Culture Purity Assessments and Morphological Dissociation in the Pleomorphic Microorganism Bacterionema matruchotii
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APPLIED MICROBIOLOGY, June 1974, p. 1134-1141 Vol. 27, No. 6
Copyright @ 1974 American Society for MicrobIlology Printed in U.S.A.
Culture Purity Assessments and Morphological Dissociation
in the Pleomorphic Microorganism
Bacterionema matruchotii
MARION N. GILMOUR AND GWENDOLYN TURNER
Eastman Dental Center, Rochester, New York 14603
Received for publication 13 December 1973
The inherent pleomorphism of Bacterionema matruchotii resulting from its
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mode of reproduction was enhanced by temporal effects of culture age and growth
condition and by the more lasting effects of rough to intermediate to smooth
morphological dissociation. Routine morphological observations with a single
growth condition were inadequate to permit unambiguous judgements of culture
purity. Multiple criteria were required. Pleomorphism and the undetected
presence of contaminants in primary and successive cultures of B. matruchotii
can explain the emergence of unrelated bacteria from B. matruchotii reported
previously by others and ascribed to genetic instability.
Morphological variability exhibited by Bacte- pure culture assessments and correlative ge-
rionema matruchotii, a filamentous microorga- netic factors and taxonomic concepts would be
nism indigenous to the oral flora, is a conse- highly complicated. However, with more than
quence of its mode of reproduction (9), cultural 200 aerobically derived and tested strains iso-
environment (10), and an apparent rough to lated in the U.S.A., England, and Japan, there
intermediate to smooth (R -_ I _ S) morphologi- was a remarkable degree of homogeneity and
cal dissociation (2). This pleomorphism should reproducibility in some 28 simple biochemical
reduce the accuracy of culture purity assess- tests, and only one serotype has been reported
ments which depend upon routine observations (M. N. Gilmour, In R. Buchanan and N. Gib-
of colonial and cell morphologies in a restricted bons, ed., Bergey's Manual of Determinative
number of cultural conditions. Bacteriology, 8th ed., in press). Also, the de-
Recent reports describe the emergence of velopment of "variants" as described by Streck-
genetically different bacillary and coccal "vari- fuss and Smith (20) has not been seen during
ants" from B. matruchotii (7, 19, 20). observations of the growth of more than 500
Gram-negative rod elements released during single cells to microcolonies (9; M. N. Gilmour
serial broth transfer were associated with the and G. Turner, unpublished data).
development of diphtheroid "variants" which These discrepancies instigated the current
formed flat small colonies on plates (20; J. L. studies. (i) The effects of cultivation conditions
Streckfuss, W. N. Smith, A. C. Taylor, and J. and R -_ S transitions on morphology and other
Ennever, Int. Ass. Dent. Res. Abstr., no. 192, characteristics of B. matruchotii were re-
1971). The diphtheroid "variants" were consid- examined. (ii) The emergence of coccal and
ered to be a genetically unstable intermediate bacillary "variants" was studied using the exact
phase because those which were subculturable experimental conditions of Streckfuss and
yielded different and stable variants, some of Smith (20), since culture instability usually
which were identified as Streptococcus sp. correlates with culture history and handling (3).
(mutans, sanguis, and mitis) and Lactobacillus (iii) The colony-forming unit (CFU) recoveries
sp. (W. N. Smith, J. L. Streckfuss, and L. of B. matruchotii and a diphtheroid similar to
R. Brown, Int. Ass. Dent. Res. Abstr., no. 59, the above-described intermediate diphtheroid
1971). "Variant" formation was proposed to "variant" were compared in pure and in mixed
occur as a result of genetic instability in B. cultures. (iv) Culture purity assessments based
matruchotii rather than from contamination or on conventional observations were shown to be
the interaction between species and was termed inadequate, and the need for nonroutine proce-
"morphological dissociation" (20). dure is discussed.
This interpretation of unique genetic instabil-
ity in B. matruchotii implies that extreme MATERIALS AND METHODS
variability should occur both between and Cultures and maintenance. Table 1 lists cultures,
within pure culture isolates. If this were true, which were kindly provided by J. L. Streckfuss, and
1134VoL 27, 1974 CULTURE PURITY ASSESSMENTS IN B. MATRUCHOTII 1135
their lineage and history with respect to "variant" per ml (13); modified tryptone with 0.5% glucose (13);
production. Original isolation of the two ATCC cul- and brain heart infusion (BHI, Difco). The gaseous
tures was by Gilmour (10), and that of strain 13 was incubation environments were 95% H3 plus 5% CO, in
by Richardson and Schmidt (18). The cultures re- Brewer jars, air, and air supplemented with 5% CO.
ceived were tested for purity immediately upon open- Appropriate variations of these conditions were em-
ing and again 3 days later. Additional isolates and ployed to subculture colonies other than B.
strains utilized were obtained from dental calculus or matruchotii which were encountered.
plaque and were similar to previous descriptions of Colonial morphologies were assessed microscop-
them (9-12). Working stock cultures were maintained ically both for typical mature colonies, which can vary
on a half-strength brain heart infusion agar (HBHI) in appearance (2, 11, 12), and for immature spider-
(10) by incubating 2 days in air supplemented with 5% microcolonies (12), which were found herein and
CO, storing at 3 C, and transferring at 2-week elsewhere (M. N. Gilmour, unpublished data) to be a
intervals. For long-term storage, suspensions of early universal characteristic of all known strains of B.
stationary-phase cultures in HBHI supplemented matruchotii. Cell morphologies were examined at
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with 10% glycerol were heat-sealed in glass vials and x 1,000 (numerical aperture [N.A. l 1.35) utilizing (i)
stored at -75 C. plates with microcolonies which were covered with
Culture purification and purity testing. Unless cleaned cover slips, and (ii) smears prepared with
otherwise stated, purification was accomplished by: filtered stains.
(i) serially streaking microcolonies (30 to 50 cells) Final purity assessments included observations of
three times using UBHI with 20-h carbon dioxide- growth in microcultures (9) and biochemical testing
supplemented aerobic incubation and/or HBHI- (10). The microcultures were modified by using a
hemin with 30-h anaerobic incubation, followed by medium prepared by mixing equal volumes of sepa-
(ii) cultivation of approximately 40 tubes, each con- rately sterilized double-strength HBHI broth and 4%
taining 1 ml of a broth suspension homogenized Ionagar no. 2 (Colab Laboratories, Inc., Glenwood,
lightly to disperse the cells and diluted to contain less Ill.) in water (pH 7.4) and adding glucose to 0.5%.
than 0.5 CFU/ml, (iii) recovery from cultures with 1 Growth from single cells was observed with phase-
granule or the least amount of growth for a second or contrast microscopy (x900, N.A. 1.35).
third cycle of streaking and dilution as above, and (iv) "Morphological dissociation." Experiments
a final round of serial streaking as in (i). were performed as described by Streckfuss and
Cultures were routinely tested for purity by micro- Smith (20) with pure cultures of strains 1-F, P. and
scopic examination of colonial and cellular morpholo- 15-65 P3. Freshly prepared BHI broth or agar was
gies of simultaneously prepared multiple cultures. To used, and all cultures and controls were incubated in
enhance the sensitivity of contaminant detection, air at 37 C. Multiple CFU of 1-F7 P. were used to
three media were employed, and each was incubated initiate serial transfer of exponential-phase cultures
in three gaseous environments. The media used were: of 1-F7 P.. To initiate cultures from a single CFU
HBHI supplemented with glucose to 0.5% and, for (20), spider-microcolonies from 20-h plates of strain
anaerobic incubation, addition of 0.05 mg of hemin 15-65 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 11-65 P2d Pd variants
ATCC 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 74uvariants
ATCC 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 variants
,
Richardson'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.1136 GILMOUR AND TURNER APPL. MICROBIOL.
and diluted, and multiple flasks were each inoculated three to six dilutions for each sample, were done.
with approximately 1 CFU which was ascertained
from viable counts and the incidence of granules in RESULTS
the broth cultures. The resulting culture was used to
initiate serial broth-to-broth passage of exponential- Morphological observations. All cultures
phase cultures. To reduce the possibility of contami- were checked for purity as described above.
nant entry (3), a duplicate culture was made at each Among those received, strain 15-65 P2 was found
passage for testing "variant" production. These cul- to contain typical B. matruchotii and large
tures were incubated for 40 h and four serial dilutions populations of a diphtheroid and a coccus in
from them were plated. After 5 days of incubation, all both smears and cultures. The latter two were
plates, dilution tubes, original cultures, and subcul- eliminated by means of rigorous purification
tures from them were observed for any colonial and
cell morphologies differing from B. matruchotii. and were not encountered over a subsequent
Mixed cultures. The effect of mixed culture 2-year interval, except when attributable to
contaminant entry ("morphological dissocia-
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growth on CFU recoveries was studied with B.
matruchotii strain 15-65 P2 and a flat diphtheroid tion" experiments below, Table 2). Both of the
colony which was isolated from the morphological cultures derived from ATCC 14266 (Table 1) ap-
dissociation experiment broth culture 2 of 1F,-P. peared to contain minor diphtheroid and coccal
(Table 2) and which was similar to the diphtheroid population components which also were effec-
variant described by Streckfuss and Smith (20). tively eliminated from the typical B.
Separate cultures of each were grown in BHI broth matruchotii component.
until mid-log phase, directly counted, and inoculated
into flasks of BHI broth at diphtheroid-B. matruchotii Morphological comparisons between the
cell ratios of 1:1 and 1:10. Controls included pure strains received, when pure, and our isolates
cultures of each microorganism and uninoculated showed that the four cultures derived from
broth. All flasks were incubated in air and sampled at Gilmour isolates 18 and 47 (11-65 P2, 15-65 P2,
intervals; Gram stains and viable plate counts, using 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-containing
colonies in B. matruchotii cultures
Inoculum Plating Plates with aberrant colonies
No. of plates with
Serial Fc NoofFrom
Strain Source broth
tested
Operatorb maskc
ms No.tf All Flat
plts aberrant diph-
dilution
tube no.
types theroids
15-65 P2 CFU 1 2 B - 12 1 1 1
3 B - 12 1 0 1
CFU 3 1 B - 8 1 0 3
CFU 7 1 B - 12 1 0 4c
2 B - 12 1 0 3
7 B - 12 2d 0 1
8 B - 12 0 0
1-F7 P6 Culture received 1 B - 12 0 0
after serial streak- 1 B - 9 0 0
ing 2 B - 12 3 1 1,4
3 B - 12 1 0 2
4 B - 12 9d 1 1,2,3,4
Sterile broth 1 B - 100 25d 10
2 B + 100 12d 7
3 C + 100 4 1
a
The first culture of the serially transferred exponential-phase broth cultures was started with single CFU of
15-65 P2 and multiple CFU of 1-F7 PI.
'Operator B was a trained technician; operator C was a highly trained microbiologist. Symbols: -, no face
mask 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.VOL. 27, 1974 CULTURE PURITY ASSESSMENTS IN B. MATRUCHOTH11137
our cultures of 18 and 47 when they were serially fact, resembled those described between rough
transferred on BHI and incubated aerobically. to intermediate and smooth colony variants
Aerobic mature colonies on BHI were raised described by Bibby and Berry (2).
with a high convoluted surface resembling a To substantiate the occurrence of an R - I -
molar tooth. They were white, opaque, and S transition, 20 different pure culture strains
friable, and tended to have a lobate edge and which exhibited the R morphology in primary
sometimes a fine lacerate halo. The cells were culture were simultaneously maintained aero-
shorter, some filaments had bulbous ends which bically and anaerobically on various media for
eventually lysed, and branching and pleo- approximately 30 transfers. The results con-
morphic forms were more common than when firmed that colony transitions, with concomi-
cultivated on other media or under reduced tant cellular morphology changes, occurred in
oxygen tension. With anaerobic incubation on an R (tough, adherent, and, under anaerobic
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HBHI, mature colonies were tough, filamen- conditions, flat, filamentous, and lacerate and
tous, lacerate, and flat to umbonate, and cellu- composed of long filaments) through intermedi-
lar components included many long filaments ate (crumbly, less adherent) to S (smooth, soft,
attached to or separated from wider bacillary nonadherent, short cells) sequence as detailed
elements. Under all conditions, very young by B. G. Bibby (Ph.D. thesis, University of
immature colonies were spider-like. Growth in Rochester, Rochester, N.Y., 1935). The R - I -
broth was granular or in loose flakes and was S transition was dependent upon cultural con-
readily dispersed with light homogenization. ditions. R colonies were maintained with an-
The mode of reproduction, observed in mi- aerobiosis, particularly in neutral or alkaline
crocultures, and biochemical reactions tested at environments; I colonies were found most fre-
three different times were as previously de- quently after repeated aerobic transfers and
scribed (9, 10). readily reverted to the R form under anaerobio-
Cultures 1-F7 P. and 2-F7 P,, which derived sis; and S colonies developed after prolonged
from Richardson's strain 13 (Table 1), had the aerobic incubation, particularly in acid environ-
same biochemical characteristics and mode of ments or on BHI. The 1-F7 and 2-F7 cultures
reproduction, which resulted in spider-like mi- were similar to S-colony variants previously
crocolonies, but had certain morphological dif- illustrated by Bibby and Berry (2).
ferences. Broth culture growth was diffuse. Broth culture appearance and cellular mor-
Aerobic mature colonies on BHI were butyrous, phologies were also affected by this R - S
low convex, and usually with an entire edge and transition as well as by culture age and growth
were composed of diphtheroid-like cells, as condition. R cultures were granular and con-
illustrated for the RF7 bacillary "variant" by tained longer cells than S cultures where growth
Streckfuss and Smith (see Fig. 4 and 5, ref. 20). was diffuse. Pleomorphism increased after log
The incidence of long rods and oval bacillary phase; it was enhanced by aerobic incubation,
elements with deeply staining large intracellu- as illustrated by Bibby and Berry (2), and it
lar inclusions varied with incubation time, and became particularly marked in aerobic BHI
branching and bizarre pleomorphic morpholo- cultures. Such cultures of 15-65 P2, 1-F P,, and
gies increased markedly with culture age. Long 50 other strains had a preponderance of
filaments and the typical whip-handle cells gram-negative cells with intracellular gram-
were extremely rare, which was consistent with positive inclusions, an increase in pleomorphic,
microculture observations of aerobically main- sometimes bizarre cell forms, and considerable
tained and incubated cultures wherein frag- cellular debris including minute gram-negative
mentation occurred earlier than usual, thereby rod-like and gram-positive spherical elements.
preventing the formation of long filaments. A There was also an associated loss of viability:
change to a more typical cellular morphology decreased plate counts, turbidities, and
occurred in very young cultures and, with serial transferability.
transfer, under anaerobic conditions on HBHI "Morphological dissociation" yielding
where colonies eventually became more fila- coccal and bacillary "variants." Morpho-
mentous and less butyrous and comprised logical dissociation experiments were per-
higher proportions of longer filaments and formed with serially transferred broth cultures
whip-handle cells. These differences in colo- of strains 15-65 P2 and 1-F7 P. considered to be
nial and cellular morphologies between 1-F7 or pure by the criteria given in methods above.
2-F7 and the other four cultures received were Colonies other than B. matruchotii were found
more stable than the temporal effects resulting on many of the plates, and their incidence was
from culture age and growth condition and, in independent of initiation from single or multiple1138 GILMOUR AND TURNER APPL. MICROBIOL.
CFU (Table 2). In general, one plate from each
series had one such colony on it, but some
plates had many which originated from carry-
over by the spreader to successive plates.
There was no correlation between the sequence
of the dilution tube plated and the occurrence
of non-B. matruchotii colonies, including those
similar to the diphtheroid "variants" described
by Streckfuss and Smith (see Fig. 1, ref. 20).
The occurrence of such colonies could be dis-
continuous among the broth passages, the
colony type found varied between and within FIG. 1. Effect of mixed culture growth with varying
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plates from the individual broth cultures, and inocula sizes on viable counts of B. matruchotii and a
there was no relationship between the fre- diphtheroid contaminant.
quencies with which non-B. matruchotii
colonies occurred and either the percentage of
viable count or the B. matruchotii densities. the 1: 1 mixed culture than those in the 1: 10,
In fact, the overall incidence of non-B. indicating growth inhibition by B. matruchotii.
matruchotii colonies on the morphological dis- Neither the growth rate nor the CFU yields of B.
sociation test plates approximated that found matruchotii were affected by the diphtheroid.
on plates inoculated with sterile broth (operator The relationship between the growth rate of
B, no face mask, Table 2), and the types of both organisms was such that the diphtheroid
colonies on both sets of plates were similar. The could be carried by serial transfer of exponen-
source of these contaminants was indicated to tial-phase cultures, but the ability to reliably
be operator-related, because the incidence of detect it with the large B. matruchotii inocula
contaminated control plates was significantly ratio required the use of many plates per
reduced to 12% (P < .05) when operator B wore dilution plated to minimize sampling error
a face mask, and it was further significantly problems, and even this procedure eventually
reduced to 4% (P < .05) when operator C was became unsatisfactory. Plates from the un-
employed for plate preparation and streaking inoculated broth controls were uncontaminated
(Table 2). Moreover, comparisons of the relative throughout.
proportions of small, flat, translucent diphthe- In smears prepared at each sampling time,
roid colonies with gram-positive, coccal-con- the diphtheroid was readily differentiated from
taining colonies among the contaminants and in B. matruchotii when each was grown singly,
the nares populations were similar within each because the diphtheroid tended to occur in loose
of the operators B and C, but were different aggregates of four to twenty cells. However, in
between them. the mixtures at 21 h, only one aggregate was
Stains and subcultures of each of the small, observable in 0.005 ml of the undiluted 1:1
flat, translucent colonies encountered indicated mixture and none was found in the 1: 10 culture.
that the majority were diphtheroids which had At this time, the diphtheroid viable counts were
colony and cellular morphologies exactly as highest (Fig. 1), indicating that the CFU must
illustrated for the unstable diphtheroidal "vari- have occurred primarily as single cells. Their
ant" (see Fig. 1 and 3, ref. 20). These were appearance in pure cultures closely resembled
transferable and, when purified, did not pro- some of the rods in the B. matruchotii culture at
duce variants of differing genera. The remain- 21 h or later, and therefore their discrimination
der were streptococci, some of which did not by stained smears was unreliable.
subculture on BHI under aerobic conditions. Effect of pleomorphism on culture purity
CFU recoveries from mixed cultures. Be- assessments. These results showed that routine
cause of these results, the plating recoveries morphological examinations of growth from one
from mixed broth cultures of a diphtheroid cultural condition were inadequate for assessing
similar to the described morphological dissocia- culture purity. This was also found during
tion "variant" and B. matruchotii were studied. purification of 188 primary isolates over a 4-year
The input inoculum ratio sizes of diphtheroid- interval. Each isolate had typical B.
B. matruchotii were 1: 1 and 1: 10 from direct matruchotii colonial morphology in primary
counts and approximated 1: 40 and 1: 260 from pour plates (12). Inocula of crushed cell suspen-
viable plate counts (Fig. 1). Yields of the sions were serially streaked with the simultane-
diphtheroid in pure cultures were higher than ous use of multiple growth conditions through-
those in the mixtures, and they were greater in out, and morphologies of colonies and of cellularVOL. 27, 1974 CULTURE PURITY ASSESSMENTS IN B. MATRUCHOTII 1139
components in Gram-stained smears were purification of the B. matruchotii, errors were
monitored microscopically. Although the major made in assessing culture purity unless multiple
cellular components in the primary colonies criteria were employed. Those cultures having
were attributable to B. matruchotii, approxi- the following characteristics remained pure for
mately 95% had minor suspicious coccal, diph- more than 6 years. (i) Under a variety of
theroidal, or actinomyces-like elements in cultural conditions, all colonial and cellular
stained smears, and >50% exhibited contami- morphologies found were typical of B.
nant colonies when first transferred. matruchotii. (ii) Tests of a range of biochemical
Consistent aerobic cultivation on BHI re- characteristics were reproducible when re-
sulted in a 50% loss of cultures, 38% of which peated a minimum of three times with different
resulted from cessation of growth and 12% from inocula. (iii) The reproduction patterns from
overgrowth by contaminants. Curiously, these single cells and the resultant cell groupings were
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losses occurred nonrandomly with respect to consistent with those normally found (9).
transfer number from primary isolation (Table
3), and they could not be associated with known DISCUSSION
experimental variables. The previously reported morphological varia-
Among the cultures which were not lost, 10% bility in B. matruchotii, resulting from mode of
of the original isolates which appeared pure by reproduction, culture age, and cultivation con-
colony morphology and stained smears at the dition, was confirmed, as was the occurrence of
consecutive fifth, sixth, and seventh aerobic an R - I - S morphological dissociation. The
transfer on BHI subsequently were found to be data suggest that morphological dissociation
contaminated when subcultured under different occurs from mutant selection as reported for
conditions (Table 3). In each of these cases, the other systems (14). Primary isolates from the
contaminant encountered was similar to that natural habitat of dental plaque or calculus
which had been present in the homologous generally form the distinctive R colonies, al-
primary isolate. The most frequent were diph- though S colonies may occur (2; B. G. Bibby,
theroids, although Actinomyces sp. and a strep- Ph.D. thesis, University of Rochester, Roch-
tococcus were also involved. Their eradication ester, N.Y., 1935).
was successfully accomplished by (i) inoculat- However, "morphological dissociation,"
ing with microcolonies which dispersed better wherein B. matruchotii was reported to give rise
than mature colonies, (ii) changing the medium to a variety of genera and species (20; J. L.
to HBHI and incubating in reduced oxygen Streckfuss, W. N. Smith, A. C. Taylor, and J.
atmospheres, and (iii) utilizing cycles of streak- Ennever, Int. Ass. Dent. Res. Abstr. no. 192,
ing and dilution of broth suspensions. During 1971; W. N. Smith, J. L. Streckfuss, and L. R.
Brown, Int. Ass. Dent. Res. Abstr. no. 59, 1971)
TABLE 3. Percentages of culture losses and detection could not be confirmed. Although some aspects
of contaminants in apparently pure cultures' at each of the phenomenon were observed, these were
transfer number of B. matruchotii primary isolates attributable to pleomorphism and contaminant
under aerobic culture on BHI entry and, therefore, do not support the pro-
Transfer no.
posed extreme genetic instability for this micro-
Class- organism. Further, to date there is no evidence
1 2 3 4-6 7 8 9 12 |>14 which would allow attribution of the observa-
tions to endoparasitic bacterial symbionts.
Losses' The data presented, in conjunction with L-
Non-transferable 15 7 7 1 5 4 11140 GILMOUR AlNID TURNER APPL. MICROBIOL.
eradication (5, 8, 15). Moreover, growth initia- ble criteria include antigenic composition and
tion from a single cell is not attainable with deoxyribonucleic acid analyses. For B. matru-
some microorganisms (e.g., spirochetes), nor chotii, the percentage of guanine plus cytosine
are micromanipulators and/or the technique for (G + C) is 55 to 57 (L. R. Page and G. N.
utilizing them available to all. Krywolap, Abstr. Annu. Meet. Amer. Soc. Mi-
The criteria selected for culture purity assess- crobiol., 1973, G101, p. 43) and 53 (B. F. Ham-
ment are therefore important. Cellular and mond, personal communication). In contrast,
colonial morphologies are used routinely. The the G+C mole percent values ranged from 35 to
former is unreliable when applied to cultures of 41 for the various subspecies of Streptococcus
microorganisms which have variable cellular mutans (6), one of the reported "variants"
morphologies or where the contaminant and the isolated from B. matruchotii.
desired culture have similar morphologies. In
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either case, contaminant discrimination by rou- ACKNOWLEDGMENTS
tine means then relies solely upon colonial This work was supported by Public Health Service grants
morphology differences. The latter is of limited DE-370 and 2452 from the National Institute for Dental
value when cultivation conditions are subopti- Research.
mal for the contaminant or when the technique The authors thank J. M. Slack and S. S. Socransky for
employed is insensitive to low contaminant helpful discussions and review of the manuscript and Helen
Cselenyi and R. E. Berman for excellent technical assistance.
levels. These morphological characteristics are
therefore often as inadequate for culture purity LITERATURE CITED
assessment, particularly with pleomorphic mi-
croorganisms, as they are recognized to be for 1. Barile, M. F., and R. A. Delguidise. 1972. Isolations of
Mycoplasmas and their rapid identification by plate
classification purposes. Other characteristics epi-immunofluorescence, p. 165-181. In Pathogenic
must be employed in conjunction with them. mycoplasmas, CIBA Foundation Symposium. Elsevier,
These should include a minimum of two attri- New York.
butes which reflect different cellular properties 2. Bibby, B. G., and G. P. Berry. 1939. A cultural study of
filamentous bacteria obtained from the human mouth.
in order to allow for the normal variability J. Bacteriol. 38:263-274.
which will occur in the large populations being 3. Clasener, H. 1972. Pathogenicity of the L-phase of bacte-
studied. For example, deoxyribonucleic acid ria. Annu. Rev. Microbiol. 26:55-84.
assays may be affected by plasmid, prophage, or 4. Darlow, H. M. 1969. Safety in the microbiological labora-
tory, p. 169-203. In J. R. Norris and D. W. Ribbons
intracellular bacterial parasite content. Al- (ed.), Methods in microbiology, vol. 1. Academic Press
though purity assessments based upon Inc., New York.
reproducibility of major characteristics and 5. Doyle, J. E., and R. R. Ernst. 1969. Alcohol flaming-a
consistent colonial and cellular morphologies possible source of contamination in sterility testing.
Amer. J. Clin. Pathol. 51:407-408.
under a variety of growth conditions will mini- 6. Dunny, G. M., T. Hausner, and D. B. Clewell, 1972.
mize errors, the successful use of these criteria Buoyant densities of DNA from various strains of
remains dependent upon a judicious regard for Streptococcus mutans. Arch. Oral Biol. 17:1001-1003.
systematic contaminant entry sources, techni- 7. Ennever, J., L. Streckfuss, and I. Takazoe. 1973. Calcifi-
cation of bacillary and streptococcal variants of Bacte-
cal limitations, and symbiotic relationships be- rionema matruchotii. J. Dent. Res. 52:305-308.
tween mixtures and pleomorphism. However, 8. Ernst, R. R., and A. P. Kretz, Jr. 1964. Compatability of
with their proper application to cultures which sterilization and contamination control with applica-
appear to indicate extremely novel biological tion to spacecraft assembly. J. Amer. Ass. Contam.
Contr. 10:3-7.
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