VARIATION IN PIGMENTATION AND MORPHOLOGY OF COLONIESOF 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 ofChromobacterium strains from soil by a simpletechnique (Corpe, 1951). When these strainswere streaked onto the surface of plates of plainnutrient agar or of other common media, aconsiderable vaiiation in colonial form and pig-mentation appeared. The character of growth ofthese isolates in broth cultures as well as on agardiffered considerablv from strains from othersources that have been studied by the writer andfrom most of the strains described in the litera-ture.

This paper is concerned with the variation inpigmentation and colonial morphology of someof the soil isolates. A cultural, physiological, andmorphological comparison of the soil isolateswith strains fiom other sources will appear else-where.

MATERIALS AND METHODS

The Chromobacterium stIains studied were iso-lated from soil after enrichmenlt on sterile ricegrains in distilled water. The cultures were identi-fied as Chromobacteriutm species according toBergey's Manual (Breed et al., 1948). The iso-lates were maintained on 1 per cent tryptone(Difco) agar slants or broth. Stock transferswere made every two weeks into fresh media.After growth at 25 C for 48 hours, the cultureswere kept at 2 to 4 C until the next transfer.The cultures selected for study possessed vari-

ous special characteristics which in most in-stances were probably of minor taxonomic im-portance.

Strains la and 2 were selected because theyhad lost their ability to produce violet pigmentin the medium on which they were isolated,namely, plain nutrient agar. Furthermore stockcultures of these strains had failed to form pig-ment 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 revertto the original pigmented condition. Stirain 2grew poorly on the plain nutrient agar and hada greater ability to ferment sugars and hydrolyzestarch than the other strains.

Strain D3 was selected because it could growat a higher temperature than the majority of thesoil strains and produce three types of colonies:both wrinkled and smooth colonies which weretough, gelatinous and raised, and a flat non-gelatinous type.

Strain 21 a and L2a were selected because theydissociated readily into gelatinous and nonge-latinous colonial types.The stabilities of these colonial variants were

studied as follows: The cultures were inoculatedinto 1 per cent tryptone broth and incubated at25 C for 24 hours. These broth cultures thenwere streaked onto 1 per cent tryptone agarplates. As soon as colonial differences appearedat about 3 days at 25 C, different colonial typeswere picked into 1 per cent tryptone broth andthe process was repeated until strains showingdefinite colonial differences had been isolated.The variants were transferied into 1 per centtryptone broth at weekly intervals, incubatedfor 24 hours, and refrigerated until the nexttransfer. From time to time these strains werestreaked on 1 per cent tryptone agar to makesure of their purity.

Strains which failed to produce violet pigmenton 1 pel cent tryptone nevertheless grew luxuri-antly on that medium, and in old cultures thegrowth itself and the medium immediately sur-rounding it became tinted with a yellowish sub-stance. This characteristic was not noticed instrains producing violet pigment on tryptoneagar. Plain nutrient agar, containing 5 g ofpeptone (Difco) and 3 g of beef extract (Difco)per liter, supported the same type of growth andthe pioduction of the yellowish substance.

In an earlier experiment it was noted that the

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yellowish substance was not produced in plaingelatin by nonpigmented gelatinous variants.This observation suggested that the protein orprotein hydrolyzate used in the preparation ofthe medium might be a critical factor in pigmentproduction and might play a role in the variationin pigmentation of colonies observed.

Several proteins, peptones, and hydrolyzatesof proteinaceous substances were prepared in 1per cent concentrations, the reaction adjustedto pH 7.0, and 1.5 per cent agar added. Thepeptones and hydrolyzates (all Difco products)used were: peptone, tryptone, tryptose, protone,neopeptone, casitone, soytone, and proteose pep-tone (2, 3, 4). Gelatin (Difco) and purified casein2were the proteins used. Sterile plates containingthese media were streaked with variant strains,incubated at 25 C for 2 weeks, and observedperiodically for the production of violet pigmentand the gelatinous and nongelatinous colonies.

Gelatin is known to lack tyrosine and tryp-tophan. Difco peptone contains a smaller quan-tity of these amino acids than does tryptone(Difco Manual, 1953). One per cent peptoneagar plates containing from 1 X 10-6 to 1 percent of added tryptophan and tyrosine werestreaked with the nonpigmented variants. Thecultures were observed and compared with 1per cent peptone and 1 per cent tryptone con-trols, for a period of one week, for the develop-ment of pigment.

Beef extract and yeast extract also were tested,in various concentrations, in 1 per cent peptoneto determine their effect on pigmentation andcolonial form. One per cent peptone agar platescontaining from 0.01 per cent to 0.5 per centof beef extract and yeast extract were prepared.The surface of the plates were dried of excessmoisture. Twenty-four hour tryptone brothcultures of strains la, D3, L2a, and 21a werediluted to 1 X 105 and 1 X 106. One-tenthof a ml of each dilution was spread carefully overthe surface of the plates described above alongwith 1 per cent peptone agar controls. Platingswere done in triplicate. The inoculated plateswere incubated at 25 C for 5 days, and the per-centage of pigmented and nonpigmented, gelat-inous and nongelatinous colonies was deter-mined.

2 Coleman-Bell purified casein was dissolved in1 N NaOH, neutralized with 1 N HCl, and the pHadjusted to pH 7.0.

A synthetic medium (Wilson and Knight,1947) with the following composition was usedto ascertain whether or not amino nitrogen wasrequired for the production of the gelatinousmaterial and pigment: Na(NH4)HPO4, 1.0 g;MgSO4.7H20, 0.2 g; CaCl2, 0.1 g; K2HPO4, 1.0g; FeSO4, 0.1 g; distilled water, 1,000 ml. Onemilliliter of yeast extract was added for growthfactors, and the pH was adjusted to 7.0. Glucosewas added to this basal medium aseptically aftersterilization.To ascertain whether any other characteris-

tics were associated with the observed colonialvariation, a number of physiological tests wereperformed on parent strains and their variants.Tests were made for hydrolysis of starch, gelatin,and casein, action in litmus milk, and fermenta-tion of glucose, fructose, mannose, maltose,and trehalose. Measurements were made of celldimensions.

RESULTS

The chromobacteria commonly formed a thick,gelatinous, violet growth on 1 per cent tryp-tone agar or on plain nutrient agar strokes, whenincubated at 25 C for a week. After severaltransfers on these media most strains began tolose some pigment producing ability or appearedto develop pigmented and nonpigmented areasin the streak. Three strains lost their pigmentproducing ability altogether in transfer on 1 percent tryptone agar.

All of the soil strains isolated produced a verytough, gelatinous growth on plain nutrient agarand broth after isolation. The growth on agarcould be removed only if forcibly peeled away,and in broth a very tough pellicle was produced.The pellicle could often be lifted intact from thealmost clear medium beneath. The texture of thepellicle resembled the tough film or "motherof vinegar" formed by species of Acetobacter.The gelatinous nature of the growth variedconsiderably from one transfer to the nextof a single culture, at times being almost lackingand on other occasions being so tough thatremoval of growth on a culture loop was im-possible.

Characteristics of parent and variant strainson agar and in broth are listed in table 1. Figures1 to 7 show several of the various types of coloniesobserved.

Strains lacking in violet pigment on 1 per cent

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WILLIAM A. CORPE

TABLE 1The cultural characteristics of strains of Chromo-

bacterium in 1 per cent tryptone

STRAIN BROTH AGAR SLANTNO.

Thick ring or pel-licle, white be-coming yellowwith age, slightlycloudy.

Thin, membrane-like, very toughpellicle, no violetpigment, brothclear.

Thick, violet, fre-quently wrinkledpellicle, usuallyvery tough,slightly cloudy.

Thick, violet, oftenwrinkled gelati-nous pellicle,slightly cloudy,unstable.

Violet, thin ring or

light pellicle, verycloudy through-out.

Thick pellicle, withor without violetpigment, brothslightly cloudy.

Thick slightly yel-lowish pellicle,usually tough andgelatinous, no vi-olet pigment, veryslightly cloudy.

Thin ring or lightpellicle, pigmentnot released intobroth, brothcloudy.

Raised growth,white becomingyellowish, non-

gelatinous.

STRAINNO.

L2a

L2aa

Flat, tough, dirtywhite becomingyellowish, no vi-olet pigment.

Well raised, gelat-inous, tough,usually wrinkledwith deep fur-rows, well pig-mented.

Well raised, gelati-nous, very tough,heavily wrinkled,unstable.

Slightly raised,nongelatinous,not wrinkled.

Raised, smooth,usually gelati-nous and tough,with or withoutviolet pigment.

Well raised, tough,gelatinous,smooth, no violetpigment.

Slightly raised,smooth, deep vio-let pigment,growth smears

when touchedwith a wireneedle.

L2ab

TABLE 1-Continued

Thick, violet, tough,gelatinous pel-licle, brothslightly cloudy.

Heavy ring anddeep violet, thinpellicle, pigmen-ted growth re-tained flocculentchanges whengrowth is dis-turbed. Brothslightly cloudy.

Thick tough, violetpellicle, brothslightly cloudy.

AGAR SLANT

Raised, tough, vio-let growth withlightly and in-tensely pigmen-ted areas.

Slightly raised, in-tensely pig-mented, smoothbutyrous smearswhen touchedwith "I wireneedle.

Well raised anid ge-latinous, smoothsurface, slightlyyellowish tinige atthe edge of thegrowth.

tryptone produced moderate amounts of violetpigment on peptone, neopeptone, purified casein,and gelatin. Growth on these media was not as

luxuriant as on 1 per cent tryptone. Coloniesinitially were white, but within 48 houirs theybecame pigmented in the center and on furtherincubation at 25 C the pigment spread rapidlyto the periphery of the colony. The yellowishsubstance, previously described, was not pro-duced by these strains in visible amounts inpeptone, neopeptone, casein, and gelatin. Strainsthat were pigmented on tryptone produced very

intensely pigmented growth on peptone, neo-

peptone, casein, and gelatin, but again growthwas not as luxuriant as on 1 per cent trvptone.Pigmented strains also produced moderateamounts of pigment in the other hydrolyzatestested.

Strain 2 did not produce pigment under theconditions found to be favorable for pigmentformation by the other strains which were non-

pigmented on tryptone. This strain had lost itsability to produce violet pigment soon after iso-lation from soil. Furthermore, it grewv verv poorlyon peptone and tryptone and had to be trans-ferred often on these media to remain viable. ODmedia containing soluble st-rch, such as Difco

la

2

D3

D3a

D3d

21a

21aa

21ab

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starch agar, excellent growth was obtained withthis strain as with the other strains and theproperty of pigment production could be re-called. It was found further that the incorporationof 1 per cent glucose in peptone agar permittedpigment production by this strain althoughtryptone could not replace peptone for thispurpose. Pigment production and texture of thegrowth on the various media employed are sum-marized in table 2.

Strain L2a dissociated into gelatinous andnongelatinous tvpes. Both of these are pigmentedon tryptone but more intensely on peptone.All attempts to isolate nonpigmented variantsof this strain failed. The gelatinous type was

of D3 cultures failed to increase the proportionof nongelatinous strains.

It was found that the incorporation of 0.5per cent yeast extract or beef extract in 1 percent peptone caused the gelatinous variants of21a and L2a to become wrinkled and have amulberry appearance. Gelatinous strains of D3became convoluted even more heavily undersuch conditions.

Twenty-four hour peptone broth cultures werediluted to 1 X 105, and 0.1 ml was spread evenlyover the surface of agar plates containing vari-ous concentrations of beef extract and yeastextract. These substances did not affect thecolonial texture since the percentage of non-

TABLE 2The effect of several agar media on pigment production by variant strains of Chromobacterium

AGAR MEDIA*

1% peptone 1% peptone+ 1% +0.3%glucose Ibeef extract

++

+++

+++

++

++

++

+++

1% tryptone1% try+ one 1% starch gelatin

agar (Difco)

++

++

++

+++

++

++

++

+++

-t++++

++

++

+++

++

+++

+++

1% casein

+++

++

+++

iSyn-thetict

+++++++

* +++ Good pigmentation, ++ fair pigmentation, + faintly pigmented, 4h pigmented with non-

pigmented sectors, - nonpigmented.t Wilson and Knight medium described in the text.

pigmented only lightly on tryptone and plainnutrient agar, but when transferred back topeptone intense pigmentation returned.

In most experiments L2a was similar to 21ain respect to the texture of the colonies and ratioof gelatinous to nongelatinous strains.

Strain D3 also produced gelatinous and non-

gelatinous variants. Although the former were

predominant, the latter appeared with regular-ity. A large proportion of the colonies producedby D3 in media containing amino nitrogen was

wrinkled and uniformly gelatinous. The wrinkledcondition of the colonies varied from lightdimpling to very deep convolutions. All attemptsto select a wrinkled variant which producednothing but wrinkled colonies failed. The aging

gelatinous colonies formed was essentially thesame as in the control plates. Beef extract andyeast extract exerted a depressing effect on

pigment production in concentrations above0.05 per cent in peptone or tryptone agar. Theeffect of 0.5 per cent beef extract is shown intable 3.The amino acids, tyrosine and tryptophan, are

lacking in gelatin and present in lower concentra-tion in Difco peptone than in Difco tryptone.When 0.5 per cent to 1 per cent of tryptophanwere added to peptone, the gelatinous strainsdeveloped a yellowish substance comparablewith that formed in 1 per cent tryptone broth.Experiments showed, however, that neither in-creased concentrations of tryptophan or tyrosine

1% peptone

++

+++

+++

STRAINNO.

la2D3D3aD3d21a21aa21abL2aL2aaL2ab

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474 WILLIAM A. CORPE, [

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CHROMOBACTERIUM SPECIES FROM SOIL

or combinations of the two in peptone had anysignificant effect on production of violet pigmentor the gelatinous nature of growth.No pronounced differences were noted in cell

measurements or in the physiological reactionsproduced by the variant and parent cultures.The nature of the gelatinous colony. A gelatinous

colony of strain 21a was placed in a test tube andcrushed with a glass rod till small pieces had beentorn away and floated free of the mass. A few

TABLE 3The effect of 0.5 per- cent beef extract on variation inpigmentation and colonial morphology of

several strains of Chromobacterium

1% PEPTONE AGAR 1% PEPTONE AGAR + 0.5%PERCENTAGE OF BEEF EXTRACT PERCENTAGE OF

STRAINNO. Pigmented Non- Pigmented Nonpigmented

*G NonG G NonG G NonG G NonG

21a 1.2 98.8 0 0 0 96.1 3.9 0la 0 100.010 0 0 0 0 100.0L2a 33.41 64.61 0 0 29.1 10.7 0 60.2D3 94.81 5.2i 0 0 56.1 8.4 35.5 0

* G = gelatinous; NonG = nongelatinous.

of the small pieces were deposited onto a scrupu-lously clean glass slide and stained by the flagellastaining technique of Leifson (1951). The gelat-inous material was stained a light pink. Thebacteria stained a deep pink or red and wereembedded haphazardly in the gelatinous matrix.The edges of the pieces of gelatinous materialwere frayed, and when observed closely seemedto be made up of millions of tiny threads, muchfiner than the flagella which were monotrichousand polar. Occasionally, bacteria could be seen

lying free of the large mass. Some of these ap-peared to retain bits of fibrous mateirial, but mostof them were clean of this material.

Gelatinous colonies picked from the surfaceof ]. per cent tryptone agar were placed in testtubes and subjected to various solvents (table4). The gelatinous material in the colonies wasnot dissolved by the neutral solvents tested buitwas soluble in both acid and alkaline solutions

TABLE 4The solubility of gelatinous colonies of soil chromo-

bacteria

SOLVENT SOLUBILITY

Alcohol, 50% ................ _Alcohol, 95% ................ _Ether........Carbon tetrachloride....... -

NaOH, 0.1 N................ + 15 minNaOH, 1.0 N ................ + 5minNaOH, concentrated .......... + immediatelyHCl, 0.1 N ................ -

HCl, 1.0 N ................ -

HCI, concentrated ............ + 4 minCold distilled water........... -

Boiling distilled water . + 5 min

and boiling water. Several colonies were hydro-lyzed in boiling, dilute HCl, neutralized andtested for carbohydrates by the Fehling test.Negative tests were obtained.

DISCUSSION

It is perhaps of some importance that the soilstrains have showvn the characteristic of produc-ing gelatinous variants while these were not en-countered in strains from other sources. Someof 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 pig-mented.

Figure 3. Strain 2 on 1 per cent peptone-glucose agar. The dark ring that appears in the colony isviolet. The rest of the colony is nonpigmented.

Figure 4. Strain D3 on glucose-peptone agar, showing wrinkled gelatinous colonies and smooth non-gelatinous, 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.

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WILLIAM A. CORPE

sources (principally water) do have the abilityto produce a membrane-like growth in brothcultures which at times holds together verytenaciously. Most of these cultures were labeledChromobacterium amethystinum. They also showedvariation in pigmentation and colonial form, butin every instance they failed to produce thegelatinous tvpe of growth described in this paper.This difference is not considered to be sufficientlygreat to warrant considering the soil strains asseparate species.Ward (1898) described violet pigmented bacilli

isolated from the Thames which produced toughgrowth on agar and frequently failed to developpigment on artificial media.The variations described occurred at approxi-

mately optimum temperature for growth, whichfor most strains was 25 to 28 C and might beconsidered as "normal variation" in contrast tothe type of variation induced by specific chemi-cals (Hutchinson and Kelner, 1942; Kelner,1947) or special environmental conditions im-posed on the culture during growth.The soil strains studied, since their isolation,

have changed considerably as far as pigmentationand colonial morphology are concerned. Theresults at least partially explain the loss of pig-ment producing ability bv la and 2. Whenstrain la was isolated initially from soil, it waspigmented uniformly and was gelatinous onplain mntrient agar. Sometime during routinetransfer in tryptone broth it lost its ability toproduce pigment and still later lost its abilityto produce the tough, gelatinous colony. Oneother strain (not reported here), isolated fromthe same plot of soil, has maintained its gelatinousnature though nonpigmented variants exist intryptone broth culture.Somewhat the same changes seem to have oc-

curred in strain 2. This organism initially waswell pigmented and grew well on nutrient agar.During cultivation it lost its ability to producepigment and to grow well in tryptone or peptonemedium. It grew well on synthetic media withglucose as the source of carbon and ammoniumsalts as the source of nitrogen. It also grew welland 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 tryptonecontained some substance lacking in gelatinwhich inhibited pigmentation in strain 2 andin the other strains nonpigmented on tryptone.

Strain 21a dissociated readily into gelatinousand nongelatinous, pigmented and nonpigmentedcolonies on tryptone. When first isolated thisstrain also produced a uniformly violet gelatinoustype of growth. The first sign of variation ob-served was in the failure of the strain to produceall gelatinous colonies. Later however, it disso-ciated further to produce nonpigmented, gelat-inous variants. Strain L2a at present seems tobe undergoing the same pattern of changes. Asreported here it produced pigmented, gelatinousand pigmented, nongelatinous variants.

Strain D3 appeared to follow the same patternof dissociation shown bv L2a except that in mostof the media tested D3 produced a large num-ber of wrinkled or rugose colonies.

Pigmentation of the gelatinous strains atleast has been shown to be influenced by themedium, but the tough gelatinous nature ofthe colonies seems to be more stable. The latterwas altered somewhat in the degree of toughnessas the colony aged, possibly due to a release ofautolytic enzymes by the bacteria, causing abreakdown in the gelatinous material. Gelat-inous colonies showed no tendency to revert tothe nongelatinous state under experimentalconditions although la lost this ability for anunexplained reason. Pigmentation in nonpig-mented gelatinous strains, however, could berecalled on media containing 1 per cent peptoneor gelatin.

Studies on the nutrition of soil chromobacteriaprobably would be helpful in elucidating someof these observations.

ACKNOWLEDGMENT

The author wishes to express his appreciationto Dr. K. F. Gregory of the Department ofBacteriology, Dalhousie University, Halifax,Nova Scotia, for his suggestions and criticismof the manuscript.

SUMMARY

Variations in pigmentation, texture of growth,and colonial form of several strains of Chromo-bacteritum species are described. Pigmentation wasinfluenced by the type of medium used. All soilchromobacteria dissociated into gelatinous andnongelatinous strains. Beef extract, yeast extract,tryptone, and a number of other peptones andprotein hydrolyzates were found to discouragethe formation of pigment by all strains although

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the gelatinous strains were affected most. In-hibitory substances weie not evident in peptone,neopeptone, and gelatin in 1 per cent concentra-tion. The substance inhibiting pigmentation intryptone is neither tyrosine nor tryptophan.Tryptophan was shown to be the source of thediffusible vellowish substance produced in tryp-tone broth.No significant differences wXere found in the

biochemical characteristics and cell measure-ments between parent strains and colonialvariants. The colonies could be dissolved by acidand alkaline solutions and by boiling distilledwater but not by cold distilled water and neutralsolvents The large gelatinous colony typical ofsoil strains was not seen in cultures from sourcesother than soil.

REFERENCES

BREED, R. S., MIURRAY, E. G. D., AND HITCHENS,A. P. 1948 Bergey's manual of determinativebacteriology. 6th ed. The Williams andWilkins Co., Baltimore, Md.

CORPE, W. A. 1951 A study of the widespreaddistribution of chromobacterium species insoil by a simple technique. J. Bact., 62,515-517.

HUTCHINSON, W. G., AND KELNER, A. 1942 Astudy of secondary colonies of Chromobac-teriiun violaceutm. J. Bact., 43, 29-30. (Ab-stract)

KELNER, A. 1947 Secondary colonies of bacteriainduced by salts of alkali metals, with specialreference to Chromnobacteriumn violaceum andother bacteria on lithium chloride agar. Am.J. Botany, 34, 105-112.

LEIFSON, E. 1951 Staining, shape, and arrange-ment of bacterial flagella. J. Bact., 62,377-389.

Mlanual of dehydrated culture miiedia and reagents.9th ed. 1953 Difco Laboratories, Inc.,Detroit.

WARD, H. MI. 1898 A violet bacillus from theThames. Ann. Botany, 12, 59-76.

WILSON, P. W., AND KNIGHT, S. G. 1947 Experi-ments in bacterial physiology. Burgess Pub-lishing Co., Minneapolis, Minn.

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