mixture is added to tubes containing serum dilutions immediately before reading to remove air bub- bles which will cause falsely high readings.

The standard curve for Bl2 is conveniently plotted on 3-cycle semi-log paper. The BIZ concentration is plotted on the logarithmic side and the turbidity reading on the linear side. Corrections must be made for

Reading and calculating results.

108 VITAMIN BI2-Ass~y WITH EUGLENA

turbid or colored test fluids. To do this, the turbidity is first read and then after sedimentation of the alga (immobilized by caprylic alcohol), or after centrifugation, the turbidity of the supernate is read. This value is subtracted from the total turbidity.

A regular increase or decrease (“drift”) in calcu- lated BI2 value with increasing dilutions of test fluid indcicates tlhe presence of inhibitory or stimulating substances in the sample which are interfering with the assay, e.g. serum and urine more concentrated than 1 in 10 are usually inhibitory; with a series of higher dilutions, however, comparable assay values are usually found and the concentration of B12 in the sample can be taken as the mean concentration of the results with these higher dilutions.

bacillaris and z strains in both the old and the new medium are shown in Fig. 2.

laris strain in the previous medium(21). Growth is more rapid and luxuriant and there is better differentiation between the amounts of growth in the presence of very low concentrations of BIZ.

The advantage of the z strain over the bacillaris strain in degree of growth is more apparent in the new than in the old medium (Fig. 2). In fact in several tests there was little difference in growth of the two strains in the old medium at 30°C. in light.

Effect of washing inoculum. The sensitivity, rapid- ity, and accuracy of assay is further increased by using a washed inoculum of Euglena cells. We have confirmed the observation( 19) that the supernatant fluid of well-grown Euglena cultures contains material which inhibits growth of the Euglena. Our preliminary ex-

Representative standard curves for B12, for the periments show that the material is highly potent, being able to exert measurable inhibition of growth when the supernatant fluid is present in as low a concentration as 1 in 2000. I t is removed by Seitz filtration but not by dialysis. I t appears to act by binding free BIZ and so making it unavailable for growth of Euglena cells in the inoculum. The fact that it is completely inactivated by heating at I O O O C . for 15 minutes and

RESULTS The z strain and new basal medium. The z strain

of Euglena gracilis used with the new basal medium gives more satisfactory Bls assays than does the bacil-

NEW BASAL MEDIUM - PH - M NaOH

c

-

g 5

-

U

1

100 150 200 250

- 8

- 7

- 6

- 4

3 300

PH

Fig. 2 . ( a ) Effect of growth of I” and bacillaris strains of Euglena gvacilis on pH of old and new basal medium after 7 days’ incubation. ( b ) The amount of alkali produced in 4-ml. volumes has been litrated against turbidity of growth by addition of sufficient 0.1 N KaOH to raise the pH to the pK of phenolphthalein.

In the new medium there has been very little rise in pH with profuse growth.

VITAMIN B I ?-ASSAY WITH EUGLENA 109

is unaffected by heating a t 56°C. for 30 minutes indi- cates a similarity in behavior with the B12-binding material normally present in such body fluids as serum and gastric juice.

Because the new medium is richer than the old one there is greater agreement between results with higher and lower concentrations of nutritionally- rich test material such as serum. With the more de- ficient old medium higher values were often found with, for example, 1:20 serum than 1:40 or 1:80. This is presumably because of growth-stimulating substances in serum other than B12 and because of a slight rise in pH due to the relative alkalinity and buffer- ing power of serum.

This effect is much reduced in the new richer and better-buffered medium but is still not always entirely excluded. With addition of normal 1:20 serum, the pH of the basal medium may be immediately raised from 3.6 to 3.8 and after incubation in the assay there may be a greater rise in p H in dilutions of serum than in dilutions of equivalent amounts of BIZ in water. With very heavy growth in 1:20 serum, the pH has been found to rise to pH 5 or more with consequent precipitation of protein. Provided sera are diluted, for example 1 : 40 or 1 : 80, to contain a final concentration of no more than 10 ppg. BIJml., this effect of the rise in pH does not occur and comparison of results in different dilutions is very satisfactory.

In a series of comparisons of assays of 40 sera in 10 different assay batches by the old method(21) against the new method described in this paper, set up in parallel, the mean results agreed to within 10%. I t should be noted, however, that values in test fluids may occasionally vary from one assay batch to another by as much as 25%. The accuracy of each batch can be readily assessed by always including in it samples of two sera of known BIZ content; several hundred ml. of each of these control sera are stored in 2 ml. amounts a t -20°C. and a fresh 2 ml. amount of each serum is set up in each assay batch. To obtain highly accurate results it would seem desirable to standardize the assay procedure as much as possible, particularly by using inocula of constant age and size and using a constant temperature and time of incubation for all assay batches.

Spwd of assay. With certain sera there is a slight tendency for growth to be a little more rapid than with water containing equal amounts of B12. However, ap- proximate assay results can be obtained after only 24 to 48 hours of incubation. T o obtain these rapid results it is necessary to wash the inoculum very thoroughly and to use a heavy inoculum-for example with a final O.D. reading of about 5 or 6 . I t is not suggested, however, that incubation should routinely be as short as this-batches vary in speed of growth

Accuracy.

due to such factors as temperature and degree of illumination during incubation and size and potency of the inoculum. It seems likely that more reproducible results are found when incubation is allowed to con- tinue until all B13 in both test fluids and standards is completely used up and growth has ceased. We be- lieve therefore that reducing the time of incubation is likely to reduce accuracy because of substances in test fluids which either stimulate or depress the speed of growth. Incubation for 5 or 6 days, however, is usually satisfactory.

Addition of cyanide in the assay. Preliminary treatment of samples for BIZ assay with cyanide (NaCN or KCN) is widely employed. Cyanide converts cobalamins such BIZ, (hydroxycobalamin) and BIZ, (nitroso- cobalamin) to the more stable BIZ (cyanocobalamin) . Because cobalamins without cyanide are inactivated to some extent during autoclaving in the preparation of the assay batch, assays with organisms such as Lacto- bacillus leichmannii or Orhromonas may be grossly low if cyanide or some reducing agent such as thiogly- collic acid is omitted. With the Euglena assay of B12

in body fluids, however, it has not appeared necessary to add cyanide. As light tends to remove the CN from the B1? molecule, it is probable that in the assay in light, and during growth of the organism in nature in water exposed to sunlight, Euglena is accustomed to utilize much BIZ in cobalamin form without CN, for example a s BIZ,. Certainly in tests with Blo and Br2:, there is little difference in growth response, and also with incubation in light or in darkness the growth rate is similar in suitable media.

The introduction of cyanide into the assay is complicated by the fact that the effects of the same concentration of cyanide in standards containing BIZ in distilled water and in dilutions of serum or urine may be different. High concentrations of cyanide are more inhibitory in the B12 standards and other fluids without protein, such as urine, than they are in serum. In standards set up with 0, 2, 10, 50, or 250 ppg. KCN/ ml., growth is usually slightly greater with either 2 or 10 ppg. than with 0 or 50 ppg./ml. while a concentration of 2 5 0 ppg./ml. is markedly inhibitory. Cer- tainly if cyanide is introduced it should not be used in a final concentration of more than 10 ppg./ml. and the effect of its use should be carefully controlled.

ifddition of p-aminobenzoic acid to the basal medium. Lear, Harris, Castle & Fleming( 14) reported that sera containing high therapeutic concentrations of sulfadiazine or sulfisoxazole were inhibitory to Eu- glena. Sulfonamide therapy should, therefore, be considered as a possible cause of an unexpectedly low serum BIZ concentration. This inhibition was over- come by including p-aminobenzoic acid in the medium (at a final concentration of 1.25 pg./ml.), but not

110 VITAMIN B].-AssAY WITH EUGLENA

folic acid or citrovorum factor. We have confirmed that this is a satisfactory concentration of p-aminobenzoic acid to use for this purpose.

DISCUSSION Euglena for assays of vitamin B I Z . The various test

organisms currently in use for the assay of BIZ differ in pattern of specificity(7). The most specific is Ochromonas malhamensis. So far no divergence by it from the vertebrate pattern has been reported. Och- romonas is slightly less sensitive than Euglena. For the assay of B12 in serum with it, the B12 must first be separated from the serum proteins by heat-coagula- tion. This is because the organism grows poorly at a pH of less than 5 and therefore the proteins in whole serum precipitate during incubation in the assay. This prevents the separate measurement of combined and uncombined B12; it is not known how well its greater digestive ability allows it to utilize combined B12. Some bacterial methods, such as those based on Es- cherichia coli 113-3, and on thermophilic bacilli, are simpler and faster, but these bacteria are much less specific for B12, responding to methionine or the pigmented portion of the B12 molecule severed from the dimethylbenzimidazole nucleotide-like portion. The Lactobacillus methods are faster but less sensitive, require complicated basal media, and respond to de- oxyribosides and to ‘.pseudo-B~2’~’~, i.e. cobalamins in which the dimethylbenzimidazole portion is replaced by other bases; these pseudo Bin's are not BIZ-active for mammals and birds.

Euglena is slightly less specific than Ochromonas in that it responds to pseudovitamin B12, Factor A, and Factor C2, but is more specific than the E . coli and Lactobacillus methods. In comparison with these two bacteria1 methods it suffers only in respect to the time of incubation. As already stated, by the use of the z strain of E. gracilis, the richer medium and a heavy washed inoculum, it is possible to make satisfactory readings in 2 to 3 days when rapid results are needed. In speed therefore there is now much less difference between the various assay methods. In several other respects there is advantage in the Euglena method. For example, the necessity for strict sterility is reduced by the acid pH of the medium which makes contamination very rare even when unheated materials are used; both combined and uncombined BIZ can be measured; the basal medium is simple to prepare and keeps certainly for many months; results are easily reproduced, provided glassware is very clean and the temperature of incubation does not rise above about 30°C. In respect to this last point, an elaborate system of incubation a t fixed temperature with special illumination is not essential-incubation in a warm room near any bright even light, is effective. Further-

more, workers assaying B12 in the blood of patients who are undergoing treatment with antibiotics have noted another advantage: Euglena is unaffected by nearly all the antibiotics, e.g. penicillin, streptomycin, chlortetracycline, chloramphenicol, and p-aminosali- cylic acid, a t concentrations that interfere with the bacterial assay methods(21,22).

Therefore, we recommend this new Euglena technique for the assay of BI2 as more satisfactory than that which we previously described ( 1 1,2 1 ) .

The diverse patterns of specificity of the various organisms used for BIZ assay, while helping to define the unique position of Euglena as an analytical reagent, also point to deep- seated molecular differences that may prove to be de- pendable characters for tracing phylogenetic affinities. The agreement in preliminary experiments between the BIZ patterns for the bacillaris and z strains raises ques- tions: does the Euglena pattern extend to the B12- requiring members of the Volvocales and Chlorococ- cales(7)?. Is the pattern in the green-pigmented flagellates opposed to that in Ochromonas; does the Ochromonas pattern extend to other BIZ-requiring brown-pigmented algae as well as to metazoa?

BIZ appears to be totally absent in green plants and most fungi. This does not prove, though, that BI:! does not figure in the formation of plant tissues. The few experiments with E. gracilis given minimal amounts of BIZ indicate that B12 is quickly transformed into some form which is inactive for the rat and for all the presently available microbiological methods. The presence, then, of B12 in protist groups of doubtful affinity may imply that such protists should not be assigned to the “green stock” or the presumably related fungi. Preliminary assays of Tetrahymena pyriformis and Crithidia fasciculata by both Euglena and Ochronzonas indicate that they contain little if any B12. These results cannot be fitted into a larger framework until more work is done on the specificity of the BIZ patterns in these and other microorganisms; it may turn out that besides green-pigmented plants and fungi, other groups may be devoid of BIZ, and so negative results may not imply any specially close relation to the green plant-fungi line. A further pit- fall which is recognized from this analysis of the Euglena-blood serum system is that negative results are not conclusive: the BIZ may be bound in a form such that more drastic treatments are needed to re- lease i t ; BIZ in sow’s milk is a case in point( 7 ) .

Can it, as a phago- troph, and unlike Euglena, directly utilize the “bound” B12 in blood serum? The Blz-serum system would appear to be a good test system, a t least for the eugle- noid series, for deciding whether the assumed superior digestive ability of the phagotrophs, as compared with

Euglena as a phylogenetic tool.

Peranema requires B l ~ ( 2 4 ) .

VITAMIN B I 2 - A s s ~ ~ WITH EUGLENA 111

their osmostrophic relatives, extends to a superior ability to utilize vitamins in high-molecular combination.

We have emphasized that use of Euglena enables one to distinguish between free and combined BIZ in blood serum. This brings forward a problem in comparative biochemistry: is Blz in serum and in the gastric mucosa bound by the same kind of compound as in the Euglena body and in supernates of Euglena cultures? Or, stated more broadly: are “intrinsic factors” and the BI2-binding substance(s) in blood serum representatives of a class of chemically similar compounds which includes all the B12-binding compounds of protists? Binding of B12 by Euglena may be con- ceived as having two phases: ( a ) initial binding; (b) transformation of bound BIZ into an unassayable form. I t should be interesting therefore to apply to Euglena itself, both bodies and culture supernate, the kind of procedure described for the differential analysis of free and combined B12 in blood serum. One of us (G. I. M. R.) is now using radioactive BIZ to study its uptake and metabolism by Euglena cells; this work will be published elsewhere.

Sugar utilization. How Euglena uses sugar is un- known. This is being studied in other laboratories and so only brief remarks are justified here. The acid produced from sugar-ven in light-remains to be identified. In unpublished experiments (New York) , we found that in acid media (pH 3.9) lactic and gly- colic acids are readily utilized; perhaps this is con- nected with sugar utilization. We have been unable to demonstrate the utilization of citric acid even, to favor penetration, in media as acid as pH 2.5. Citric acid was not utilized directly as a substrate nor did it promote sugar utilization under test conditions sensitive enough to be positive for 0.01% succinic acid or 0.001 % L-asparagine. Danforth (6) , using homogen- ates of a streptomycin-bleached strain of E . gracilis var. bacillaris, adduced much evidence that it uses the tricarboxylic acid cycle but did not mention whether “condensing enzyme” or other enzymes immediately concerned with citrate utilization were present. We had hoped that activation of sugar utilization by citrate would help tell whether sugar activation was bound up with the functioning of the Krebs cycle. Experiments with filter-sterilized phosphorylated sug- ars ( g1ucose-6-phosphate7 fructose-1 , 6-diphosphate), with the bacillaris strain in acid media where, as with citric acid, penetration would be favored, were incon- clusive.

Pringsheim proposes that sugar-utilizing strains of E. gracilis, all belonging to his group 2, be designated as var. sactharophiZa(20). This seems a useful way to designate the extremely heterotrophic strains of the bacillaris-t-z type. The poor growth of the z strain in

simple sugar media suggests that it will be of taxonomic as well as physiological interest to know precisely which compounds activate sugar utilization in z-type as compared with t-type strains.

The apparent need, in sugar activation, for abun- dantly available N and, a t least for the z strain, a Krebs cycle intermediate (or auxiliary substrates readily convertible to one), may not be unique. For example, in the mucoraceous mold Zygorhynchw moelleri, NH4, glutamate, and asparagine increased the rate of oxidation of glucose( 18).

ACKNOWLEDGEMENTS Besides the workers mentioned in the text, we owe

much to Professor Pringsheim, who suggested the use of the t strain and so enabled us to avoid the pitfalls in working solely with bacillaris. E. L. George, cura- tor of the Cambridge Culture Collection of Algae and Protozoa, was most helpful in providing strains of E . gracilis. Harlan Lane helped with the survey experiments with different strains. M. K. Gddberg as- sisted in development of the “tray-solution” fungicide. The late R. A. Hutner participated in the survey work on N-sources and the last experiments on temperature effects. T. J. Cayle helped with early experiments on the basal medium. In applying the new method to the assay of BIZ in serum, Dr. D. L. Mollin, Miss B. Anderson, and Miss G. Coats helped greatly.

REFERENCES 1. Baker, H., Aaronson, S., Rodriguez, E., Petersen, R. A,,

Hustner, R. A. & Hutner, S. H. (1955). Cobalamin, thiamine, “chrysomonad suspension factor” and other enhanced requirements of Ochromonas grown at elevated temperatures. J . Pro- tozool. (Proc.), 2, 10.

2 . Baker, H., Huttner, S. H. & Sobotka, H. (1955). Nutri- tional factors in thermophily: a comparative study of bacilli and Euglena. Ann. N . Y . Acad. Sci., 62, 349-376.

3. Coates, M. E. & Ford, J. E. (1955). Methods of measurement of vitamin B I ~ , in Williams, R. T., The Biochemistry of Vitamin BIZ, Biochem. Soc. Symposia no. 8, 36-51.

4. Cowperthwaite, J., Weber, M . M., Packer, L. & Hutner, S. H. (1953). Nutrition of Herpetomonas (Strigomonas) cu- licidarum. Ann. N . Y . Acad. Sci., 56, 972-981.

5 . Cramer, M. & Myers, J. (1952). Growth and photosyn- thetic characteristics of Euglena gracilis. Arch. Mikrobiol., 17,

6. Danforth, W. (1953). Oxidative metabolism of Euglena. Arch. Biochem. Biophys., 46, 164-173.

7. Ford, J. E. & Hutner, S. H. (1955). Role of vitamin Blz in the metabolism of microorganisms. Vitamins and Hormones, 13, 101-136.

8. Hoff-Jfirgensen, E. (1954). Microbiological assay of vitamin BIZ, in Glick, B., Methods of Biochemical Analysis, l , 81-113.

Comparative biochemistry of flagellates, in Hutner, S. H. & Lwoff, A. Bio- chemistry and Physiology of Protozoa, Vol. 11, Academic Press, Inc., New York, 17-43.

10. Hutner. S. H.. Provasoli. L. & Filfus. 1. (1953). Nutri-

384-402.

9. Hutner, S. H . & Provasoli, L. (1955).

I _ ,

tion of some phagotrophic fresh-water chrysomonads. N . Y . Acad. Sci., 56, 852-862.

Ann.

11. Hutner, S . H., Provasoli, L., Schatz, A. & Haskins, C. P. (1950). Some approaches to the study of the role of metals in

112 Tetrahymena WITH A CAUDAL CILIUM

the metabolism of microorganisms. Proc. A m . Phil. Soc., 94, 152 -1 70.

12. Killander, A. (1955). The assay of vitamin BIZ in blood serum and urine. Acta Chein. Scand., 9, 1045.

13. Lascelles, J. & Cross, M. J. (1955). The function of vitamin 31, in micro-organisms, in Williams, R. T., The Bio- chemistry of Vitamin BIZ, Biochem. SOC. Symposie no. 8, 109-123.

14. Lear, A. 24., Harris, J. W., Castle, W. B. & Fleming, E. M. (1954). . The serum vitamin BI2 concentration in pernicious anemia. J . Lab. Clin. Med., 44, 715-723.

15. Macek. T. J . & Feller, B. A. (1952). Crystalline vitamin B,, in pharmaceutical preparations. J . A m . Pharm. Assoc., 41, 285-288.

16. Mollin, D. L. & Ross, G. I. M. (1952). The vitamin BIZ concentrations of serum and urine of normals and of patients with megaloblastic anaemias and other diseases. J . Clin. Pathol., 5, 129-139.

17. __ (1955). Serum Byr concentrations in leukaemia and in some haematological conditions. Brit. J . Haeinatol., 1, 155-172.

18. Moses, V. (1954). The effect of ammonia on the oxida-

tion of glucose by Zygorhynchus inoelleri. Biochem. J., 57,

19. Qstergaard Kristensen, H. P. (1954). Investigations into the Euglena gracilis method for quantitative assay of vitamin BIZ. Acta Physiol. Scand., 33, 232-237.

20. Pringsheim, E. G. (1955). Kleine Mitteilungen iiber Flagellaten und Algen. 11. Euglena gracilis var. saccharophila n. var., und eine vereinfachte Nahrlosung zur Vitamin B12- Bestimmung. Arch. Mikrobiol, 21, 414-419.

21. Ross, G. I. M. (1952). Vitamin BIZ assay in body fluids using Euglena gracilis. J . Clin. Path., 5, 250-256.

22. Sakai, H., Miyazawa, S. & Terada, 0. (1953). The microbiological assay of vitamin BIZ. Comparative study of various assay organisms. J . Agr. Chem. SOC. Japan, 27, 377-381.

23. Stokstad, E. L. R., Broquist, H. P. & Sloane, N. H. (1955). Nutrition of micro-organisms. Ann. Rev. Microbiol., 9, 111-144.

24. Storm, J . & Hutner, S. H. (1953). Nutrition of Pera- xenia. Ann. N . Y . Acad. Sci., 56, 901-909.

25. Ungley, C. C. (1955). The chemotherapeutic action of vitamin BIZ.

26. Williams, R. T. (ed.) (1955). The Biochemistry of Vita- min BIZ. Biochent. SOC. Syniposia no. 8, 123 pp.

547-556.

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J . PROTOZoOL., 3, 112-118 (1956).

Tetrahymena setifera n.sp., a Member of the Genus Tetrahymena with a Caudal Cilium*

GEORGE G. HOLZ, JR. and JOHN 0. CORLISS

University of Illinois, Urbana, Illinois Depautnzents of Zoology, Syracuse University, Syracuse, X e w F o r k , and

SUMMARY. A new species of Tetrahyinrna is described, the ninth to be allocated to this genus. Most significant morphologically and taxonomically is its possession of a single, slender caudal cilium, twice the length of the surrounding somatic ciliature. The presence of this inconspicuous organelle, quite novel to the genus, is also detectable in the infraciliature where an argentophilic “polar basal granule-complex” appears exactly at the posterior pole of the body. The diagnosis of the genus Tetrahymena is expanded to embrace species with such a caudal appendage.

SMALL holotrichous ciliate collected from pond A water by the senior author in June, 1954, for use in nutritional research, appeared, in preliminary ob- servations of its morphology, to resemble very strik- ingly Tetrahyena pyriformis, the well-known type species of that hymenostome genus. More extensive subsequent investigations, however, employing in par- ticular the nearly indispensable technique of silver im- pregnation, revealed that it could ‘be distinguished from T . pyrifornzis, and from the other known congeneric species as well. I ts single, outstanding feature of greatest differential value appeared to be a characteristic quite novel, a t the time of our initial studies, to Tetra-

* This investigation was supported in part by a research grant (E-797) to the senior author from the National Micro- biological Institute of the National Institutes of Health, U. s. Public Health Service. Much of the work of the junior author was carried out, as recipient of a National Science Foundation postdoctoral research award, during the summer of 1955 a t the University of \lrginia Biological Station, Mountain Lake, Vir- ginia.

hywzena : the new ciliate possessed a single, well-defined caudal cilium arising directly a t the posterior pole of the body.

At the same time, working quite independently, Jesse C. Thompson, Jr., discovered another new hymenostome, which he described as T . corlissi in a preliminary communication published in the summer of 1955 (23)) also characterized by possession of a caudal cilium of similar nature. Upon mutual sharing of much of our data, it became apparent to all of us working with these ciliates that an organism which is practically indistinguishable, physiologically (in major respects) and ecologically as well as anatomic- ally, from other species in a given genus should not arbitrarily be excluded from that genus because of its having a single unique feature, unless there is a basis for considering such a characteristic to be of distinct differential value in the taxonomy of this and neigh- boring genera. The single, relatively inconspicuous caudal cilium borne by the ciliates mentioned above