determine the extent and nature of the effect of the ph of the According to Dernby (1921) B. subtilis grows through the wide

Transcription

1 THE EFFECT OF THE REACTION OF THE MEDIUM ON THE CHARACTERISTICS OF BACTERIA II. BEHAVIOR OF BACILLUS SUBTILIS ESTHER WAGNER STEARN AND ALLEN E. STEARN Divisions of Medical Bacteriology and of Physical Chemistry, University of Missouri, Columbia, Missouri Received for publication, September 13, 1932 According to Dernby (1921) B. subtilis grows through the wide ph range 4.5 to 8.5, with optimum limits between 6.0 and 7.5. In 1927 Stearn and Stearn attempted, by culturing this organism alternately in broth at ph 5.2 and on neutral agar plates, taking care to transplant from the latter medium only colonies which showed a preponderance of Gram-negative rods, to obtain a pure variant. This technic produced a strain which showed a shift in isoelectric point, a decreased sensitivity to gentian violet in broth, and a significant loss in Gram-positiveness. In that study, as well as in the one recorded here, single cell cultures, obtained by the Orskov technic and purified by the usual agar plate method, were used. It was noted that the acid broth cultures showed decided differences from neutral and alkaline broth cultures, and this observation initiated a long series of experiments, not only on B. subtilis but also on several other organisms, in an effort to determine the extent and nature of the effect of the ph of the medium on the character of these organisms. In the first paper of this series the experimental methods of approach and the observed changes in certain other organisms have been described and discussed (Stearn and Stearn (1933)). In this paper an effort is made to record the results obtained with B. subtilis, results which were found to be very suggestive. 37

2 38 ESTHER WAGNER S'TrEtARN AND ALLEN E. STEARN ORGANISAI, MNEI)IA AND STAIN-ING TECHN-I(C The organism used for this study was a strain of the American Type Culture Collection, obtained from the American Museum of Natural History. By means of a modified Orskov technic a single cell culture was obtained. On agar slants it grows as a dry, attached, weakly-refractive spreading membrane with a crenate margin. On agar plates it forms medusa-head colonies, which are dry and attached. It is strongly Gram-positive, and forms central spores. Rods are long, with rounded edges. In neutral beef-extract broth pellicle formation is preceded by a dense turbidity and some flocculated growth. As the time of incubation is increased to forty-eight hours a grayish sediment forms, the broth becomes clear, and a firm pellicle floats on the surface. On glycerol agar the organism forms a glistening, brownish-red, moist growth, less spreading than that on nutrient agar. Mledia were prepared, adjusted to the desired ph with HCl or NaOH, and enough phosphate buffer added to make a total phosphate concentration of M/15. They were allowed to boil for a few minutes, after which they were cooled, filtered, tubed in 10 cc. portions and sterilized in the autoclave. To insure sterility the tubes were incubated for forty-eight hours at 37'. Before using, the ph of the broth was determined electrometrically. A twenty-hour agar or broth culture of the original B. subtilis culture was used as a control oin each slide when staining behavior was studied. The usual classic Gram technic was employed unless otherwise specified. 1. Types of growth in M/15 phosphate nutrient broth at three ph values One drop of a twenty-four-hour broth culture was inoculated into tubes at the various ph's, incubated for twenty-four hours and then transferred to new tubes at the same ph. In table 1 are given descriptions of the appearance of the broth cultures after twenty-four hours' incubation. They are given for 5 consecutive transfers.

3 EFFECT OF REACTION OF MEDIUM ON BACTERIA Within twenty-four hours pellicle formation and the clearing of the medium occurred at ph 7.8, while at ph 7.0 a small pellicle eventually formed though some growth remained diffused. At ph 5.4 no pellicle formed and the growth remained dispersed through the broth. Cultures streaked from the last tubes (after the fifth transfer as of table 1) to neutral nutrient agar showed the following changes: 1. The culture from the acid tube, ph 5.4, developed colonies with round white centers, moist but non-viscid, and with fuzzy edges. TABLE 1 CULTURE TRANSFER ph 5.4 ph 7.0 ph No pellicle; diffuse No pellicle; diffuse Pellicle; sparse diffuse growth; stringy sed- growth; slight sedi- growth; coagulated iment ment sediment 2 No pellicle; diffuse No pellicle; diffuse Pellicle; clear growth; stringy sed- growth; slight sediiment ment 3 No pellicle; diffuse Small pellicle; diffuse, Pellicle; stringy, flocgrowth; stringy sed- stringy growth culating growth iment 4 No pellicle; diffuse Small pellicle; diffuse, Pellicle; stringy, flocgrowth; stringy sed- stringy growth culating growth iment 5 No pellicle; diffuse Small pellicle; diffuse, Pellicle; stringy, flocgrowth; stringy sed- stringy growth culating growth iment 2. That from the neutral broth gave small shrivelled firmedged dull colonies. 3. That from the alkaline tube gave moist, glistening colonies, which resembled those from the acid culture rather than those from the neutral culture. 2. Effect of continued subculturing on acid and alkaline miedia B. subtilis was subcultured for four months in nutrient broth buffered with phosphate mixtures, of M/15 in total phosphate, and adjusted, in 1 case, to ph 5.2 and, in the other, to ph

4 40 ESTHER WAGNER STEARN AND ALLEN E. STEARN After forty-eight hours, transfers were made to agar slants adjusted to the same ph as the broth from which the transplant was made, incubated for twenty-four hours at 370 and then returned to broth. The strain kept at ph 5.2 will be called the acid strain and the other one the alkaline strain. Cultural differences could be regularly noted, the acid strain growing diffusely with the broth becoming nearly colorless, the alkaline strain growing with a clearing of the broth as a waxy pellicle formed. It was noted that this waxy pellicle in the alkaline tubes turned to brownish red when the tubes were kept at room temperature for a week. TABLE 2 Comparative sensitivity of the acid and alkaline strains to gentian violet DYE DILUTION ACID STRAIN ALKALINE STRAIN ph 5.2 ph 6.2 ph 7.7 ph 5.2 ph 6.2 ph 7.7 1:1,000,000{ :2,000,000{ + i i :4000,000{ :6,000, At the end of four months, when the two strains were streaked to 2 per cent nutrient agar of ph 7.6, the acid strain developed as smooth regular rounded moist colonies, while the alkaline strain gave irregular fuzzy-edged dry colonies. The alkaline strain was either Gram-positive or else showed Gram-positive granules enclosed in Gram-negative ectoplasm. The acid strain showed a large proportion of greatly shortened Gram-negative rods. Difference in sensitivity to gentian violet was determined by adding one drop of a saline suspension of twenty-hour acid agar plates (i.e., of the smooth colonies), or of twenty-hour alkaline agar plates (fuzzy colonies) to buffered gentian violet

5 EFFECT OF REACTION OF MEDIUM ON BACTERIA nutrient broth. Results for both twenty-four and seventy-twohour incubation periods are given in table 2. The greater sensitivity of the alkaline strain is quite marked. 3. Further experiments on the changes involved in response to the ph of the medium After the preliminary work of colony selection the organism was transferred to tubes containing meat infusion broth buffered with M/15 phosphate mixtures. The ph of the acid tubes was, in the first part of the work, adjusted to a value of 5.6, that of the neutral tubes to 7.0, and that of the alkaline tubes to 8.0. Although differences could be noted at the end of the first twenty-four hours, they became more pronounced after fortyeight hours. Streaked to neutral agar there was at this time no change noted between these strains. A series of transfers to new tubes was made every twenty-four hours, streaking to agar plates to determine differences in colony formation. After the seventeenth transfer three strains were obtained as described in table 3. Though the organism was subcultured for five weeks longer, no further change was noted in the strains either in the tubes or the colonies obtained from them. 4. Reversal of the acid strain Reversal of the acid strain to the original was studied by: 1. Transplanting into neutral nutrient broth; 2. Transplanting into alkaline nutrient broth; 3. Transplanting onto neutral nutrient agar. In neutral nutrient broth there is a slow change toward the original strain. In the first transplant, at the end of twentyfour hours the rods vary greatly in size and Gram character, though most of the rods are still short and Gram-negative. In the eighth culture of the series the broth still shows no pellicle formation, the growth remaining evenly dispersed with some sedimentation, though the rods are normal in size. Continued cultivation will, however, restore their normal Gram character, their ability to form a pellicle, and their typical colonies on agar. 41

6 42 ESTHER WAGNER STEARN AND ALLEN E. STEARN In alkaline broth the acid strain was found to have reverted to the original in every respect by the time of the twelfth transplant. By transplanting directly from neutral agar to neutral agar the acid strain of colony after the thirteenth transplant gives organisms which have reached normal size and have attained a Gram- TABLE 3 Description of strains produced at various values of ph STRAIN CULTURED AT STRAIN CULTURED AT STRAIN CULTURED AT CHAIIACTER p)h 5.6 pli 7.0 ph 8.0 Appearance in broth Colonies on 2 per cent neutral agar Morphology Motility of 20 hour broth culture Sensitivity to gentian violet tested on 2 per cent gentian violet meat infusion plates Dye dilution: 1: 500,000 1:3,000,000 1:4,000,000 Hlomogeneous clouding, no pellicle Glistening, regular edges; moist, easily removed Short, thick, Gram-negative Sluggish + Diffuse, flocculating, fragile pellicle Weakly refractive, spreading, (medusa head), grayish, dry, attached Normal size, Gram-positive I + I + Clearing, flocculating gelatinous sediment; waxy pellicle More spreading than those from the neutral strain Long, thin, Grampositive + positive character. In this reversion there seems to be a gradual change, and the strain shows a stage in which the organisms are beaded, the rods containing Gram-positive granules in a Gramnegative ectoplasmic zone, which, in turn, gradually changes to solid-staining normal subtilis rods after additional transfers.

7 EFFECT OF REACTION OF MEDIUM ON BACTERIA 5. Two types of colonies obtained by streaking the alkaline strain to acid agar (ph 5.6) B. subtilis, grown for a week in broth at ph 7.8 and then streaked to 1.5 per cent agar adjusted to ph 5.6 gave two kinds of colonies: 1. A spreading, translucent, dull growth, called type B; 2. White, moist, viscous, discrete secondary colonies, called type A. Results obtained with type B may be thus summarized: Planted to acid agar (1): Less spreading than B itself, more translucent. From acid agar (1) to acid broth (2): Pellicle formed with clearing. From acid broth (2) to acid broth (3): Pellicle formation with turbidity. From acid broth (3) to acid agar (4): Colonies with hollow, lytic centers surrounded by a raised and curved shell with a spreading pale outer periphery. Lytic areas. Long slender rods mostly Gram-negative showing decreased sensitivity to gentian violet. Results indicate that type B planted to media adjusted to ph 5.2 forms colonies with lysed centers from which the acid type strain is obtained. Planted to alkaline agar (1): More translucent and spreading than on acid agar. Long Gram-positive rods. From alkaline agar (1) to alkaline broth (2): Pellicle formation with clearing and flocculated growth. From alkaline broth (2) to alkaline broth (3): Pellicle formation with clearing and flocculated growth. From alkaline broth (3) to alkaline agar (4): Spreading translucent growth. These latter results show that in alkaline media colony type B is preserved. In the same way results obtained with type A may be summarized: Planted to acid agar (1): Discrete, viscous, smooth edged colonies. From acid agar (1) to acid broth (2): Diffuse growth, no pellicle. 43

8 44 ESTHER WAGNER STEARN AND ALLEN E. STEARN From acid broth (2) to acid broth (3): Diffuse growth, no pellicle. From acid broth (3) to acid agar (4): Same as A above, white, with roughened centers. Short and thickened rods, mostly Gram-negative. Thus on acid media the colony type A is retained. Planted to alkaline agar (1): Same appearance as on the acid agar. Mixed Gram-positive and Gram-negative rods. From alkaline agar (1) to alkaline broth (2): Pellicle formation with clearing and flocculated growth. From alkaline broth (2) to alkaline broth (3): Pellicle formation with clearing and flocculated growth. From alkaline broth (3) to alkaline agar (4): Spreading translucent growth. Thus, on alkaline media colony type A is seen to change to colony type B. 6. Resuilts obtained by using technic based on Qtlirk's mnethod for obtaining S and R forms of Bacillus subtilis The original culture was again purified by successive plate culture, and during this procedure no differences in colony form were noted. A needle transfer of a young agar culture was made to unbuffered meat infusion broth adjusted to ph's 5.15, 5.38, 5.45, 5.60, 5.65, 5.67, 5.8, 5.95, 6.0, 6.13, 6.3, 6.6, 6.8, 6.9, 7.05, 7.2, 7.6, 7.8, 7.9 and 8.2. These values were obtained electrometrically when broth tubes, kept for forty-eight hours at room temperature after sterilization, were tested. The inoculated tubes were allowed to stand at room temperature for thirty minutes, after which one loopful from each tube was transferred to another broth tube of the same ph. These latter tubes were incubated for twenty-four hours at room temperature, after which they were again planted twice successively (as before) to tubes of corresponding ph's. These last tubes were incubated at room temperature for twenty-four hours before streaking to 1.5 per cent meat infusion agar plates at corresponding ph's. This procedure was based on that suggested by Quirk (1931).

9 EFFECT OF REACTION OF MEDIUM ON BACTERIA a. Broth cultures at different ph's: ph range Description 5.15 Slow growth; granular and sedimentary Large fluffy floccules, otherwise clear; no pellicle in six days; sediment easily dispersed Turbid waxy pellicle, flaky sediment Uniform turbidity, gelatinous strings and a gelatinous sediment With an increase in ph from 5.67 to about 8 there is a greater and earlier formation of flakes which settle out, and a larger and more waxy pellicle forms. b. Colonies on agar at different ph's: ph range Description 5.15 Opaque, dry, rugose, mealy, center easily removed (fig. 1) Opaque, white, smooth or finely granular, easily removed from agar, glistening, faintly iridescent, showing clearing areas on continued incubation. (There is a well-defined margin, though after forty-eight hours the colony may become somewhat spreading. They never approach the appearance of the other colonies even after two to three weeks of incubation) (fig. 2) Intermediate, easily removed, somewhat mealy, slightly glistening Spreading, flat, slightly glistening and moist, center adherent, thin and membranous, medusahead (figs. 3 and 4) c. Transplants from broth to agar slants at corresponding ph's. When a loop or needle transfer is made to agar slants at varying ph's, marked changes, similar to those observed on agar plates, are noted. ph range Description 5.2 Opaque, metallic, raised, somewhat iridescent, easily removed 6.0 Opaque, gray, flat; generally intermediate 7.0 Thin, membranous, adherent, spreading, glistening 8.4 Generally similar to those at ph 7.0 but more spreading and translucent (fig. 5) d. Morphology. Twenty-four hour cultures, both agar and broth, show the following forms predominantly: ph range Description 5.15 Decidedly short and plump; Gram-negative with some granular; occurring both in long chains and in clumps 45

10 46 ESTHER WAGNTER STEAR-N AND ALLEN E. STEARN 5.6 Curved and varying greatly in length; long chains from the broth cultures 6.8 Normal Gram-positive rods; long and short chains in the broth From very short to long Gram-positive rods; short chains and single rods in broth 8.2 Very long and slender rods, homogeneous, Gram-positive; a few short chains but mostly single rods e. Motility. Broth and agar cultures of nine to thirty hours were found through the ph range 5.15 to 8.2 to be non-motile when incubated at room temperature. 'When incubated at 370, sluggish motility was observed in broth cultures at ph's 5.5 to 6.8 but not at 7.9 to 8.0. It is possible that the reason for the apparent lack of motility in the case of the acid strains is the fact of their growth in such long chains and large clumps. f. Spore formnation. At all ph's, i.e., from 5.15 to 7.9 spores seem to be formed equally readily, and they germinate throughout that range. At and above ph 8 the rods show a distinct tendency to form large Gram-positive granules, and later to become evacuolated without forming spores, although spore formation occurs to a certain extent. g. Suspensions in physiological salt solution. Growth from twenty-four-hour agar cultures at the ph's designated below showed great differences when transfered to physiological salt solution. ph range Description Easily emulsified, homogeneous suspensions, do not flocculate during twenty-four hours Less easily emulsified and non-homogeneous, some flocculation during twenty-four hours Very difficult to emulsify, flocculent; complete flocculation 8.0 Most resistant to emulsification, remain flaky The difference in the ease of emulsification and in the stability of the suspensions in salt solution- is progressive from low to high ph's. h. Cultural characters. Cultures obtained from three sets of tubes at ph's 5.5, 6.8 to 6.9, and 7.9 to 8.0, were used to determine whether any change in cultural reactions could be noted. Transplants were made into the various media from twelve-hourbroth cultures and from eight-hour and twenty-four-hour agar cultures.

11 EFFECT OF REACTION OF MEDIUM ON BACTERIA 4 All three strains gave similar results as follows: Glucose: Acid; no gas. Lactose: No fermentation. Maltose: Acid; no gas. Mannitol: Acid; no gas. Sucrose: Acid; no gas. Nutrient gelatin: White surface growth with liquefaction. Two-tenths per cent soluble starch broth: Complete hydrolysis with the formation of pink vesicles on the pellicle. Potato slants: Cheese-like, moist pink vesicles. The organism, though cultured through widely differing ph ranges, exhibits no change in enzyme activity with respect to the nutritive substances examined. We are thus not dealing with degenerated cultures. i. Effect of transplanting from agar cultures at varying ph's to neutral broth. Needle transfers were made from agar cultures at different ph's to neutral broth tubes. These were incubated for twenty-four hours at 37. ph range Description 5.15 Stringy, flocculating growth, producing a gelatinous sediment; no pellicle (morphology-long chains of long rods; sluggish motility) 5.6 Flocculated growth and flaky sediment (morphology-long chains; sluggish motility) 6.8 Turbid, with sediment which is easily suspended (morphologyshort chains and single cells; non-motile) Cloudy, with gelatinous sediment (morphology-few chains, rods single and short; non-motile) Thus it seems that the changes induced by the differing ph's of the media are more than temporary. DISCUSSION B. subtilis is generally cultured in media adjusted to ph's near the neutral point, and since it forms alkali readily it seldom exists in an acid environment. It can, however, grow readily in media of low ph's. Changes in appearance of cultures are thought to be accompanied by distinct differences in the composition and structure 47

12 N AND ALLEN E. STEARN of the bacterial cell, which can be observed during successive generations. Dissociants are of this nature. Two investigators in particular have studied the dissociants of B. subtilis. Soule (1928) studied three forms. His S form was motile, Gram-positive, and formed large oval spores near the middle. At 33 this form grew, at first, diffusely in broth, though later a granular precipitate formed at the side and, after twenty-four to thirtysix hours, a tough tenacious pellicle. On agar plates the S colonies were friable, whitish, slightly glistening, moist, smooth and regular. The R form was rarely motile; it was Gram-positive, forming large oval spores near the middle. In broth, this form gave diffuse growth in three to four hours, yielding a bulky flocculant precipitate in about twenty-four hours. After a longer incubation period the broth became clear, a tough pellicle forming. The R colonies on agar plates were usually twice the size of the S colonies. They were of medusa-head type and viscous. He studied the effect of ph on the dissociation of his S and R forms by inoculating them into beef-tea cultures adjusted to ph's ranging from 6.0 to 8.4, and incubating at 33 for one hundred sixty-eight hours, with examination of the tubes at twentyfour-hour intervals. He found, contrary to our finding, that very little if any multiplication took place at ph 8 to 8.4; that no S forms appeared in any of the tubes inoculated with R forms; that in broth with an initial reaction neutral or slightly acid a somewhat larger amount of dissociation of S to R took place. It should be pointed out that in beef-extract broth there is a possibility that the initial ph may have been decidedly altered during this extraordinarily long incubation period. Graham (1930) obtained four distinct variants of B. subtilis by plating broth cultures on agar. His variants had the characteristics given in table 4. According to Graham, his variants I and II correspond to the S and R forms of Soule. Non-motile variants III and IV have acquired the ability to produce a "mucoid material." The ability of variant II to grow as threads and chains accounts for the irregular medusa-head form of the colonies. The loss of motility and of the H antigen appears to be accompanied by an

13 EFFECT OF REACTION OF MEDIUM ON BACTERIA increase in mucoid material. All four variants possess the same heat-stable (1000C.) somatic antigen. Graham points out the difficulty in calling variant I the S variant, since none of the variants described (except a certain saltsensitive form of one strain) have any true R characters in contrast with the "smooth" variants I and III. Concomitant with the evidence of dissociation involved in colony form and appearance of broth culture and agar slants are TABLE 4 VARIANT I VARIANT II VARIANT III VARIANT IV Appearance Smooth, glis- Dull, uneven, Smooth, domed Like II but of colony tening larger than translucent, shiny, granopaque, de- I, dry, gran- mucoid, de- ular center, fined margin ular, medusa fined margin waxy margin head Broth cul- Turbid; pel- Same but Uniform turture licle in 5 to 6 coarser sedi- bidity days ment Morphology Short, occur- Longer than I, Similar to I Between I and ring singly chains and II or in pairs threads Motility + :4 Suspension Easily emulsi- More difficult Difficult to Stable suspenin 0.5 per fied, perma- to emulsify emulsify be- sion cent salt nent suspen- permanent cause of sion suspension slimy threads H antigen + + other evidences of change in the organism when cultured at ph's which are distinctly acid. The dissociants, if that term may be here applied, differ in Gram character, in sensitivity to gentian violet, in isoelectric point, in motility, and in their suspensions in physiological salt solutions. This behavior indicates that the changes brought about are more or less fundamental. In this respect the ph is an auxiliary rather than a primary factor, and thus differs in the type of 49

14 50 ESTHER WAGNER STEARN AND ALLEN E. STEARN influence it exerts from its influence on staining or stasis behavior where it is probably a primary factor. That is, it affects directly the chemical state of both the bacterial cell and the staining or stasis agent and thus their mutual action, the latter being the phenomenon directly under investigation. In culture work the ph effect is probably brought about by its influence on the chemical state of the bacterial cell and the various classes of food materials, thus affecting their relative availabilities and, indirectly, the nature of the resulting organism. These mutual reactions between organism and foodstuffs are usually not directly under investigation, but changes are noted only indirectly as certain behavior characteristics of the organism are observed. Pampana (1931) has devised a test for distinguishing between the inagglutinable smooth and the agglutinable rough forms in working with the typhoid-paratyphoid, dysentery and Brucella groups. This test is carried out by mixing a 1:500 solution of trypaflavine in saline with a fairly thick suspension of a twentyfour-hour agar culture and incubating for six hours at 370 and overnight at room temperature. He states that if a culture which apparently consists of smooth forms is agglutinated, the probability is that it contains a considerable portion of the rough form. In the study reported in the present paper a few experiments were tried employing this procedure and using twenty-four-hour growth from agar at different ph's with the following results: ph Results denotes that flocculation occurred; -that the suspension remained turbid. One cubic centimeter of dye solution and 0.5 cc. of bacterial suspension were mixed in these tests. Five different determinations were made and the above results are merely suggestive. There are several objections to rigid interpretation. It is extremely difficult to get comparable suspensions before adding them to the dye, and furthermore the ph of the suspensions is probably not identical. There is no question, however, that the organisms at ph's 5.8 to 8.0 show an increasing tendency to flocculate as the ph of the culture increases.

15 EFFECT OF REACTION OF MEDIUM ON BACTERIA 51 SUMMARY 1. At sufficiently low ph, B. subtilis develops a variant which differs from the strain produced by culturing at high ph in colony form, pellicle formation, broth clearing, Gram character, motility, isoelectric point, and sensitivity to gentian violet. 2. There seems to be a fairly critical ph above which the "acid" strain will not develop. 3. By altering cultural conditions one can bring about reversion to the original type, or, if one desires, a reversal of the "acid" and "alkaline" strains. 4. The ph is probably an auxillary rather than a primary factor in these changes, as original variation, reversion or reversal does not result at once in response to ph change. REFERENCES DERNBY, K. G Ann. Inst. Pasteur, 35, 277. GRAHAM, N. C Jour. Path. and Bact., 33, 664. PAMPANA, E. J Ann. d'igiene, 41, 537. QUIRK, A. R Science, N. S., 74, 461. SOULE, M. H Jour. Inf. Dis., 42, 93. STEARN, E. W., AND STEARN, A. E Jour. Bact., 26, 9.

16 52 ESTHER WAGNER STEARN AND ALLEN E. STEARN PLATE 1 FIGS. 1 TO 5 FIG. 1. B. subtilis after five days of incubation on 1.5 per cent beef infusion agar at ph X. FIG. 2. B. subtilis after four days of incubation on 1.5 per cent beef infusion agar at ph 5.5 (showing clearing areas). 4X. FIG. 3. B. subtilis after four days of incubation on 1.5 per cent beef infusion agar at ph X. FIG. 4. B. subtilis after four days of incubation on 1.5 per cent beef infusion agar at ph 7.9 to X.

17 ,, *, :.... *.'. '--... 'e:-...,.,;. <,, -. :.,-,.- :.... ;. '-. ;: :.! tk... W. JOURNAL OF BACTERIOLOGY, VOL XXVI ^''-' i. r ',.:., '' ;v"":,..: S '; '; ':.,.;,....;!: ' <.:,',: X Z<S" 7v.." A.',.'C,', at X ^s ; >$B- $ e v r,.t; <X0';'5 >''t">''' PLATE 1 Downloaded from on August 26, 2018 by guest (Esther Wagnier Stearm anid Alleni E'. Stearn: E-ffeet of reaction of iiiedi uni oni bacteria.

18 54 ESTIIERt AVA(.NER 'STEARN AND) ALLEN E. ST'IEAIRN PI1,.A'I1' 2 F1i SUBTI IS AFTER FORITY-EIGHIT HI OURS on- N ITRI (;NIA t. 1X Tlube 1, pil 5.2. lube 2'. p1 t.o. lube 3, )1l 7.0. 'ltue 4, p

19 JOURNAL OF BACTERIOLOGY, VOL. XXVI PLATE 2 Downloaded from / TeS, (Estiter Wagner Stear aiid Alleni E. Stearim: Effect of reactionl of medium on hacteria ) on August 26, 2018 by guest