PLOIDY STUDIES ON THE LARGE CELLS OF MICROCOCCUS AUREUS'

J. B. CLARK AND R. B. WEBB

Department of Plant Sciences, University of Oklahoma, Norman, Oklahoma

Received for publication April 25, 1955

The determination of ploidy in bacterial cellshas been unsatisfactory in most cases because thecriteria of ploidy in higher forms can not as yetbe applied directly to bacteria. The best evidenceof diploidy in bacteria has been in the various"Het" strains of Escherichia coli strain K-I2 de-veloped by Lederberg et al. (1951). Unfortunately,genetic recombination has not been found inmany bacteria, so this means of ploidy determina-tion has been very limited in use. Genetic andradiobiological studies indicate that bacteria areusually haploid (Lea, 1947); however, severalinstances have been reported in which cytologicalinformation suggests a diploid phase (Bisset,1950).

Lindegren (1942) presented cytological evi-dence for a meiotic division in Micrococcusorchraceous. Webb and Clark (1954) found evi-dence of a diploid or polyploid phase in a lifecycle of M. aureus. These large cells appearedcytologically to undergo a meiotic division toyield normal haploid cells. This paper is a con-tinuation of the studies of the large, apparentlydiploid cells of M. aureus.

MATERIALS AND METHODS

The organisms used in this study were M.pyogenes var. aureus strain FDA 209, nine otherstrains of M. aureus, and thirteen other cocci,all obtained from the University of OklahomaCulture Collection. These organisms were main-tained in stock on nutrient agar (Difco), and allexperiments were performed on nutrient agarunless otherwise noted.

Radiation experiments were performed using aGeneral Electric low-pressure mercury vaporultraviolet light and a Picker Army Field X-rayof 30 ma output. In the ultraviolet studies ir-

1 This investigation was supported in part bygrants from the Faculty Research Committee ofthe University of Oklahoma and from the MedicalResearch and Development Board, Office of theSurgeon General, Department of the Army undercontract No. DA-49-007-MD-319.

radiation was done at a distance of 14 inchesfrom a cell suspension in 0.85 per cent saline orfrom the agar of a petri dish which had beensurface plated with approximately 300 viablecells. In X-ray experiments cell suspensions in0.85 per cent saline were irradiated with the tubeoperating at 75 kv and 16 ma at a target distanceof 5 inches.

In order to eliminate misleading results due toclumping, cell suspensions were shaken vigorouslyfor 5 to 10 minutes with glass beads in a screwcap test tube. This procedure yielded suspensionsof 95 per cent single cells as revealed by the phasemicroscope. Cross septation was further checkedin all suspensions by using a cell wall stain(Webb, 1954).Nuclear studies were performed using the

crystal violet nuclear stain (Chance, 1952; Clarkand Webb, 1955a) the acid giemsa stain (Mur-ray et al., 1950) and the thionin-SO2 technic(DeLamater, 1951). Phase studies were per-formed with American Optical dark contrastmedium and B-minus contrast medium ob-jectives.

RESULTS

Conditions offormation. In order to adequatelyinvestigate the large cells of M. aureus it wasnecessary to determine the conditions of culturewhich caused the maximum formation of thesecells, since cultures containing at least 75 percent large cells were desired. Under the usualcultural conditions the occurrence of large cellswas erratic. They were found to appear at amaximum incidence after 8 hours of growth andto disappear rapidly after 12 hours of incubation(table 1). It was also found that the presence ofexcess moisture on the nutrient agar surfacecaused an increased incidence. Few, if any, largecells would form on a dried agar surface. Thiswas not directly associated with medium agesince old, partially dried agar would yield a highpercentage of the cells after it had been meltedand resolidified to yield a moist surface.

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The presence of carbohydrates interfered withlarge cell formation. Only a few such cells wereobserved when any carbohydrate was incor-porated in the nutrient agar. The stage of growthof the inoculum was also significant in that themaximum yield of large cells was noted whenresting cells were used.Another significant condition for large cell

formation was the temperature of incubation. Asshown in table 1, the maximum yield of suchcells occurred when the cultures were incubatedat 25 C. By fulfilling conditions of temperature,moisture, inoculum age, and time, it was possibleto consistently produce cultures containing 70 to90 per cent large cells, but this percentagediminished rapidly as the culture aged.

It was also found that there was a correlationbetween the incubation temperature and thedegree of clumping. A high incidence of clumpedcells was found in cultures incubated at 37 C, andthe proportion of clumps decreased as incubationtemperature was decreased.

Incidence of formation. Nine strains of M.aureus were examined to see if the formation oflarge cells was a restricted phenomenon. All ninestrains were found to produce these cells withvarying incidence under similar cultural con-ditions. The FDA 209 strain yielded the highestpercentage of large cells under the conditionstested, but it appeared probable that the con-ditions for maximum formation varied from strainto strain. A similar large cell formation was alsoobserved in other cocci (table 2). These cultureswere observed at both 9 hours and 7 days, andif large cells were observed the culture wasrecorded as positive. Seven of the 13 various cocci

TABLE 1Effect of time of incubation on the occutrrence of

of large cells in Micrococcus aureus

Percentage Large Cells at IncubationTime of 'Temperatures of

Incubation20 C 25 C 30 C 37 C

hr

0 0 0 0 05 38 50 1 18 65 80 1 110 65 75 1 112 50 69 1 114 36 55 1 124 36 1 0 0

TABLE 2The occurrence of large cells in various cocci

Large CellsPresent

Organism9-hr 7-day

culture culture

Micrococcus flava................... -

M. perflava ........................ - +M. candidus ....................... -

M. ureae........................... +M. citreus......................... - -

M. pyogenes var. albus............. + iUnknown hemolytic micrococcus + +Streptococcus lactis................. + +S. pyogenes .................... -

Diplococcus pneumoniae............ + +Sarcina lutea...................... - -

Sarcina flava....................... + +Gaffkya tetragena................... +

cultures tested were found to contain large cellsafter 9 hours' incubation. In the case of M.perflava, large cells were found only in oldcultures.Mechanism offormation. Repeated studies were

made on living cultures of M. aureus using thephase microscope in an effort to determine theorigin of the large cells. In no case was anycellular fusion seen, and in several instancesisolated normal cells were observed to increase insize and divide as large cells. Although no nuclearactivity was observable in those cases with thephase microscope, this is suggestive of anautogamy or endopolyploidy. An intracellularnuclear fusion would cause a ploidy increase, andthere is some cytological evidence for such inbacteria (Bisset, 1950).The large cells were studied cytologically using

the crystal violet nuclear stain (Chance, 1952),the acid giemsa stain (Murray et al., 1950), thethionin-SO2 stains (DeLamater, 1951), and a cellwall stain (Webb, 1954).

In the thionin-CO2 staining procedure thefreeze-dehydration process was omitted and thepreparations were examined and photographedas temporary water mounts (DeLamater, 1951).It should be recognized that the freeze-dehydra-tion procedure is not an essential part of thestaining reaction but rather functions to yieldpermanent slides with perhaps a slight increase innuclear definition.

Large cells were produced by streaking an

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inoculum of a 4-day-old culture on the surface ofa petri plate and incubating the plate at 25 Cunder semihumid conditions for 6 to 8 hours.Under these conditions only a small portion of

the cells were found to be the large type, but theincidence was sufficient for cytological studies.The acid giemsa stain yielded results which ap-

peared very similar to that of the crystal violet

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Figures 1, 3, 6, 7, and 9 are photographs of large cells stained with the thionin-SO2 method and show-ing 4 or 6 nuclear components.

Figures 2, 4, 6, 8 and 10 are drawings of the significant cells in the photographs to better reveal theappearance of the nuclear structure.Note in figures 2 and 3 the normal Micrococcus aureus cells with two internal structures in non-septate

cells. All photographs magnified 3250X.

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nuclear stain (Webb and Clark, 1954) exceptthat no cell plates were stained. The nucleus ap-peared elliptical in resting cells and becameround prior to division. Nuclei of the large cellswere approximately twice the diameter of smallcell nuclei. The nuclei were less discrete than whenstained by the crystal violet nuclear stain.The thionin-SO2 procedure also gave results

similar to that of the crystal violet nuclear stainbut, as in acid giemsa, no cell plates were stained.The nuclei of the large cells were much largerthan those of the small cells and were in manycases more discrete. Occasionally classicalmitosis-like figures were observed; however,these observations were not considered sufficientevidence for such a mitosis in bacteria. In manyof the large cells four, and sometimes six smallrods or spots were seen, and in the small cellsoften two spots could be observed in cases wherea 2-celled condition could be eliminated (figures1-10). Since not all of the structures observed inthe cells were in the same plane of focus, repre-sentative drawings are used to clarify the photo-graphs. If these spots were chromosomes, forwhich there is little evidence, the ploidy of thelarge and small cells would be established. Thesestructures could be called chromosomes but, inconsideration of the present knowledge of bac-terial cytology and the lack of understanding ofthe precise specificity and mechanism of thestain reaction, there is at present little justifica-tion in such nomenclature.

In no case was any clear evidence of "classicalmitosis or meiosis" observed, and the additionalstain work supported the earlier concept (Webband Clark, 1954) that the nuclear membranenormally remains intact during nuclear divisionin bacteria. A similar concept has also beenpostulated by DeLamater (1954). This intactnuclear membrane normally obscures the in-ternal nuclear structure and thus makes it im-possible to determine the exact mechanism ofdivision by using the presently available technics.From a genetical viewpoint the cell must undergomitosis since there is normally an even distribu-tion of genetic determinants to the daughtercells. However, this does not infer that a "classicalcytological mitosis" is responsible for the geneticdivision as assumed by DeLamater (1954).

Effect of chemical and physical agents on forma-tion. In the course of experiments to determinethe conditions under which the large cells formed,

TABLE 3The effect of ultraviolet radiations on the occurrence

of large cells of Micrococcus aureus

Age of Culture at Exposure Time PercentageTime of Irradiation Large Cells

hr sec

O O 10 2.5 30 10 150 15 33O 20 450 30 300.5 20 351.0 20 254.0 20 1

it was noted that several chemicals and radiationstimulated the formation of these cells. Most ofthese stimulating agents have been classified asradiomimetic in that they may induce haploidiza-tion in diploid cells of E. coli (Lederberg et al.,1951). In this case the opposite effect was found,since the agents apparently induced diploidizationin haploid cells.The effect of x-rays and ultraviolet light on the

formation of large cells was investigated by ir-radiating surface inoculated nutrient agar dishesfor varying lengths of time at different culturalages. The dishes were then incubated at 37 C for8 hours and the organisms checked by examiningnuclear stained smears from each petri dish. Asshown in table 3, a 20-second exposure to ultra-violet light at the time of inoculation producedthe maximum yield of typical appearing largecells under the experimental conditions used. Ir-radiation of cultures ;1 to 1 hour old was lesseffective, but it also resulted in increased largecell formation. It was concluded that resting cellsor early lag phase cells could be utilized ef-fectively in stimulation experiments, but logphase cells were ineffective.

X-radiation at 75 kv and 15 ma at a targetdistance of 8 inches was ineffective in increasingthe incidence of large cells. This was determinedfor times of radiation varying up to 4 minutes.The effect of formaldehyde was tested by the

gradient plate technic (Szybalski, 1952) and bybroth cultures containing varying concentrationsof formaldehyde. The surfaces of gradient platescontaining 0.01 per cent formaldehyde werespread with a moderate suspension of restingcells. After 48 hr several colonies appeared in the

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TABLE 4

Effect of physical and chemical agents onthe occurrence of large cells in

Micrococcus aureus

AgentProductionof TypicalLargeCells

MaximumPercentageof TypicalLarge CellsObserved

Productionof Atypical

LargeCells

Ultra violet light + 60X-ray ................ 00Formaldehyde.±.......+ 100Hydrogen peroxide-... 0Terramycin .......... + 20Aureomycin .......... + 40Bacitracin ........... + 20Penicillin ............ 5Chloromycetin ....... 10Dihydrostrepto-mycin .............. 0

Polymyxin B.. 0 -

edge of the zone of inhibition. These colonieswere found to be composed entirely of large cellswhich were cytologically indistinguishable fromthe normal large cells. These large cells tended torevert to normal after transfer to nutrient agar.

Hydrogen peroxide was tested in the same

manner as formaldehyde, but no large cells were

detected under the experimental conditions used.The effects of antibiotics were determined

through the use of Difco sensitivity disks. Col-onies growing inside the zone of inhibition were

checked for cytological and morphological ap-

pearance through the use of the crystal violetnuclear stain and the phase contrast microscope.The action of the antibiotics listed was also ob-served on slide cultures with the phase contrastmicroscope.Most of the cells enlarged greatly from ex-

posure to the antibiotic; however, most cellsfailed to divide in the presence of the antibiotic.A few of the enlarged cells proceeded to divide,forming micro colonies of these cells. The coloniespicked from zones of inhibition also representedcells dividing in the presence of the antibiotic.

Results of these experiments are summarizedin table 4. The atypical large cells produced bypenicillin and chloromycetin were 2 to 4 , indiameter with nuclei varying from a granular,lobed condition to an evenly staining sphericalappearance. The significance of these forms isunknown.

The appearances of various induced large formswhen stained with the crystal violet nuclear stainare shown in figures 11 to 21.The exact relation of typical large cells pro-

duced by the chemical and physical agents withthose occurring naturally has not yet been de-termined, although there are suggestions that inboth cases a change in ploidy may be involved. Itis also uncertain whether selection or induction isinvolved in the action of these agents since, eventhough the treatment apparently induces an in-creased formation of large cells, the environmentresulting is strongly selective in favor of thesecells.

Radiobiological response. Many factors havebeen found to influence the rate of killing of abacterial culture when exposed to lethal radia-tions (unpublished experiments). These includeclumping of the cells, multinuclearity, and"multicellularity." In order to eliminate thesevariables and thus validate interpretations ofradiobiological response based on ploidy, severalprecautions were observed. Clumps were brokenup as indicated in "Materials and Methods" andno culture was used in an irradiation experimentunless it revealed at least 90 per cent single cells."Multicellularity" has been shown to occur inM. aureus and multinuclearity also can exist(Webb and Clark, 1954). The extent of "multi-cellularity" was checked on all suspensions by thecell wall stain and was found to be less than 1per cent after 24 hours' incubation, and normallyfrom 5 to 20 per cent in young cultures; however,under certain conditions the percentage wasfound to exceed 50. The presence of even 20 percent such cells was found to exert no extremeeffect on the final shape of the killing curve. Theextent of multinuclearity was checked with thecrystal violet nuclear stain and found to benegligible.

Survival curves from ultraviolet irradiation forM. aureus were found to be basically exponentialunder the usual laboratory conditions (figure 22).Age of culture was found to considerably alter theresistance to ultraviolet light, and young cellswere shown to be more resistant than older cellswhich differs from findings previously reported(Gates, 1929). M. aureus has been reported asshowing a sigmoidal killing rate by several in-vestigators (Lea, 1947). Failure to eliminateclumping may be the basis of these results; alarge degree of "multicellularity" would also give

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Figure 11. Normal Micrococcus aureus cells stained with the crystal violet nuclear stain (this stainused in all photographs on this plate).

Figure 12. Normally occurring large cells of M. aureus.Figure 13. Formaldehyde-induced large cells.Figure 14. Aureomycin-induced large cells.Figure 15. Atypical large cells caused by aureomycin.Figures 16 and 17. Atypical large cells induced by chloromycetin.Figure 18. Atypical large cells induced by dihydrostreptomycin.Figures 19 and 20. Atypical cells induced by penicillin.Figure 21. Atypical large cell induced by terramycin.All photographs magnified 3250X.

rise to spurious results. It was noted that thesurvival curves of the 7- and 18-hour normalcells deviated slightly from the true exponentialtype of curve, which was probably due to thelevel of "multicellularity" and to the presence ofdividing cells.

Large cells were produced for radiation studiesunder conditions which resulted in the greatestpercentage of large cells together with a low levelof "multicellularity." To stimulate the productionof large cells, cultures were incubated at 25 C forthe first 8 hours and then removed to room tem-

perature until harvested at 11 to 15 hours.Usually cultures composed of principally largecells would revert to small cells at 12 to 18 hours,giving rise to a highly "multicellular" population;thus it was necessary to microscopically examineeach culture before irradiation. Under the con-ditions of the experiments "multicellularity" ofthe large cells was not found to exceed the"multicellularity" in the normal small cells of thesame age, so the radiobiological response curvesare directly comparable.The survival curve for large cells is strongly

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Figure 22. Ultraviolet irradiation survival re-

sponse of normal and large cells of Micrococcusaureus.

sigmoidal (figure 22), which is typical of the"multiple hit" type of curve. The ultravioletsurvival curve is approximately a 2-hit curve as

compared to a hit multiplicity of 8 or more withx-ray. This together with the converging slopesof the normal killing curve and the exponential

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section of the sigmoid curve are probably indica-tive of a secondary effect of the ultraviolet light.These results provide evidence in addition todirect observation that the large cells are at leastdiploid-whereas the small normal cells, whichare killed exponentially, are haploid.

Cultures of both large and small cells were sub-jected to x-radiation. The results were completelyin accord with the findings from ultraviolet ir-radiation experiments (figure 23). The small cellswere killed exponentially whereas large cellsshowed a sigmoidal survival curve. The differencein resistance between the large and small cellswas considerably greater from x-radiation thanultraviolet radiation, probably because of a sec-ondary effect of the ultraviolet light.X-ray and ultraviolet survival curves of

formaldehyde-resistant large cells were found tobe sigmoidal. The survival curves were verysimilar to those obtained from normally oc-curring M. aureus large cells (figure 24). Theresults suggest that formaldehyde stimulates theproduction of large cells which are at leastdiploid and which show increased resistance tothe chemical agent.Attempts to produce an ultraviolet-resistant

strain of M. aureus were only partially successfulsince all strains thus far obtained have rapidly

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TIME OF IRRADIATION (SECONDS)

Figure 24. Ultraviolet irradiation survival re-sponse of normal and formaldehyde-resistant largecells of Micrococcus aureus.

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reverted back to the sensitive condition. Theultraviolet survival curve of a 4-day-old re-sistant culture was decidedly sigmoidal and wasquite similar to the survival curve of large cells.Similar results were reported in M. albus, inwhich the ultraviolet survival curves of the re-sistant forms weie sigmoidal and the survivalcurves of the nonresistant forms were exponential(Rentschler and Nagy, 1942). Witkin (1947)reported that ultraviolet-resistant strains of E.coli strain B showed a sigmoidal survival curvein contrast to the exponential type curve for thenonresistant forms. These findings are suggestiveof an association of radiation resistance anddiploidy or polyploidy in many of the bacteria.However, substantiation of an association be-tween a persistent diploid condition and radiationresistance in bacteria must await clarification ofsome of the conflicting reports relative to thissubject.

DISCUSSION

The occurrence of life cycles in bacteria hasbeen postulated for many years. The more sig-nificant of these references are covered by Dubos(1945) and Bisset (1950). The existence of suchcycles has in many cases been based on question-able observations, and it is apparent that cyclicschemes could be postulated on the basis of mor-phological changes induced by varied culturalconditions or by population pressures in the de-veloping culture. If the concept of a life cycle inmicroorganisms is restricted to mean those in-stances in which a change in ploidy is involved(with or without a proven sexual process), thenthere is doubt if many of the earlier observationsactually involved such a cyclic event. Althoughmuch cytological evidence has been presented(Bisset, 1950) for nuclear fusion and reductiondivision in bacteria, there has been little support-ing evidence of a physiological or genetical nature.Direct cytological observations in bacteria willbe of limited value without additional supportingevidence until the mechanism of stain reactionsand specificity, the significance of artifacts, andthe role of fixation procedures are better under-stood.The determination of ploidy in the bacterial

cell cannot at present be based on cytologicalobservations alone. In the case reported here itmight be concluded that the nuclear structuressometimes observed were chromosomes and thus

a direct chromosome count would be valid.Further work may show these to be bacterialchromosomes, but proof is as yet lacking. Thesestructures are somewhat similar to the nuclearconfiguration referred to as a "compoundnucleus" by Knaysi (1955). Ploidy can also bedetermined by genetic analysis but, except in thecase of E. coli, this is at present impossible inbacteria. The determination of the induced rateof recessive mutations would also be an indicationof ploidy. In most cases of suspected or provendiploidy in bacteria, it has been found that thediploid state is unstable and the mutagenic treat-ment causes increased segregation (Lederberget al., 1951; unpublished data). This in additionto the complicating factors of "multicellularity,"clumping, and multinuclearity can obscure theexpression of a recessive allele.

Radiobiological lethal response can also be ofaid in ploidy determinations. If the lethal actionof ultraviolet light or x-ray is due primarily tolethal mutation or some similar single mechanism,then a haploid cell would give a survivor inci-dence that is exponential in relation to dose(Zirkle, 1952; Tobias, 1952). This assumes thatonly one "hit" or "event" at any of severalsensitive sites would result in death of the cell.Since lethal mutations have been found to berecessive in many forms of life, it can be con-sidered that such would also be the case in bac-teria and the survivor response would be indicatedby a sigmoidal or multi-hit curve in a diploid cell.The number of hits required in a diploid cellcould be as small as 2 if the lethal action involvesonly chromosomal hits or if excessive segregationoccurred, or it could be a large number if onlyrecessive lethal alleles were involved. The poly-ploid series of yeasts has correlated nicely withkilling curve shape (Latarjet, 1952), and it ap-pears reasonable that bacterial cells should be-have similarly. Unfortunately several factors caneffect the shape of a killing curve and, althoughthese are being investigated at present, their fullsignificance is as yet unknown. A "multicellular"condition of a haploid cell would yield a sig-moidal response to lethal radiations since thetechnics used are based on the single-celled entity(Clark and Webb, 1955b). Similarly a multi-nuclear state of haploid nuclei would also show ahit multiplicity of at least 2 (Atwood and Nor-man, 1949). Therefore precautions were taken inthese experiments to make sure that cultures con-

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sisting predominantly of single-celled, single-nucleated units were used. This aided in validat-ing interpretation of survival curves in terms ofploidy.The interpretation of radiation-induced events

which lead to inhibition of cell division in terms ofgenetic entities within the cell does not depend onthe formulation of any theory as to the possibledirect or indirect action of the radiation. Thekinetics of the determined reactions is dependenton the number of events which occur and forpresent purposes is independent of the precisenature of this event. It could be the hitting of aspecific target area within the cell; it could also bethe end result of a chain of reactions occurringwithin the cell.Although it has not been established definitely

that the large cell of M. aureus is a result of anincrease in ploidy, all confirming tests performedso far indicate this to be the case. Thus the inter-pretation of cytological evidence is strengthenedby confirming physiological and genetical ex-periments.The predominant appearance of these large

cells in the lag phase of growth suggests thatthese might be the normal enlarged cell usuallyassociated with such growth phase. However,care must be taken in distinguishing between thetype of large cell reported here which involves adefinite nuclear change and the type of enlargedcell which results from protoplasmic synthesisexceeding the rate of cell division. The large cellsinvolving apparent ploidy change in many casespersist long after the lag phase of growth.

In dealing with large populations of cells suchas found in a bacterial culture, a homogeneity ofcells cannot be assumed. Cytological investigationon the large cells of M. aureus reveals severaltypes of structures which could correspond tohaploid, diploid, and tetraploid cells, all withinthe same culture. Thus the terms diploid or poly-ploid are not used in the strict sense of their spe-cific meaning when used in referring to the ploidyconditions of a culture. To overcome the problemof incorrectly using well-established terminology,we propose that the term, mixoploid, be used inreferring to such cultures. A condition of mixo-ploidy would refer to a population of cells inwhich a mixture of ploidy occurred with themajority of cells being diploid or greater inploidy.

SUMMARY

The large cells of the apparent life cycle ofMicrococcus aureus were investigated, and theconditions were found under which a large per-centage of such cells formed. Cytological studieswith various stains and with phase microscopydid not reveal the precise origin of the large cells,but information was found which suggested anintracellular nuclear fusion to yield a diploid orpolyploid nucleus which would then undergomeiosis and result again in normal haploid cells.The diploid nature of the large cells was sub-stantiated by radiobiological studies. In these,the dose-survivor relationship was exponentialin the normal cells with both x-ray or ultravioletradiations. This single-hit type of response canbe interpreted as a criterion of haploidy. Thedose-survivor curve of the large cells when ir-radiated by x-ray or ultraviolet light was sig-moidal, showing a hit-multiplicity of two or more-thus indicating the diploid or polyploid natureof these celLs. Factors of clumping, multi-nuclearity, and multicellularity were investigatedin order to validate ploidy interpretations of theradiobiological responses.

It was found that ultraviolet light and certainchemicals would cause an increased incidence inthe large cells. In many cases the large cells thusproduced were indistinguishable from those oc-curring naturally, but some of the chemicalstested caused atypical-appearing large cells toform.The appearance of the large cells was found to

be a fairly generalized phenomenon, since theywere found in most of the coccus cultures whichwere tested. Thus the existence of a simple lifecycle involving a ploidy change may be commonin the bacteria.

It was suggested that the term, mixoploid, beused in referring to populations such as bacterialcultures in which a mixture of ploidy is foundwith the majority of the cells diploid or greater.

REFERENCES

ATwOOD, K. C., AND NORMAN, A. 1949 On theinterpretation of multi-hit survival curves.Proc. Natl. Acad. Sci. U. S., 85, 696-709.

BIsSET, K. A. 1950 The cytology and life historyof bacteria. The Williams & Wilkins Co.,Baltimore.

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CHANCE, H. L. 1952 Crystal violet as a nuclearstain for Gaffkya tetragena and other bacteria.Stain Technol., 27, 253-258.

CLARK, J. B., AND WEBB, R. B. 1955a A com-parison of the crystal violet nuclear stain withother technics. Stain Technol., 30, 73-78.

CLARK, J. B., AND WEBB, R. B. 1955b Radi-ation response as an indication of ploidy inbacteria. Bacteriol. Proc., 1955, 36.

DELAMATER, E. D. 1951 A staining and de-hydration procedure for the handling of micro-organisms. Stain Technol., 26, 199-204.

DELAMATER, E. D. 1954 Cytology of bacteria.II. The bacterial nucleus. Ann. Rev. Micro-biol., 8, 23-46.

DUIBOs, R. J. 1945 The bacterial cell. HarvardUniv. Press, Cambridge.

GATES, F. L. 1929 A study of the bactericidalaction of ultra violet light. I. The reactionto monochromatic radiations. J. Gen. Phy-siol., 13, 231-248.

KNAYSI, G. 1955 The structure, composition,and behavior of the nucleus in Bacillus cereus.J. Bacteriol., 69, 117-129.

LATARJET, R. 1952 Some factors influencingcell radiosensitivity by acting at the level ofthe primary biochemical action. Symposiumon Radiobiol., pp. 241-257.

LEA, D. E. 1947 Actions of radiations on livingcells. Cambridge Univ. Press, England.

LEDERBERG, J., LEDERBERG, E. M., ZINDER, N. D.,

AND LIVELY, E. R. 1951 Recombinationanalysis of bacterial heredity. Cold SpringHarbor Symposia Quant. Biol., 16, 413-443.

LINDEGREN, C. C. 1942 Nuclear apparatus andsexual mechanism in a micrococcus. IowaState Coll. J. Sci., 16, 307-318.

MURRAY, R. G. E., GILLEN, 0. H., AND HEAGY,F. C. 1950 Cytological changes in Escheri-chia coli produced by infection with phageT2. J. Bacteriol., 59, 603-615.

RENTSCHLER, H. C., AND NAGY, R. 1942 Bac-tericidal action of ultraviolet radiation onair-borne organisms. J. Bacteriol., 44, 85-94.

SZYBALSKI, W. 1952 Gradient plate techniquefor the study of bacterial resistance. Science,116, 45-51.

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