APPLIED MICROBIOLOGY, June 1974, p. 1134-1141Copyright @ 1974 American Society for MicrobIlology

Vol. 27, No. 6Printed in U.S.A.

Culture Purity Assessments and Morphological Dissociationin the Pleomorphic Microorganism

Bacterionema matruchotiiMARION N. GILMOUR AND GWENDOLYN TURNEREastman Dental Center, Rochester, New York 14603

Received for publication 13 December 1973

The inherent pleomorphism of Bacterionema matruchotii resulting from itsmode of reproduction was enhanced by temporal effects of culture age and growthcondition and by the more lasting effects of rough to intermediate to smoothmorphological dissociation. Routine morphological observations with a singlegrowth condition were inadequate to permit unambiguous judgements of culturepurity. Multiple criteria were required. Pleomorphism and the undetectedpresence of contaminants in primary and successive cultures of B. matruchotiican explain the emergence of unrelated bacteria from B. matruchotii reportedpreviously by others and ascribed to genetic instability.

Morphological variability exhibited by Bacte-rionema matruchotii, a filamentous microorga-nism indigenous to the oral flora, is a conse-quence of its mode of reproduction (9), culturalenvironment (10), and an apparent rough tointermediate to smooth (R -_ I _ S) morphologi-cal dissociation (2). This pleomorphism shouldreduce the accuracy of culture purity assess-ments which depend upon routine observationsof colonial and cell morphologies in a restrictednumber of cultural conditions.Recent reports describe the emergence of

genetically different bacillary and coccal "vari-ants" from B. matruchotii (7, 19, 20).Gram-negative rod elements released duringserial broth transfer were associated with thedevelopment of diphtheroid "variants" whichformed flat small colonies on plates (20; J. L.Streckfuss, W. N. Smith, A. C. Taylor, and J.Ennever, Int. Ass. Dent. Res. Abstr., no. 192,1971). The diphtheroid "variants" were consid-ered to be a genetically unstable intermediatephase because those which were subculturableyielded different and stable variants, some ofwhich were identified as Streptococcus sp.(mutans, sanguis, and mitis) and Lactobacillussp. (W. N. Smith, J. L. Streckfuss, and L.R. Brown, Int. Ass. Dent. Res. Abstr., no. 59,1971). "Variant" formation was proposed tooccur as a result of genetic instability in B.matruchotii rather than from contamination orthe interaction between species and was termed"morphological dissociation" (20).This interpretation of unique genetic instabil-

ity in B. matruchotii implies that extremevariability should occur both between andwithin pure culture isolates. If this were true,

pure culture assessments and correlative ge-netic factors and taxonomic concepts would behighly complicated. However, with more than200 aerobically derived and tested strains iso-lated in the U.S.A., England, and Japan, therewas a remarkable degree of homogeneity andreproducibility in some 28 simple biochemicaltests, and only one serotype has been reported(M. N. Gilmour, In R. Buchanan and N. Gib-bons, ed., Bergey's Manual of DeterminativeBacteriology, 8th ed., in press). Also, the de-velopment of "variants" as described by Streck-fuss and Smith (20) has not been seen duringobservations of the growth of more than 500single cells to microcolonies (9; M. N. Gilmourand G. Turner, unpublished data).These discrepancies instigated the current

studies. (i) The effects of cultivation conditionsand R -_ S transitions on morphology and othercharacteristics of B. matruchotii were re-examined. (ii) The emergence of coccal andbacillary "variants" was studied using the exactexperimental conditions of Streckfuss andSmith (20), since culture instability usuallycorrelates with culture history and handling (3).(iii) The colony-forming unit (CFU) recoveriesof B. matruchotii and a diphtheroid similar tothe above-described intermediate diphtheroid"variant" were compared in pure and in mixedcultures. (iv) Culture purity assessments basedon conventional observations were shown to beinadequate, and the need for nonroutine proce-dure is discussed.

MATERIALS AND METHODSCultures and maintenance. Table 1 lists cultures,

which were kindly provided by J. L. Streckfuss, and

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their lineage and history with respect to "variant"production. Original isolation of the two ATCC cul-tures was by Gilmour (10), and that of strain 13 was

by Richardson and Schmidt (18). The cultures re-ceived were tested for purity immediately upon open-ing and again 3 days later. Additional isolates andstrains utilized were obtained from dental calculus orplaque and were similar to previous descriptions ofthem (9-12). Working stock cultures were maintainedon a half-strength brain heart infusion agar (HBHI)(10) by incubating 2 days in air supplemented with 5%CO, storing at 3 C, and transferring at 2-weekintervals. For long-term storage, suspensions of earlystationary-phase cultures in HBHI supplementedwith 10% glycerol were heat-sealed in glass vials andstored at -75 C.

Culture purification and purity testing. Unlessotherwise stated, purification was accomplished by:(i) serially streaking microcolonies (30 to 50 cells)three times using UBHI with 20-h carbon dioxide-supplemented aerobic incubation and/or HBHI-hemin with 30-h anaerobic incubation, followed by(ii) cultivation of approximately 40 tubes, each con-taining 1 ml of a broth suspension homogenizedlightly to disperse the cells and diluted to contain lessthan 0.5 CFU/ml, (iii) recovery from cultures with 1granule or the least amount of growth for a second orthird cycle of streaking and dilution as above, and (iv)a final round of serial streaking as in (i).

Cultures were routinely tested for purity by micro-scopic examination of colonial and cellular morpholo-gies of simultaneously prepared multiple cultures. Toenhance the sensitivity of contaminant detection,three media were employed, and each was incubatedin three gaseous environments. The media used were:

HBHI supplemented with glucose to 0.5% and, foranaerobic incubation, addition of 0.05 mg of hemin

per ml (13); modified tryptone with 0.5% glucose (13);and brain heart infusion (BHI, Difco). The gaseousincubation environments were 95% H3 plus 5% CO, inBrewer jars, air, and air supplemented with 5% CO.Appropriate variations of these conditions were em-ployed to subculture colonies other than B.matruchotii which were encountered.

Colonial morphologies were assessed microscop-ically both for typical mature colonies, which can varyin appearance (2, 11, 12), and for immature spider-microcolonies (12), which were found herein andelsewhere (M. N. Gilmour, unpublished data) to be auniversal characteristic of all known strains of B.matruchotii. Cell morphologies were examined atx 1,000 (numerical aperture [N.A. l 1.35) utilizing (i)plates with microcolonies which were covered withcleaned cover slips, and (ii) smears prepared withfiltered stains.

Final purity assessments included observations ofgrowth in microcultures (9) and biochemical testing(10). The microcultures were modified by using a

medium prepared by mixing equal volumes of sepa-

rately sterilized double-strength HBHI broth and 4%Ionagar no. 2 (Colab Laboratories, Inc., Glenwood,Ill.) in water (pH 7.4) and adding glucose to 0.5%.Growth from single cells was observed with phase-contrast microscopy (x900, N.A. 1.35).

"Morphological dissociation." Experimentswere performed as described by Streckfuss andSmith (20) with pure cultures of strains 1-F, P. and15-65 P3. Freshly prepared BHI broth or agar wasused, and all cultures and controls were incubated inair at 37 C. Multiple CFU of 1-F7 P. were used toinitiate serial transfer of exponential-phase culturesof 1-F7 P.. To initiate cultures from a single CFU(20), spider-microcolonies from 20-h plates of strain15-65 P3 were suspended in broth, vigorously shaken,

TABLE 1. Culture derivation and morphological dissociation historya

Culture derivation Culture History of flat colony variant productionc

CFU 11 11-65P2d Pd variantsATCC 14265- CFU 1

(M.N.G., no. 18) ( 1-65) _CFU 15 15-65 P2 P1 - variants (1-65 74 variants)

CFU 17 17-66 P2 P1 and P2 74uvariantsATCC 14266

(M.N.G., no. 47) ,CFU 19 19-66 P2 P1 and P2 74 variants

CFU 1 1-F7 P, P. /- variants, but P,0 , variantsRichardson's - CFU 7 (by

strain 13 dilution)CFU 2 2-F7 PI P,-P,,, /- variants

aData from J. L. Streckfuss and W. N. Smith (personal communication).b CFU refers to the designation of the CFU isolated either by micromanipulation or dilution.c The occurrence of variant colonies was low among the cultures tested, different between strains and be-

tween CFU isolates from the same strain, and possibly independent of the number of serial broth passages, be-cause variants were found as early as the first broth passage of the CFU 15-65 but not until the tenth with 1-F,.

d P, serial exponential-phase broth passages numbered consecutively.

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and diluted, and multiple flasks were each inoculatedwith approximately 1 CFU which was ascertainedfrom viable counts and the incidence of granules inthe broth cultures. The resulting culture was used toinitiate serial broth-to-broth passage of exponential-phase cultures. To reduce the possibility of contami-nant entry (3), a duplicate culture was made at eachpassage for testing "variant" production. These cul-tures were incubated for 40 h and four serial dilutionsfrom them were plated. After 5 days of incubation, allplates, dilution tubes, original cultures, and subcul-tures from them were observed for any colonial andcell morphologies differing from B. matruchotii.Mixed cultures. The effect of mixed culture

growth on CFU recoveries was studied with B.matruchotii strain 15-65 P2 and a flat diphtheroidcolony which was isolated from the morphologicaldissociation experiment broth culture 2 of 1F,-P.(Table 2) and which was similar to the diphtheroidvariant described by Streckfuss and Smith (20).Separate cultures of each were grown in BHI brothuntil mid-log phase, directly counted, and inoculatedinto flasks of BHI broth at diphtheroid-B. matruchotiicell ratios of 1:1 and 1:10. Controls included pure

cultures of each microorganism and uninoculatedbroth. All flasks were incubated in air and sampled atintervals; Gram stains and viable plate counts, using

three to six dilutions for each sample, were done.

RESULTSMorphological observations. All cultures

were checked for purity as described above.Among those received, strain 15-65 P2 was foundto contain typical B. matruchotii and largepopulations of a diphtheroid and a coccus inboth smears and cultures. The latter two wereeliminated by means of rigorous purificationand were not encountered over a subsequent2-year interval, except when attributable tocontaminant entry ("morphological dissocia-tion" experiments below, Table 2). Both of thecultures derived from ATCC 14266 (Table 1) ap-peared to contain minor diphtheroid and coccalpopulation components which also were effec-tively eliminated from the typical B.matruchotii component.Morphological comparisons between the

strains received, when pure, and our isolatesshowed that the four cultures derived fromGilmour isolates 18 and 47 (11-65 P2, 15-65 P2,17-66 P2, and 19-66 P2, Table 1) were similar to

TABLE 2. Relationship between plating errors and occurrence of aberrant and flat diphtheroid-containingcolonies in B. matruchotii cultures

Inoculum Plating Plates with aberrant colonies

No. of plates withSerial Fc NoofFromStrain Source broth Operatorb maskc No.tf All Flat dilutiontested ms plts aberrant diph- tube no.

types theroids

15-65P2 CFU 1 2 B - 12 1 1 13 B - 12 1 0 1

CFU 3 1 B - 8 1 0 3CFU 7 1 B - 12 1 0 4c

2 B - 12 1 0 37 B - 12 2d 0 18 B - 12 0 0

1-F7 P6 Culture received 1 B - 12 0 0after serial streak- 1 B - 9 0 0ing 2 B - 12 3 1 1,4

3 B - 12 1 0 24 B - 12 9d 1 1,2,3,4

Sterile broth 1 B - 100 25d 102 B + 100 12d 73 C + 100 4 1

a The first culture of the serially transferred exponential-phase broth cultures was started with single CFU of15-65 P2 and multiple CFU of 1-F7 PI.

'Operator B was a trained technician; operator C was a highly trained microbiologist. Symbols: -, no facemask used; +, face mask used.

c Dilution tube was contaminated with the same contaminant as on the plate.d Contaminant present on one uninoculated plate was obviously spread and carried on the spreader to other

plates.

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our cultures of 18 and 47 when they were seriallytransferred on BHI and incubated aerobically.Aerobic mature colonies on BHI were raisedwith a high convoluted surface resembling amolar tooth. They were white, opaque, andfriable, and tended to have a lobate edge andsometimes a fine lacerate halo. The cells wereshorter, some filaments had bulbous ends whicheventually lysed, and branching and pleo-morphic forms were more common than whencultivated on other media or under reducedoxygen tension. With anaerobic incubation onHBHI, mature colonies were tough, filamen-tous, lacerate, and flat to umbonate, and cellu-lar components included many long filamentsattached to or separated from wider bacillaryelements. Under all conditions, very youngimmature colonies were spider-like. Growth inbroth was granular or in loose flakes and wasreadily dispersed with light homogenization.The mode of reproduction, observed in mi-crocultures, and biochemical reactions tested atthree different times were as previously de-scribed (9, 10).

Cultures 1-F7 P. and 2-F7 P,, which derivedfrom Richardson's strain 13 (Table 1), had thesame biochemical characteristics and mode ofreproduction, which resulted in spider-like mi-crocolonies, but had certain morphological dif-ferences. Broth culture growth was diffuse.Aerobic mature colonies on BHI were butyrous,low convex, and usually with an entire edge andwere composed of diphtheroid-like cells, asillustrated for the RF7 bacillary "variant" byStreckfuss and Smith (see Fig. 4 and 5, ref. 20).The incidence of long rods and oval bacillaryelements with deeply staining large intracellu-lar inclusions varied with incubation time, andbranching and bizarre pleomorphic morpholo-gies increased markedly with culture age. Longfilaments and the typical whip-handle cellswere extremely rare, which was consistent withmicroculture observations of aerobically main-tained and incubated cultures wherein frag-mentation occurred earlier than usual, therebypreventing the formation of long filaments. Achange to a more typical cellular morphologyoccurred in very young cultures and, with serialtransfer, under anaerobic conditions on HBHIwhere colonies eventually became more fila-mentous and less butyrous and comprisedhigher proportions of longer filaments andwhip-handle cells. These differences in colo-nial and cellular morphologies between 1-F7 or2-F7 and the other four cultures received weremore stable than the temporal effects resultingfrom culture age and growth condition and, in

fact, resembled those described between roughto intermediate and smooth colony variantsdescribed by Bibby and Berry (2).To substantiate the occurrence of an R - I -

S transition, 20 different pure culture strainswhich exhibited the R morphology in primaryculture were simultaneously maintained aero-bically and anaerobically on various media forapproximately 30 transfers. The results con-firmed that colony transitions, with concomi-tant cellular morphology changes, occurred inan R (tough, adherent, and, under anaerobicconditions, flat, filamentous, and lacerate andcomposed of long filaments) through intermedi-ate (crumbly, less adherent) to S (smooth, soft,nonadherent, short cells) sequence as detailedby B. G. Bibby (Ph.D. thesis, University ofRochester, Rochester, N.Y., 1935). The R - I -S transition was dependent upon cultural con-ditions. R colonies were maintained with an-aerobiosis, particularly in neutral or alkalineenvironments; I colonies were found most fre-quently after repeated aerobic transfers andreadily reverted to the R form under anaerobio-sis; and S colonies developed after prolongedaerobic incubation, particularly in acid environ-ments or on BHI. The 1-F7 and 2-F7 cultureswere similar to S-colony variants previouslyillustrated by Bibby and Berry (2).Broth culture appearance and cellular mor-

phologies were also affected by this R - Stransition as well as by culture age and growthcondition. R cultures were granular and con-tained longer cells than S cultures where growthwas diffuse. Pleomorphism increased after logphase; it was enhanced by aerobic incubation,as illustrated by Bibby and Berry (2), and itbecame particularly marked in aerobic BHIcultures. Such cultures of 15-65 P2, 1-F P,, and50 other strains had a preponderance ofgram-negative cells with intracellular gram-positive inclusions, an increase in pleomorphic,sometimes bizarre cell forms, and considerablecellular debris including minute gram-negativerod-like and gram-positive spherical elements.There was also an associated loss of viability:decreased plate counts, turbidities, andtransferability."Morphological dissociation" yielding

coccal and bacillary "variants." Morpho-logical dissociation experiments were per-formed with serially transferred broth culturesof strains 15-65 P2 and 1-F7 P. considered to bepure by the criteria given in methods above.Colonies other than B. matruchotii were foundon many of the plates, and their incidence wasindependent of initiation from single or multiple

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CFU (Table 2). In general, one plate from eachseries had one such colony on it, but someplates had many which originated from carry-over by the spreader to successive plates.There was no correlation between the sequenceof the dilution tube plated and the occurrenceof non-B. matruchotii colonies, including thosesimilar to the diphtheroid "variants" describedby Streckfuss and Smith (see Fig. 1, ref. 20).The occurrence of such colonies could be dis-continuous among the broth passages, thecolony type found varied between and withinplates from the individual broth cultures, andthere was no relationship between the fre-quencies with which non-B. matruchotiicolonies occurred and either the percentage ofviable count or the B. matruchotii densities.

In fact, the overall incidence of non-B.matruchotii colonies on the morphological dis-sociation test plates approximated that foundon plates inoculated with sterile broth (operatorB, no face mask, Table 2), and the types ofcolonies on both sets of plates were similar. Thesource of these contaminants was indicated tobe operator-related, because the incidence ofcontaminated control plates was significantlyreduced to 12% (P < .05) when operator B worea face mask, and it was further significantlyreduced to 4% (P < .05) when operator C wasemployed for plate preparation and streaking(Table 2). Moreover, comparisons of the relativeproportions of small, flat, translucent diphthe-roid colonies with gram-positive, coccal-con-taining colonies among the contaminants and inthe nares populations were similar within eachof the operators B and C, but were differentbetween them.

Stains and subcultures of each of the small,flat, translucent colonies encountered indicatedthat the majority were diphtheroids which hadcolony and cellular morphologies exactly asillustrated for the unstable diphtheroidal "vari-ant" (see Fig. 1 and 3, ref. 20). These weretransferable and, when purified, did not pro-duce variants of differing genera. The remain-der were streptococci, some of which did notsubculture on BHI under aerobic conditions.CFU recoveries from mixed cultures. Be-

cause of these results, the plating recoveriesfrom mixed broth cultures of a diphtheroidsimilar to the described morphological dissocia-tion "variant" and B. matruchotii were studied.The input inoculum ratio sizes of diphtheroid-B. matruchotii were 1: 1 and 1: 10 from directcounts and approximated 1: 40 and 1: 260 fromviable plate counts (Fig. 1). Yields of thediphtheroid in pure cultures were higher thanthose in the mixtures, and they were greater in

FIG. 1. Effect of mixed culture growth with varyinginocula sizes on viable counts of B. matruchotii and adiphtheroid contaminant.

the 1: 1 mixed culture than those in the 1: 10,indicating growth inhibition by B. matruchotii.Neither the growth rate nor the CFU yields of B.matruchotii were affected by the diphtheroid.The relationship between the growth rate ofboth organisms was such that the diphtheroidcould be carried by serial transfer of exponen-tial-phase cultures, but the ability to reliablydetect it with the large B. matruchotii inocularatio required the use of many plates perdilution plated to minimize sampling errorproblems, and even this procedure eventuallybecame unsatisfactory. Plates from the un-inoculated broth controls were uncontaminatedthroughout.

In smears prepared at each sampling time,the diphtheroid was readily differentiated fromB. matruchotii when each was grown singly,because the diphtheroid tended to occur in looseaggregates of four to twenty cells. However, inthe mixtures at 21 h, only one aggregate wasobservable in 0.005 ml of the undiluted 1:1mixture and none was found in the 1: 10 culture.At this time, the diphtheroid viable counts werehighest (Fig. 1), indicating that the CFU musthave occurred primarily as single cells. Theirappearance in pure cultures closely resembledsome of the rods in the B. matruchotii culture at21 h or later, and therefore their discriminationby stained smears was unreliable.

Effect of pleomorphism on culture purityassessments. These results showed that routinemorphological examinations of growth from onecultural condition were inadequate for assessingculture purity. This was also found duringpurification of 188 primary isolates over a 4-yearinterval. Each isolate had typical B.matruchotii colonial morphology in primarypour plates (12). Inocula of crushed cell suspen-sions were serially streaked with the simultane-ous use of multiple growth conditions through-out, and morphologies of colonies and of cellular

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components in Gram-stained smears weremonitored microscopically. Although the majorcellular components in the primary colonieswere attributable to B. matruchotii, approxi-mately 95% had minor suspicious coccal, diph-theroidal, or actinomyces-like elements instained smears, and >50% exhibited contami-nant colonies when first transferred.

Consistent aerobic cultivation on BHI re-sulted in a 50% loss of cultures, 38% of whichresulted from cessation of growth and 12% fromovergrowth by contaminants. Curiously, theselosses occurred nonrandomly with respect totransfer number from primary isolation (Table3), and they could not be associated with knownexperimental variables.Among the cultures which were not lost, 10%

of the original isolates which appeared pure bycolony morphology and stained smears at theconsecutive fifth, sixth, and seventh aerobictransfer on BHI subsequently were found to becontaminated when subcultured under differentconditions (Table 3). In each of these cases, thecontaminant encountered was similar to thatwhich had been present in the homologousprimary isolate. The most frequent were diph-theroids, although Actinomyces sp. and a strep-tococcus were also involved. Their eradicationwas successfully accomplished by (i) inoculat-ing with microcolonies which dispersed betterthan mature colonies, (ii) changing the mediumto HBHI and incubating in reduced oxygenatmospheres, and (iii) utilizing cycles of streak-ing and dilution of broth suspensions. During

TABLE 3. Percentages of culture losses and detectionof contaminants in apparently pure cultures' at eachtransfer number of B. matruchotii primary isolates

under aerobic culture on BHI

Transfer no.Class-

1 2 3 4-6 7 8 9 12 |>14

Losses'Non-transferable 15 7 7 1 5 4 1 <1 2

Contaminant 0 1 1 <1 2 0 0 2 0over-growth(So)

Apparently pure 3 4 0 3to contaminated _

a 10% which were judged pure by routine morpho-logical examinations at three consecutive transfers(no. 5-7) subsequently-were found to be contaminatedwith a microorganism present in the primary isolate.b50% were lost because of non-transferability or

overgrowth by contaminants.

purification of the B. matruchotii, errors weremade in assessing culture purity unless multiplecriteria were employed. Those cultures havingthe following characteristics remained pure formore than 6 years. (i) Under a variety ofcultural conditions, all colonial and cellularmorphologies found were typical of B.matruchotii. (ii) Tests of a range of biochemicalcharacteristics were reproducible when re-peated a minimum of three times with differentinocula. (iii) The reproduction patterns fromsingle cells and the resultant cell groupings wereconsistent with those normally found (9).

DISCUSSIONThe previously reported morphological varia-

bility in B. matruchotii, resulting from mode ofreproduction, culture age, and cultivation con-dition, was confirmed, as was the occurrence ofan R - I - S morphological dissociation. Thedata suggest that morphological dissociationoccurs from mutant selection as reported forother systems (14). Primary isolates from thenatural habitat of dental plaque or calculusgenerally form the distinctive R colonies, al-though S colonies may occur (2; B. G. Bibby,Ph.D. thesis, University of Rochester, Roch-ester, N.Y., 1935).

However, "morphological dissociation,"wherein B. matruchotii was reported to give riseto a variety of genera and species (20; J. L.Streckfuss, W. N. Smith, A. C. Taylor, and J.Ennever, Int. Ass. Dent. Res. Abstr. no. 192,1971; W. N. Smith, J. L. Streckfuss, and L. R.Brown, Int. Ass. Dent. Res. Abstr. no. 59, 1971)could not be confirmed. Although some aspectsof the phenomenon were observed, these wereattributable to pleomorphism and contaminantentry and, therefore, do not support the pro-posed extreme genetic instability for this micro-organism. Further, to date there is no evidencewhich would allow attribution of the observa-tions to endoparasitic bacterial symbionts.The data presented, in conjunction with L-

phase and Mycoplasma studies (3, 17), showthat the establishment of pure cultures and theassessment of culture purity often presentgreater difficulties than the frequently casualtreatment of them warrants. The isolation of asingle cell by micromanipulation for cultureinitiation does not necessarily ensure purity ofsubsequent cultures; with inadequate opticssmaller adherent heterologous microorganismsmay not be detected. Contaminant entry routesare numerous, diverse (4), and often unrecog-nized (1, 3, 14), and it cannot be assumed thattechniques for entry reduction are equivalent to

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eradication (5, 8, 15). Moreover, growth initia-tion from a single cell is not attainable withsome microorganisms (e.g., spirochetes), norare micromanipulators and/or the technique forutilizing them available to all.The criteria selected for culture purity assess-

ment are therefore important. Cellular andcolonial morphologies are used routinely. Theformer is unreliable when applied to cultures ofmicroorganisms which have variable cellularmorphologies or where the contaminant and thedesired culture have similar morphologies. Ineither case, contaminant discrimination by rou-tine means then relies solely upon colonialmorphology differences. The latter is of limitedvalue when cultivation conditions are subopti-mal for the contaminant or when the techniqueemployed is insensitive to low contaminantlevels. These morphological characteristics aretherefore often as inadequate for culture purityassessment, particularly with pleomorphic mi-croorganisms, as they are recognized to be forclassification purposes. Other characteristicsmust be employed in conjunction with them.These should include a minimum of two attri-butes which reflect different cellular propertiesin order to allow for the normal variabilitywhich will occur in the large populations beingstudied. For example, deoxyribonucleic acidassays may be affected by plasmid, prophage, orintracellular bacterial parasite content. Al-though purity assessments based uponreproducibility of major characteristics andconsistent colonial and cellular morphologiesunder a variety of growth conditions will mini-mize errors, the successful use of these criteriaremains dependent upon a judicious regard forsystematic contaminant entry sources, techni-cal limitations, and symbiotic relationships be-tween mixtures and pleomorphism. However,with their proper application to cultures whichappear to indicate extremely novel biologicalphenomena, confusion in the literature will bereduced. This same restraint also applies toreports of the isolation of novel microorganismsfrom mixed natural ecosystems, where purity isoften assumed with the use of an isolated colonyon the primary or secondary plate.The criteria employed herein for B. matru-

chotii, namely morphological changes in re-sponse to growth conditions, mode of reproduc-tion, and simple fermentative and biochemicalcharacteristics, should not be regarded as eitheruniversally applicable or optimal. The first ofthese requires considerable experience with themicroorganism, and the latter two would besusceptible to species variability. Other utiliza-

NID TURNER APPL. MICROBIOL.

ble criteria include antigenic composition anddeoxyribonucleic acid analyses. For B. matru-chotii, the percentage of guanine plus cytosine(G+ C) is 55 to 57 (L. R. Page and G. N.Krywolap, Abstr. Annu. Meet. Amer. Soc. Mi-crobiol., 1973, G101, p. 43) and 53 (B. F. Ham-mond, personal communication). In contrast,the G+C mole percent values ranged from 35 to41 for the various subspecies of Streptococcusmutans (6), one of the reported "variants"isolated from B. matruchotii.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantsDE-370 and 2452 from the National Institute for DentalResearch.

The authors thank J. M. Slack and S. S. Socransky forhelpful discussions and review of the manuscript and HelenCselenyi and R. E. Berman for excellent technical assistance.

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