VARIATION IN PIGMENTATION AND MORPHOLOGY OF COLONIES

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1 VARIATION IN PIGMENTATION AND MORPHOLOGY OF COLONIES OF GELATINOUS STRAINS OF CHROMOBACTERIUM SPECIES FROM SOIL WILLIAM A. CORPE' Department of Biology, Western Kentucky State College, Bowling Green, Kentucky Received for publication April 29, 1953 The author recently isolated a number of Chromobacterium strains from soil by a simple technique (Corpe, 1951). When these strains were streaked onto the surface of plates of plain nutrient agar or of other common media, a considerable vaiiation in colonial form and pigmentation appeared. The character of growth of these isolates in broth cultures as well as on agar differed considerablv from strains from other sources that have been studied by the writer and from most of the strains described in the literature. This paper is concerned with the variation in pigmentation and colonial morphology of some of the soil isolates. A cultural, physiological, and morphological comparison of the soil isolates with strains fiom other sources will appear elsewhere. MATERIALS AND METHODS The Chromobacterium stiains studied were isolated from soil after enrichmenlt on sterile rice grains in distilled water. The cultures were identified as Chromobacteriutm species according to Bergey's Manual (Breed et al., 1948). The isolates were maintained on 1 per cent tryptone (Difco) agar slants or broth. Stock transfers were made every two weeks into fresh media. After growth at 25 C for 48 hours, the cultures were kept at 2 to 4 C until the next transfer. The cultures selected for study possessed various special characteristics which in most instances were probably of minor taxonomic importance. Strains la and 2 were selected because they had lost their ability to produce violet pigment in the medium on which they were isolated, namely, plain nutrient agar. Furthermore stock cultures of these strains had failed to form pigment on 1 per cent tryptone agar or broth for 1 Present address: Department of Bacteriology, Pennsylvania State College, State College, Pa. over a year and showed no tendency to revert to the original pigmented condition. Stirain 2 grew poorly on the plain nutrient agar and had a greater ability to ferment sugars and hydrolyze starch than the other strains. Strain D3 was selected because it could grow at a higher temperature than the majority of the soil strains and produce three types of colonies: both wrinkled and smooth colonies which were tough, gelatinous and raised, and a flat nongelatinous type. Strain 21 a and L2a were selected because they dissociated readily into gelatinous and nongelatinous colonial types. The stabilities of these colonial variants were studied as follows: The cultures were inoculated into 1 per cent tryptone broth and incubated at 25 C for 24 hours. These broth cultures then were streaked onto 1 per cent tryptone agar plates. As soon as colonial differences appeared at about 3 days at 25 C, different colonial types were picked into 1 per cent tryptone broth and the process was repeated until strains showing definite colonial differences had been isolated. The variants were transferied into 1 per cent tryptone broth at weekly intervals, incubated for 24 hours, and refrigerated until the next transfer. From time to time these strains were streaked on 1 per cent tryptone agar to make sure of their purity. Strains which failed to produce violet pigment on 1 pel cent tryptone nevertheless grew luxuriantly on that medium, and in old cultures the growth itself and the medium immediately surrounding it became tinted with a yellowish substance. This characteristic was not noticed in strains producing violet pigment on tryptone agar. Plain nutrient agar, containing 5 g of peptone (Difco) and 3 g of beef extract (Difco) per liter, supported the same type of growth and the pioduction of the yellowish substance. In an earlier experiment it was noted that the 470

2 1953] CHROMOBACTERIUM SPECIES FROM SOIL 471 yellowish substance was not produced in plain gelatin by nonpigmented gelatinous variants. This observation suggested that the protein or protein hydrolyzate used in the preparation of the medium might be a critical factor in pigment production and might play a role in the variation in pigmentation of colonies observed. Several proteins, peptones, and hydrolyzates of proteinaceous substances were prepared in 1 per cent concentrations, the reaction adjusted to ph 7.0, and 1.5 per cent agar added. The peptones and hydrolyzates (all Difco products) used were: peptone, tryptone, tryptose, protone, neopeptone, casitone, soytone, and proteose peptone (2, 3, 4). Gelatin (Difco) and purified casein2 were the proteins used. Sterile plates containing these media were streaked with variant strains, incubated at 25 C for 2 weeks, and observed periodically for the production of violet pigment and the gelatinous and nongelatinous colonies. Gelatin is known to lack tyrosine and tryptophan. Difco peptone contains a smaller quantity of these amino acids than does tryptone (Difco Manual, 1953). One per cent peptone agar plates containing from 1 X 10-6 to 1 per cent of added tryptophan and tyrosine were streaked with the nonpigmented variants. The cultures were observed and compared with 1 per cent peptone and 1 per cent tryptone controls, for a period of one week, for the development of pigment. Beef extract and yeast extract also were tested, in various concentrations, in 1 per cent peptone to determine their effect on pigmentation and colonial form. One per cent peptone agar plates containing from 0.01 per cent to 0.5 per cent of beef extract and yeast extract were prepared. The surface of the plates were dried of excess moisture. Twenty-four hour tryptone broth cultures of strains la, D3, L2a, and 21a were diluted to 1 X 105 and 1 X 106. One-tenth of a ml of each dilution was spread carefully over the surface of the plates described above along with 1 per cent peptone agar controls. Platings were done in triplicate. The inoculated plates were incubated at 25 C for 5 days, and the percentage of pigmented and nonpigmented, gelatinous and nongelatinous colonies was determined. 2 Coleman-Bell purified casein was dissolved in 1 N NaOH, neutralized with 1 N HCl, and the ph adjusted to ph 7.0. A synthetic medium (Wilson and Knight, 1947) with the following composition was used to ascertain whether or not amino nitrogen was required for the production of the gelatinous material and pigment: Na(NH4)HPO4, 1.0 g; MgSO4.7H20, 0.2 g; CaCl2, 0.1 g; K2HPO4, 1.0 g; FeSO4, 0.1 g; distilled water, 1,000 ml. One milliliter of yeast extract was added for growth factors, and the ph was adjusted to 7.0. Glucose was added to this basal medium aseptically after sterilization. To ascertain whether any other characteristics were associated with the observed colonial variation, a number of physiological tests were performed on parent strains and their variants. Tests were made for hydrolysis of starch, gelatin, and casein, action in litmus milk, and fermentation of glucose, fructose, mannose, maltose, and trehalose. Measurements were made of cell dimensions. RESULTS The chromobacteria commonly formed a thick, gelatinous, violet growth on 1 per cent tryptone agar or on plain nutrient agar strokes, when incubated at 25 C for a week. After several transfers on these media most strains began to lose some pigment producing ability or appeared to develop pigmented and nonpigmented areas in the streak. Three strains lost their pigment producing ability altogether in transfer on 1 per cent tryptone agar. All of the soil strains isolated produced a very tough, gelatinous growth on plain nutrient agar and broth after isolation. The growth on agar could be removed only if forcibly peeled away, and in broth a very tough pellicle was produced. The pellicle could often be lifted intact from the almost clear medium beneath. The texture of the pellicle resembled the tough film or "mother of vinegar" formed by species of Acetobacter. The gelatinous nature of the growth varied considerably from one transfer to the next of a single culture, at times being almost lacking and on other occasions being so tough that removal of growth on a culture loop was impossible. Characteristics of parent and variant strains on agar and in broth are listed in table 1. Figures 1 to 7 show several of the various types of colonies observed. Strains lacking in violet pigment on 1 per cent

3 472 WILLIAM A. CORPE [VOL. 66 TABLE 1 The cultural characteristics of strains of Chromobacterium in 1 per cent tryptone STRAIN BROTH AGAR SLANT NO. la 2 D3 D3a D3d 21a 21aa 21ab Thick ring or pellicle, white becoming yellow with age, slightly cloudy. Thin, membranelike, very tough pellicle, no violet pigment, broth clear. Thick pellicle, with or without violet pigment, broth Thick, violet, frequently wrinkled pellicle, usually very tough, Thick, violet, often wrinkled gelatinous pellicle, slightly cloudy, unstable. Violet, thin ring or light pellicle, very cloudy throughout. Thick slightly yellowish pellicle, usually tough and gelatinous, no violet pigment, very Thin ring or light pellicle, pigment not released into broth, broth cloudy. Raised growth, white becoming yellowish, nongelatinous. Flat, tough, dirty white becoming yellowish, no violet pigment. Well raised, gelatinous, tough, usually wrinkled with deep furrows, well pigmented. Well raised, gelatinous, very tough, heavily wrinkled, unstable. Slightly raised, nongelatinous, not wrinkled. Raised, smooth, usually gelatinous and tough, with or without violet pigment. Well raised, tough, gelatinous, smooth, no violet pigment. Slightly raised, smooth, deep violet pigment, growth smears when touched with a wire needle. STRAIN NO. L2a L2aa L2ab TABLE 1-Continued Thick, violet, tough, gelatinous pellicle, broth Heavy ring and deep violet, thin pellicle, pigmented growth retained flocculent changes when growth is disturbed. Broth Thick tough, violet pellicle, broth AGAR SLANT Raised, tough, violet growth with lightly and intensely pigmented areas. Slightly raised, intensely pigmented, smooth butyrous smears when touched with "I wire needle. Well raised anid gelatinous, smooth surface, slightly yellowish tinige at the edge of the growth. tryptone produced moderate amounts of violet pigment on peptone, neopeptone, purified casein, and gelatin. Growth on these media was not as luxuriant as on 1 per cent tryptone. Colonies initially were white, but within 48 houirs they became pigmented in the center and on further incubation at 25 C the pigment spread rapidly to the periphery of the colony. The yellowish substance, previously described, was not produced by these strains in visible amounts in peptone, neopeptone, casein, and gelatin. Strains that were pigmented on tryptone produced very intensely pigmented growth on peptone, neopeptone, casein, and gelatin, but again growth was not as luxuriant as on 1 per cent trvptone. Pigmented strains also produced moderate amounts of pigment in the other hydrolyzates tested. Strain 2 did not produce pigment under the conditions found to be favorable for pigment formation by the other strains which were nonpigmented on tryptone. This strain had lost its ability to produce violet pigment soon after isolation from soil. Furthermore, it grewv verv poorly on peptone and tryptone and had to be transferred often on these media to remain viable. OD media containing soluble st-rch, such as Difco

4 1953] CHROMOBACTERIUM SPECIES FROM SOIL 473 starch agar, excellent growth was obtained with this strain as with the other strains and the property of pigment production could be recalled. It was found further that the incorporation of 1 per cent glucose in peptone agar permitted pigment production by this strain although tryptone could not replace peptone for this purpose. Pigment production and texture of the growth on the various media employed are summarized in table 2. Strain L2a dissociated into gelatinous and nongelatinous tvpes. Both of these are pigmented on tryptone but more intensely on peptone. All attempts to isolate nonpigmented variants of this strain failed. The gelatinous type was STRAIN NO. la 2 D3 D3a D3d 21a 21aa 21ab L2a L2aa L2ab of D3 cultures failed to increase the proportion of nongelatinous strains. It was found that the incorporation of 0.5 per cent yeast extract or beef extract in 1 per cent peptone caused the gelatinous variants of 21a and L2a to become wrinkled and have a mulberry appearance. Gelatinous strains of D3 became convoluted even more heavily under such conditions. Twenty-four hour peptone broth cultures were TABLE 2 The effect of several agar media on pigment production by variant strains of Chromobacterium 1% peptone 1% peptone 1% peptone 1% 0.3% glucose Ibeef extract 1% tryptone AGAR MEDIA* 1% try one 1% starch gelatin agar (Difco) -t 1% casein * Good pigmentation, fair pigmentation, faintly pigmented, 4h pigmented with nonpigmented sectors, nonpigmented. - t Wilson and Knight medium described in the text. gelatinous colonies formed was essentially the pigmented only lightly on tryptone and plain nutrient agar, but when transferred back to peptone intense pigmentation returned. In most experiments L2a was similar to 21a in respect to the texture of the colonies and ratio of gelatinous to nongelatinous strains. Strain D3 also produced gelatinous and nongelatinous variants. Although the former were predominant, the latter appeared with regularity. A large proportion of the colonies produced by D3 in media containing amino nitrogen was wrinkled and uniformly gelatinous. The wrinkled condition of the colonies varied from light dimpling to very deep convolutions. All attempts to select a wrinkled variant which produced nothing but wrinkled colonies failed. The aging diluted to 1 X 105, and 0.1 ml was spread evenly over the surface of agar plates containing various concentrations of beef extract and yeast extract. These substances did not affect the colonial texture since the percentage of non- isynthetict same as in the control plates. Beef extract and yeast extract exerted a depressing effect on pigment production in concentrations above 0.05 per cent in peptone or tryptone agar. The effect of 0.5 per cent beef extract is shown in table 3. The amino acids, tyrosine and tryptophan, are lacking in gelatin and present in lower concentration in Difco peptone than in Difco tryptone. When 0.5 per cent to 1 per cent of tryptophan were added to peptone, the gelatinous strains developed a yellowish substance comparable with that formed in 1 per cent tryptone broth. Experiments showed, however, that neither increased concentrations of tryptophan or tyrosine

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6 1953] CHROMOBACTERIUM SPECIES FROM SOIL 475 or combinations of the two in peptone had any significant effect on production of violet pigment or the gelatinous nature of growth. No pronounced differences were noted in cell measurements or in the physiological reactions produced by the variant and parent cultures. The nature of the gelatinous colony. A gelatinous colony of strain 21a was placed in a test tube and crushed with a glass rod till small pieces had been torn away and floated free of the mass. A few TABLE 3 The effect of 0.5 per- cent beef extract on variation in pigmentation and colonial morphology of several strains of Chromobacterium 1% PEPTONE AGAR 1% PEPTONE AGAR 0.5% PERCENTAGE OF BEEF EXTRACT PERCENTAGE OF STRAIN NO. Pigmented Non- Pigmented Nonpigmented *G NonG G NonG G NonG G NonG 21a la L2a D i * G = gelatinous; NonG = nongelatinous. of the small pieces were deposited onto a scrupulously clean glass slide and stained by the flagella staining technique of Leifson (1951). The gelatinous material was stained a light pink. The bacteria stained a deep pink or red and were embedded haphazardly in the gelatinous matrix. The edges of the pieces of gelatinous material were frayed, and when observed closely seemed to be made up of millions of tiny threads, much finer than the flagella which were monotrichous and polar. Occasionally, bacteria could be seen lying free of the large mass. Some of these appeared to retain bits of fibrous mateirial, but most of them were clean of this material. Gelatinous colonies picked from the surface of ]. per cent tryptone agar were placed in test tubes and subjected to various solvents (table 4). The gelatinous material in the colonies was not dissolved by the neutral solvents tested buit was soluble in both acid and alkaline solutions TABLE 4 The solubility of gelatinous colonies of soil chromobacteria SOLVENT SOLUBILITY Alcohol, 50%... _ Alcohol, 95%... _ Ether... Carbon tetrachloride... - NaOH, 0.1 N min NaOH, 1.0 N... 5 min NaOH, concentrated... immediately HCl, 0.1 N... - HCl, 1.0 N... - HCI, concentrated... 4 min Cold distilled water... - Boiling distilled water. 5 min and boiling water. Several colonies were hydrolyzed in boiling, dilute HCl, neutralized and tested for carbohydrates by the Fehling test. Negative tests were obtained. DISCUSSION It is perhaps of some importance that the soil strains have showvn the characteristic of producing gelatinous variants while these were not encountered in strains from other sources. Some of the cultures that had been isolated from other Figure 1. Strain 21a on 1 per cent tryptone agar, showing pigmented and nonpigmented colonies. Pigmented colonies are nongelatinous. Nonpigmented colonies are gelatinous. Figure 2. Strain 21a on 1 per cent peptone agar, both gelatinous and nongelatinous variants are pigmented. Figure 3. Strain 2 on 1 per cent peptone-glucose agar. The dark ring that appears in the colony is violet. The rest of the colony is nonpigmented. Figure 4. Strain D3 on glucose-peptone agar, showing wrinkled gelatinous colonies and smooth nongelatinous, pigmented colonies. Figure 5. Strain la on 1 per cent tryptone agar. The colonies are nonpigmented and nongelatinous. Figure 6. Strain la on 1 per cent peptone agar. Two of the colonies are pigmented very lightly; the others are pigmented more intensely. Figure 7. Strain L2a on glucose-tryptone agar, showing gelatinous and nongelatinous pigmented colonies.

7 476 WILLIAM A. CORPE [VOL. 66 sources (principally water) do have the ability to produce a membrane-like growth in broth cultures which at times holds together very tenaciously. Most of these cultures were labeled Chromobacterium amethystinum. They also showed variation in pigmentation and colonial form, but in every instance they failed to produce the gelatinous tvpe of growth described in this paper. This difference is not considered to be sufficiently great to warrant considering the soil strains as separate species. Ward (1898) described violet pigmented bacilli isolated from the Thames which produced tough growth on agar and frequently failed to develop pigment on artificial media. The variations described occurred at approximately optimum temperature for growth, which for most strains was 25 to 28 C and might be considered as "normal variation" in contrast to the type of variation induced by specific chemicals (Hutchinson and Kelner, 1942; Kelner, 1947) or special environmental conditions imposed on the culture during growth. The soil strains studied, since their isolation, have changed considerably as far as pigmentation and colonial morphology are concerned. The results at least partially explain the loss of pigment producing ability bv la and 2. When strain la was isolated initially from soil, it was pigmented uniformly and was gelatinous on plain mntrient agar. Sometime during routine transfer in tryptone broth it lost its ability to produce pigment and still later lost its ability to produce the tough, gelatinous colony. One other strain (not reported here), isolated from the same plot of soil, has maintained its gelatinous nature though nonpigmented variants exist in tryptone broth culture. Somewhat the same changes seem to have occurred in strain 2. This organism initially was well pigmented and grew well on nutrient agar. During cultivation it lost its ability to produce pigment and to grow well in tryptone or peptone medium. It grew well on synthetic media with glucose as the source of carbon and ammonium salts as the source of nitrogen. It also grew well and produced pigment on 1 per cent gelatin agar. Strain 2 also grew well on 1 per cent gelatin agar. It appeared then that peptone and tryptone contained some substance lacking in gelatin which inhibited pigmentation in strain 2 and in the other strains nonpigmented on tryptone. Strain 21a dissociated readily into gelatinous and nongelatinous, pigmented and nonpigmented colonies on tryptone. When first isolated this strain also produced a uniformly violet gelatinous type of growth. The first sign of variation observed was in the failure of the strain to produce all gelatinous colonies. Later however, it dissociated further to produce nonpigmented, gelatinous variants. Strain L2a at present seems to be undergoing the same pattern of changes. As reported here it produced pigmented, gelatinous and pigmented, nongelatinous variants. Strain D3 appeared to follow the same pattern of dissociation shown bv L2a except that in most of the media tested D3 produced a large number of wrinkled or rugose colonies. Pigmentation of the gelatinous strains at least has been shown to be influenced by the medium, but the tough gelatinous nature of the colonies seems to be more stable. The latter was altered somewhat in the degree of toughness as the colony aged, possibly due to a release of autolytic enzymes by the bacteria, causing a breakdown in the gelatinous material. Gelatinous colonies showed no tendency to revert to the nongelatinous state under experimental conditions although la lost this ability for an unexplained reason. Pigmentation in nonpigmented gelatinous strains, however, could be recalled on media containing 1 per cent peptone or gelatin. Studies on the nutrition of soil chromobacteria probably would be helpful in elucidating some of these observations. ACKNOWLEDGMENT The author wishes to express his appreciation to Dr. K. F. Gregory of the Department of Bacteriology, Dalhousie University, Halifax, Nova Scotia, for his suggestions and criticism of the manuscript. SUMMARY Variations in pigmentation, texture of growth, and colonial form of several strains of Chromobacteritum species are described. Pigmentation was influenced by the type of medium used. All soil chromobacteria dissociated into gelatinous and nongelatinous strains. Beef extract, yeast extract, tryptone, and a number of other peptones and protein hydrolyzates were found to discourage the formation of pigment by all strains although

8 1953] CHROMOBACTERIUM SPECIES FROM SOIL 477 the gelatinous strains were affected most. Inhibitory substances weie not evident in peptone, neopeptone, and gelatin in 1 per cent concentration. The substance inhibiting pigmentation in tryptone is neither tyrosine nor tryptophan. Tryptophan was shown to be the source of the diffusible vellowish substance produced in tryptone broth. No significant differences wxere found in the biochemical characteristics and cell measurements between parent strains and colonial variants. The colonies could be dissolved by acid and alkaline solutions and by boiling distilled water but not by cold distilled water and neutral solvents The large gelatinous colony typical of soil strains was not seen in cultures from sources other than soil. REFERENCES BREED, R. S., MIURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology. 6th ed. The Williams and Wilkins Co., Baltimore, Md. CORPE, W. A A study of the widespread distribution of chromobacterium species in soil by a simple technique. J. Bact., 62, HUTCHINSON, W. G., AND KELNER, A A study of secondary colonies of Chromobacteriiun violaceutm. J. Bact., 43, (Abstract) KELNER, A Secondary colonies of bacteria induced by salts of alkali metals, with special reference to Chromnobacteriumn violaceum and other bacteria on lithium chloride agar. Am. J. Botany, 34, LEIFSON, E Staining, shape, and arrangement of bacterial flagella. J. Bact., 62, Mlanual of dehydrated culture miiedia and reagents. 9th ed Difco Laboratories, Inc., Detroit. WARD, H. MI A violet bacillus from the Thames. Ann. Botany, 12, WILSON, P. W., AND KNIGHT, S. G Experiments in bacterial physiology. Burgess Publishing Co., Minneapolis, Minn. Downloaded from on July 15, 2018 by guest