SECONDARY COLONY FORMATION BY BACILLUS SUBTILIS ON EOSINEMETHYLENE BLUE AGAR

K. K. SHAH' AND V. N. IYER2

Microbiology Department, S. B. Garda College, Navsari, India

Received for publication November 18, 1960

Discrete secondary outgrowths on colonies ofbacteria growing on suitable indicator mediaconstitute an often observed phenomenon ofbacterial variation. Such outgrowths are pro-duced by Bacillus subtilis and several otheraerobic, sporeforming bacilli on eosine methyleneblue agar. In the instance of B. subtilis, the cellsconstituting the secondary colony do not differfrom the cells of the primary colony in theirfermentative ability. The difference lies in therate at which the acidity initially produced byfermentation is neutralized, resulting in thedevelopment of alkalinity in the medium. Inprevious studies (Lederberg, 1951; Ryan, 1952;1955) of secondary colony formation inEscherichia coli, it has been shown that secondarycolonies originate from mutants that arise in theprimary colony and which are thereafter selected.The observations recorded in this study onsecondary colony formation in B. subtilis areconsistent with a similar explanation.

MATERIALS AND METHODS

Strains. Unless otherwise stated, a strain ofB. subtilis var. niger (strain SAa) originallyisolated from a sample of commercial sugar(Bhat and Iyer, 1955) was used in all experi-ments. The other strains of sporeforming bacilliused in some of the experiments were identifiedstrains available in the laboratory.Medium. Composition of the eosine methylene

blue agar medium (EMB agar), used in all theexperiments, was as follows: peptone, 1.0 g;NaCl, 0.5 g; meat extract (Oxo Laboratories),0.3 g; glucose, 1.0 g; eosine, 0.04 g; methyleneblue, 0.0065 g; agar, 2.5 g; glass distilled water,100 ml. The glucose was sterilized separately andadded to the rest of the sterile medium beforepouring. Preliminary experiments in which the

1 Present address: Alembic Pharmaceuticals,Baroda, India.

2 Present address: Biological Laboratories,University of Rochester, Rochester, New York.

meat extract was omitted from the above mediumor where a defined medium (Demain, 1958) wasused, indicated that such media were unsuitablein eliciting adequate development of secondarycolonies.

RESULTS

Generality of the phenomenon. When a sporesuspension of strain SAa was spread on EMBagar to yield from 102 to 104 colonies, some ofthe dark colonies that were produced gave rise,on further incubation, to secondary, paler out-growths. The outgrowths were in the form ofdiscrete secondary colonies confined to the areaof the primary colony. They were of variablesize and occurred anywhere on the primary colonyfrom near its center to its periphery (Fig. la, b).They were not always well separated on thecolony surface and occasionally, some marginallylocated ones spread out beyond the area of theprimary colony. Of 15 strains of B. subtilis tested,all produced secondary colonies, although to avariable degree. Strains of B. cereus, B. me-gaterium, B. circulans, and B. sphaericus alsoproduced secondary colonies under similar con-ditions.

Development of secondary colonies in relationto the number of primary colonies and the incuba-tion period. The results of 4 experiments on theeffect of incubation period on the number andsize of primary and secondary colonies, arepresented in Table 1. Secondary colonies wereas a rule found to develop only after appreciabledevelopment of primary colonies. The numberof secondary colonies generally increased withthe incubation period reaching a maximum in3 to 4 days on plates containing discrete primarycolonies and in 5 to 6 days on plates containinga large number of confluent primary colonies.Examination of the results presented in Table1 also show that with an increase in the numberof primary colonies (or number of cells spreadon the agar surface), there is an increase in the

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Fig. 1 (a and b). Secondary colonies of strain SAa on EMB agar (X2.5).

number of secondary colonies per plate. Thisincrease is, however, not proportional to thenumber of primary colonies. For example, withan increase in the number of primary coloniesfrom about 100 in experiment 2, to 150 in experi-ment 3 (Table 1), there is a disproportionatelygreat increase in the number of secondary coloniesper plate (about four times). On the other hand,on plates containing about 450 primary colonies(experiment 4), the number of secondary coloniesthat arise is lower than to be expected on aproportionate basis.

These experiments demonstrated the influenceof the number of viable cells spread on a plate,but a question arose whether secondary colonyformation was also influenced by the number ofprimary colonies; that is, by the manner oforganization of the viable cells on the plate. Todetermine this, equal amounts of a thin sporesuspension were spread in triplicate on three setsof plates of EMB agar and also on similar setsof identical media without EMB. The plateswere incubated at 37 C and after an intervalof 2 hr, the surfaces of one triplicate set of platesof EMB agar and another of the medium withoutEMB were respread. The second set of the twomedia was respread after 5 hr and the third setleft unspread as the control. All plates, bothunspread and respread, were further incubatedfor a period of 48 hr. After this period, besidescounts of secondary colonies on EMB agar, thenumber of viable cells on the EMB-free plateswas determined by washing off the growth fromeach plate with physiological saline and deter-

mining the number of cells in such washings byplate counts on nutrient agar. As may be observedfrom Table 2, respreading did not result in anysignificant alteration in the number of viablecells on the plate, whereas the number ofsecondary colonies on respread EMB agar plateswas significantly greater. Taken together, thetwo groups of experiments (Tables 1 and 2)suggest that both the number of viable cellson a medium surface as well as their manner ofdistribution influence secondary colony for-mation.

Influence of area of inoculated medium surface.Further support for the above conclusion wasderived from experiments in which a constantnumber of viable cells was spread on varyingsurface areas of the medium. The results of 3experiments are presented in Table 3. It wasobserved that for a population size, where theinoculum permitted the development of discreteprimary colonies (100 to 200 on each plate), anincrease in the inoculated surface area was ac-companied initially by an increase in the numberof secondary colonies. Further increase in thesurface area resulted in a decline in the numberof secondary colonies (experiment 2). When theinoculum was so heavy as to result in confluentgrowth, the initial increase in the number ofsecondary colonies with increase in surface areawas again evident but detection of the subsequentdecline was not experimentally feasible (experi-ment 3). Secondary colony formation is thus afunction of the number of primary coloniesrelative to the surface area available for growth.

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TABLE 1Effect of incubation period at 37 C on number and

size of primary and secondary colonies (averageof triplicates)

Expt Incuba- Avg No. of Avg Diameter Avg No. ofNo tion Primary of Primary ColoniesPeriod Colonies Colonies per Plate

days mm

I 1 20.0 4.0 0.02 20.0 8.1 2.03 20.0 9.7 2.04 20.0 10.0 2.010 20.0 10.0 2.0

II 1 95.0 3.5 0.02 111.0 5.4 18.03 115.0 7.2 18.04 115.0 6.8 14.05 115.0 6.5 12.06 110.0 6.0 10.07 110.0 6.0 10.09 110.0 6.0 9.010 110.0 6.0 9.0

III 1 143.0 3.8 1.02 149.0 5.3 18.03 149.5 6.5 61.54 149.0 6.3 48.55 151.0 6.2 47.56 147.5 6.2 47.57 145.0 6.2 46.59 144.0 6.2 39.010 144.0 6.2 39.0

IV 1 464.5 3.4 2.02 483.0 4.7 78.03 460.0 5.0 84.54 460.0 5.0 78.05 455.0 4.9 78.06 455.0 4.9 73.57 455.0 4.8 73.59 455.0 4.8 57.510 455.0 4.8 57.5

1 45.5 X 102 No discrete 3.52 (calcu- colony 100.53 lated) 110.54 117.05 122.06 134.07 134.09 126.010 113.0

TABLE 1.-(Continued)

ExpIncuba- Avg No. of Avg Diameter Avg No. ofNo.t tion Primary of Primary ScooniesyNo, Period Colonies Colonies Colonies

days mm

1 4.5 X 104 No discrete 98.02 (calcu- colony 288.03 lated) 309.04 320.05 380.06 390.07 380.09 345.010 325.0

1 4.5 X 106 No discrete 40.02 (calcu- colony 84.03 lated) 84.04 96.05 113.06 140.07 130.010 130.0

The observed decrease in number of secondaryand primary colonies on long incubation was moreapparent than real. It was due to the growingcolonies merging with each other.

Influence of depth of medium. By varying thedepth of the medium and maintaining a constantarea of the inoculated surface, it was sought todetermine whether the volume of the mediumor amount of nutrient available to the developingcolonies could influence secondary colony forma-tion. In these experiments, amounts of EMBagar in the range of 10 to 50 ml were poured intoflat 9-cm petri dishes. Two dilutions of the sporesuspension were then plated out separately onthe media to give discrete and confluent growth,respectively. The growth of secondary colonieswas recorded after an incubation period of 4days by which time maximal development hadoccurred. The results of 4 experiments (Table4) show that an increase in the depth of themedium is accompanied by an increase in thenumber of secondary colonies, the average sizeof the colonies remaining constant.

Influence of concentration of glucose in medium.The number of secondary colonies produced onEMB agar in the presence of increasing glucoseconcentrations up to 5.0% was studied usingdifferent concentrations of cells in the inoculum

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TABLE 2Effect of respreading primary colonies on the number of secondary colonies

Plates Respread afterPlates Not Respread -

2 hr 5 hr

Number of secondary colonies (average of 3 plates)onEMB agar .................................... 48.66 108.5 325.0

Number of viable cells (average of 3 plates) on eachplate of EMB-free agar*.......................... 2.6 X 109 2.4 X 109 2.1 X 109

* Viable counts were made on suspensions washed from plates of EMB-free agar treated in an identicalmanner to each EMB agar plate.

TABLE 3Effect of area of inoculated surface of medium on number of secondary colonies formed

No. of Secondary Colonies Produced on Each of Following

Expt No. Avg No. of Viable Cells per Inoculated Surface Areas (Diameter in cm)ExptNo. ~ ml of Suspension2.0 2.5 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0

I 0.9 X 103 42 52 70 100 122II 2.9 X 103 65 104 140 300 260 240 218

III 1.4 X 106 25 45 50 94 105 121 175 230 240

(Table 5). With increasing concentrations ofglucose, there is an increase in the average sizeof primary colonies, in the number of primarycolonies giving rise to secondary colonies, andin the average number of secondary coloniesper plate (Fig. 2a to f). Secondary colonies doarise in small numbers even in the absence ofglucose though under these conditions, thedevelopment of both primary and secondarycolonies is poor. Since glucose affects the growthof primary colonies, increased secondary colonyformation must be considered as a probableresult of the interaction between the glucose andthe growing primary colony.

Differences between cells from primary andsecondary colonies. Morphological and cytologicalobservations on cells from primary and secondarycolonies revealed no apparent differences. Physio-logically, the cells ferment glucose rapidly withacid production. In preliminary experiments, itwas observed that cells from the region of thesecondary colony when inoculated into peptonewater containing glucose and an indicator,ferment the sugar as effectively as cells from therest of the colony. However, on further incubationof the tubes it was observed that after thedevelopment of acidity as judged by the indicator,the reaction gradually became alkaline. This

TABLE 4Influence of depth of medium on number of

secondary colonies

No. of Secondary ColoniesProduced on Surface of

Avg No. of Petri Dishes (DiameterExpt No. Cells Spread on 9 0 cm) Containing Varying

Each Plate Amnounts of EMB Agar (ml)

10 20 30 40 50

I 1.0 X 106 3 34 225 300 4001.0 X 102 15 20 25 25 78

II 8.0 X 106 0 90 200 400 5008.0 X 103 52 83 170 265 354

III 1.5 X 105 5 49 100 3711.5 X 102 4 83 170 265 354

IV 1.4 X 106 0 95 300 450 5501.4 X 104 80 250 4001.4 X 102 15 16 25 78

Note: No appreciable differences in the size ofsecondary colonies were observed.

reversal to an alkaline pH occurred more rapidlyin tubes inoculated from the secondary colonyarea. The observation was confirmed for severalindependently arising primary and secondary

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colonies. By serial transfer and selection of cellsfrom the primary and secondary colony area, itwas possible to isolate after approximately 60generations a primary colony type and two

TABLE 5Effect of varying concentrations of glucose on

secondary colony formation

Avg No. of Avg No. ofPrimary Primary Avg No. of

Expt Concn of Colonies with Colonies SecondaryNo. Glucose Seodr without Colonies

Scooniesy Secondary per PlateCooisColonies

g/10O ml

I 0.0 15.0 91.0 17.00.1 30.0 62.0 100.00.5 85.0 0.0 300.01.0 94.0 0.0 350.03.0 97.0 0.0 400.05.0 96.0 0.0 500.0

II 0.0 Sheet of primary 54.00.1 growth-no discrete 86.00.5 colonies 110.01.0 220.03.0 250.03.5 300.0

secondary colony types, none of which producedsecondary colonies on further subculture. Thephysiological difference observed between cellsfrom the primary and secondary colony area,persisted in these isolates. This finding demon-strated that the cells from the secondary colonywere genetically distinct from those comprisingthe rest of the primary colony.

In further experiments, 250-ml flasks con-taining 75 ml of sterile nutrient broth and 1%glucose, were inoculated separately with cellsuspensions of the isolated primary and secondarycolony types. Changes in the pH of the mediawere subsequently followed at regular intervals.It can be observed (Fig. 3) that while the initialfall in pH is similar for both primary and second-ary colony types, cells from the latter reversethe pH of the medium to an alkaline level earlierthan cells from the former.

Prevention of secondary colony formation. In anattempt to prevent the development of secondarycolonies, the EMB agar was buffered by theaddition of 1.0% calcium carbonate. Secondarycolonies were rare or absent on media containingcalcium carbonate even on prolonged incubation(up to 15 days) whereas control plates without

Fig. 2 (a-f). Influence of glucose concentration on secondary colony formation by strain SAa on EMBagar (XO.45): (a) 0.0%, (b) 0.1%, (c) 0.5%, (d) 1.0%, (e) 3.0%, (f) 5.0%.

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892 SHAH AND IYER

7.5

7.4

7.37.27.1

7.0

pH 6296.86.7

6.6S564

6.362

6.I

[VOL. 81

O --- Primary Colony typeo --- Secondary Colony type- I* --- Parent Strain-SAa

6 1024 34 4858 72 82 96 120 144 168 192 216 240264INCUBATION PERIOD (Hours)

Fig. 3. Developmental pH in nutrient broth + 1.0% glucose inoculated with isolated primary colonyand two secondary colony strains.

Fig. 4 (a and b). The ability of calcium carbonate to inhibit secondary colony formation by strainSAa on EMB agar (XO.75): (a) with calcium carbonate, (b) without calcium carbonate.

the buffer developed secondary colonies in 2 to 3 duced in a peptone medium more rapidly thandays (Fig. 4a, b). the latter. It may be reasoned that the highly

proteolytic nature of cells of B. subtilis enabledDISCUSSION them to attack the peptone in the medium rapidly

The physiological difference between cells and convert them to amino acids. Simultaneously,constituting the secondary colony and cells con- fermentation of the glucose in the medium rendersstituting the rest of the primary colony lies in the the environment acidic. In such an acidic environ-ability of the former to reverse the acidity pro- ment, the amino acids will be attacked pref-

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SECONDARY COLONY FORMATION BY B. SUBTILIS

crentially by decarboxylases to yield alkalineamines which reverse the acidity. Such a reversaloccurs more rapidly with secondary colony cellsapparently because they are endowed with greaterdecarboxylase activity. The validity of thisreasoning is supported by the experiment inwhich secondary colony formation is preventedby simply incorporating an adequate and readilyavailable buffer in the medium. The inference isthat the presence of the buffer ensures againstthe development of acidity in the environmentand thus indirectly against selection of the cellsthat have greater decarboxylase activity andwhich eventually form the secondary colony.

Consideration of the various factors found toinfluence secondary colony formation suggeststhat these are consistent with a mutation-selec-tion hypothesis although the question of whetherthe mutant cells which form the secondary colonyare selected by the environment or are inducedby the selective conditions, is not determinedexperimentally. The formation of secondarycolonies, in this particular instance, is found to beinfluenced by various complex interacting factorsamong which must be considered the age andsize of the primary colony, the number of cellsand their arrangement into colonies on the surfaceof the medium. A reasonable inference is thatas a prerequisite to secondary colony formation,(i) the primary colony must attain a populationsize sufficiently high to raise the probability ofa mutation to 1.0, (ii) the cells constituting theprimary colony must alter the growth environ-ment by the depletion of nutrients or accumula-tion of acidic metabolic products, and (iii) suchan altered environment must be specially capablefor the selective outgrowth of mutant cells. Thisselective outgrowth constitutes the secondarycolony. The production of a mutant cellpotentially capable of forming a secondary colonydoes not necessarily assure the appearance of adiscrete visible secondary colony, for phenotypicexpression is subject to various interacting factorsboth within the confines of the primary colonyand in the medium as a whole. The increase insecondary colonies produced on a plate with thenumber of cells plated is to be expected, whereasthe lack of proportionality in increase must beattributed to barriers against phenotypic expres-sion which also operate when the surface area ordepth of the medium is altered. Spreading adeveloping primary colony increases the number

of colonies as this serves to separate mutant cellsafter they have arisen. The relationship betweenthe concentration of glucose and the degree ofsecondary colony formation is understandableas an increase in glucose concentration wouldlead to the rapid production of an acidic andtherefore favorable environment for the mutantcells. Prevention of the development of such asacidic environment by means of a buffer addedto the medium is an effective way of inhibitingthe development and appearance of secondarycolonies. The fact that the cells constituting thesecondary colony can be separated away fromthe primary colony cells and that such separatedcells continue to show their physiologicaldifference on serial subculture, establishes thatthey are genetically distinct.

ACKNOWLEDGMENT

We would like to thank A. W. Ravin forsuggestions in the preparation of this manuscript.

SUMMARY

Colonies of sporeforming bacilli growing oneosine methylene blue agar produce discretesecondary outgrowths. With a strain of Bacillussubtilis var. niger these secondary colonies origi-nate from mutant cells that arise in the primarycolony. It is postulated that growth of the pri-mary colony involves the simultaneous produc-tion of acids from the glucose and amino acidsfrom the peptone in the medium, followed bydecarboxylation of the amino acids to alkalineamines that result in a reversal in hydrogen ionconcentration. Cells constituting the secondarycolony can be separated and shown to bringabout a more rapid reversal in pH as comparedto cells present in the rest of the primary colony.Furthermore, this distinctive property is retainedon subculture. Observations on various factorsinfluencing secondary colony formation, althoughconsistent with a mutation-selection hypothesis,suggest that the expression of mutant cells issubject to various interacting factors within thecolony and in the environment on the plate.

REFERENCES

BHAT, J. V., AND V. IYER 1955 Aerobic meso-philic sporeforming bacteria in Indian en-vironments. Proc. Indian Acad. Sci., 42,325-333.

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DEMAIN, A. L. 1958 Minimal media for quan- RYAN, F. J. 1952 Adaptation to use lactose bytitative studies with Bacillus 8ubtilis. J. Escherichia coli. J. Gen. Microbiol., 7, 69-Bacteriol., 75, 517-522. 88.

LEDERBERG, E. M. 1952 Allelic relationships RYAN, F. J. 1955 The direct enumeration ofand reverse mutation in Escherichia coli. spontaneous and induced mutations in bac-Genetics, 37, 469483. teria. J. Bacteriol., 69, 552-557.

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