ANTnIcIoBIAL AGENTS AND CHEMOTHERAPY, Mar. 1977, P. 482490 Copyright 0 1977 American Society for Microbiology
Vol. 11, No. 3 Printed in U.S.A.
Comparative Growth Rates of Selected Anaerobic Species in Four Commonly Used Broth Media WILLIAM SOTTILE II* Apm R. J. ZABRANSKY Microbiology Research Laboratory, Department of Pathology, Mount Sinai Medical Center, Milwaukee, Wisconsin 53201 Received for publication 1 June 1976
Generations times (gd of selected anaerobic species were compared in four culture media commonly used for anaerobic bacteria to evaluate these media for growing inocula for anaerobic antibiotic susceptibility determinations. Twentytwo clinical isolates of Bacteroides fragilis, Fusobacterium spp., Clostridium perfringens, and Clostridium spp. were tested in Schaedler broth, supplemented Lombard-Dowell broth, supplemented thioglycolate broth, and supplemented peptone-yeast extract-glucose broth. Growth curves were performed in an anaerobic chamber; changes in cell density were followed by viable count procedures and, in some cases, turbidometric procedures. The clostridia did not appear to have significantly different gt values in different media. The gt values for the five clostridial species tested ranged from 18 to 45 min. The fusobacteria also showed no apparent differences in gt values in the four media; values ranged from 51 to 60 min. The two subspecies of B. fragilis tested showed no significant differences in gt values when compared with each other in the same media. The combined data, however, indicated that the growth rate in Schaedler broth was faster than in the other media (averagegt, 45.1 min), whereas the growth rate in peptone-yeast extract-glucose was slower (average gt, 58.2 min). The growth rates in supplemented thioglycolate and supplemented Lombard-Dowell broth (average gt, 53.6 min and average gt, 51.0 min, respectively) were not significantly different.
Considerable advances have been made in routine isolation and identification of clinically significant anaerobic bacteria from clinical specimens. After isolation and identification, there are three primary techniques that may be used for antimicrobial susceptibility testing of anaerobic bacteria: (i) broth dilution, (ii) agar dilution, and (iii) agar diffusion. Each technique has numerous modifications and proponents (e.g., references 11, 16, 18, 19). Unfortunately, none of these techniques is nationally standardized as is the agar diffusion technique for aerobic susceptibility testing (National Committee for Clinical Laboratory Standards, Approved standard: ASM-2. Performance standards for antimicrobial disk susceptibility tests, Villanova, Pa. 1975). Regardless of the susceptibility testing technique used, it is important to standardize the initial inoculum because the in vitro antimicrobial susceptibility of an organism can be significantly affected by organism concentration (2, 7, 16). Currently used procedures for anaerobic susceptibility testing use a
variety of growth media and inoculum standardization methods; some investigators (1, 8, 11, 15, 16) use the no. 1 MacFarland nephelometric standard or a 50% dilution of the same standard (10) as in the aerobic disk diffusion method, whereas other investigators (4, 5, 12, 13, 17, 18) use various dilutions for 18- to 24-h broth cultures. In using a nephelometric standard to standardize inocula, one must assume that different cultures with the same turbidity have approximately the same number of colony-forming units (CFU) per milliliter. Use of a diluted 18- to 24-h culture for an inoculum source presumes that all cultures will have approximately the same CFU-per-milliliter density at the time the sample is taken. Neither possibility has been adequately documented for anaerobic bacteria. We thought, therefore, that it would be appropriate to investigate the effects of various broth culture media on the growth of anaerobic isolates routinely recovered in a clinical laboratory. It was the objective of this study to compare four commonly used broth media designed for anaerobes that could be used to grow the inoculum for subsequent antimicrobial suscep-
' Present address: Department of Pathology, Chicago Osteopathic Hosptial, Chicago, IL 60615. 482
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GROWTH RATES OF ANAEROBES IN BROTH MEDIA
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Mich.) with an atmosphere of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. Anaerobic conditions were maintained with a palladium-coated catalyst (Englehard Industries, East Newark, N.J.). All equipment was kept in the glove box for the duration ofthe study. Turbidity measurements were MATERIALS AND METHODS made within the anaerobic chamber with a KlettOrganisms. All bacterial cultures were obtained Summerson colorimeter (Klett Manufacturing Co., from the clinical microbiology laboratory at Mount New York) using a no. 66 red filter (Klett ManufacSinai Medical Center, Milwaukee, Wis., through turing Co.). In the resultant turbidity measureroutine isolation procedures for clinical specimens ment, 1 Klett unit equals 0.002 optical density units. (3, 19) except one isolate, Bacteroides fragilis subsp. Broth cultures and inoculated plates were incubated fragilis ATCC 25285, which was provided by T. Wil- at 37°C in an incubator (Thelco model Z) within the kins. Each isolate was identified by the procedures anaerobic chamber. Growth curve studies. The inoculum for each outlined by Dowell and Hawkins (6) and was assigned an accession number representing the clini- study was grown for 18 to 24 h in Schaedler broth cal specimen from which the isolate was derived. within the glove box and then diluted in saline so The organisms used in the study were as follows: that the inoculated test media would contain a 5 x Bacteroides fragilis subsp. fragilis, 5 isolates; Bacte- 10-4 dilution of the inoculum broth. In practice, the roides fragilis subsp. thetaiotaomicron, 4 isolates; inoculated test media contained about 5 x 103 CFU/ Fusobacterium species, 4 isolates; Clostridium per- ml in the late-log or early-stationary phase of fringens, 5 isolates; C. bifermentans, 1 isolate; C. growth. Two kinds of growth curve studies were undercadaveris, 1 isolate; C. ramosum, 1 isolate; and C. innoculum, 1 isolate. These particular organisms taken. For in-depth studies, representative isolates were chosen because they represent the more fre- were selected for each group, and their growth cyquently encountered, rapidly growing isolates seen cles were followed by both turbidometric and viable count procedures in each of the four broth media, in our laboratory. Media. Four media were used for comparative (these strains are indicated by an asterisk in Tables growth curve studies. They were as follows: (i) 1, 2, and 4). Turbidity measurements (Klett units) Schaedler broth (BBL) without modification; (ii) thi- in side arm flasks and samples for viable count oglycolate broth without indicator (Difco) supple- within the flasks were taken at 2-h intervals during mented with 0.5% horse serum (GIBCO) and 1% the lag, logarithmic, and early-stationary phases of vitamin K,-hemin (VK-H) solution (9); (iii) Lom- growth; longer sampling intervals were used therebard-Dowell broth supplemented with 0.5% glucose after. The in-depth growth studies were repeated (G. L. Lombard and J. C. McLaughlin, Abstr. within 1 week. The reproducibility of g, values for a Annu. Meet. Am. Soc. Microbiol. 1974, M334, p. given strain in a given medium was within 10%. Abbreviated studies were done in screw-cap 122); and (iv) peptone-yeast extract-glucose (PYG) broth supplemented with 1% VK-H solution (9). As- tubes. Only viable count procedures were used and cites fluid (16) or horse serum (19) is frequently only during the logarithmic phase of growth. In added to thioglycolate broth as a supplement to these cases, samples were taken at the appropriate enhance the growth of fastidious organisms, and we time intervals suggested by the preliminary and believed it appropriate to include such a supplement more detailed studies. in this study. VK-H solution was added to thioglySamples for determinations of CFU per milliter colate broth so that all media would contain compa- were taken with a Selecta-pipette (Clay-Adams, rable amounts of these growth supplements. Glu- New Jersey) and diluted in 0.85% saline. Blood agar cose was added to Lombard-Dowell broth so that all plates were inoculated in duplicate with 0.1 ml of a media would have comparable amounts of the same given dilution and spread over the agar surface with fermentable carbohydrate. Two-hundred milliliters a glass spreader. The time involved in sampling, ofeach broth medium was dispensed into 300-ml side diluting, and spreading was less than 7 min for each arm flasks or, alternatively, 15 ml was dispensed sample. Preliminary studies indicated that this prointo screw-cap tubes (16 by 150 mm). All media were cedure had no significant effect on the number of autoclaved for 15 min at 1210C at 15 lb/in2; after viable cells over a 7-min time period in an anaerobic cooling to 40 to 500C, all liquid media were promptly atmosphere. Calculation of gi values. Linear regression of placed in an anaerobic chamber until used. Viable counts were done on Columbia agar (BBL) sampling time (t_) on log10 CFU per mililliter obplates supplemented with 0.5% yeast extract (Difco), served for that sample was used to construct a 1% VK-H solution (9), and 5% defibrinated sheep regression line, from which the gt could be calcublood (GIBCO). After hardening, plates were lated. This was done to minimize random error due promptly placed in an anaerobic chamber until to any given CFU-per-mililliter determination. Data points for the early- and or late-logarithmic used. Both broth and plate media were allowed to become reduced for 24 h before use. phases of growth were included in the analysis as Anaerobic system and instrumentation. All pro- long as the correlation coefficient for the regression cedures (inoculation, incubation, dilution, spread remained above 0.950. The g,, defined as minutes plating, and reincubation) were done in an anaero- per generation, was calculated on the basis of the bic glove box (Coy Manufacturing, Ann Arbor, formula: gt = (tf - t1 +. (log10 CFUflml - log10
tibility testing. The criteria for comparison of each medium to support rapid growth was the generation time (g,) for selected anaerobes isolated from clinical specimens.
484
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cies appear in Table 2. Except for C. perfringens, the other clostridia were tested only once in each medium, and it cannot be stated whether the observed differences in gt values for a given organism are significantly different in different media. However, when the average gt values observed for one organism are compared with those of another organism, some significant differences can be found. Specifically, the mean gt values for C. ramosum and C. cadaveris, 32 and 33 min, respectively, are not significantly different, whereas the mean gt value for C. innoculum, 46 min, is significantly different from those observed for C. ramosum or C. catests. daveris. Furthermore, the meangt value for C. perfringens, 25 min, appears to be significantly RESULTS less than that of C. ramosum and significantly The gt values for five isolates of C. perfrin- more than that of C. bifermentans, 18 min. In contrast to C. perfringens, the two subspegens on each of the four media are shown in Table 1. Each isolate is denoted by its accession cies of B. fragilis that were studied show statisnumber in the left-hand column. Although it tically significant differences in gt values in would appear that the averagegt was longest in different media. Analysis of variance of the supplemented thioglycolate broth, the analysis observed gt values in different media. Analysis ofvariance indicates that there is no significant of variance of the observed gt values for B. difference in growth rate of C. perfringens in fragilis subsp. fragilis, listed in Table 3, indicated that one or more of the average gt values any of the four media at the 95% confidence level. Values for gt of four other clostridial spe- were significantly different from the others.
CFUJ/ml) log,02], where CFU,4ml and CFUJIml are the final and initial CFU per milliliter, respectively, and tf and tf are the times at which the final and initial samples were taken. Statistical analysis. One-way analysis of variance or Student's t test was used, where appropriate, to determine whether observed differences in mean gt values were statistically significant. The one-way analysis of variance was used to test the significance of differences between three or more sample means. Unless otherwise stated, the 95% confidence level (a = 0.05) was chosen as the threshold for determination of significant differences between mean values. The reader is referred to standard texts on statistics for details concerning the performance of these +
TABLE 1. gt values of Clostridcium perfringensa gt
Isolate accession no.
Schaedler broth (avg = 23.9, SD = 4.2) 23.6 22.1 31.2 22.4 20.4
(min/doubling) in:
Thioglycolateb (avg = 26.6, SD = 7.1)
Lombard brothb (avg = 23.6, SD = 3.8) 19.0 26.9 20.6
PGYb (avg = 24.2, SD = 0.4)
24.2 31.0 1058-1* 24.7 21.2 3708-6 23.6 36.9 3544-6 24.3 27.5 23.6 4640-1 24.1 20.5 24.1 4839-1 a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without asterisk were calculated from abbreviated studies in which only logarithmic growth was monitored.
b Plus appropriate supplements.
TABLE 2. Comparison of gt values for five Clostridium speciesa gt (min/doubling) for:
Medium
C. innoccum (avg = 45.8, SD = 1.8)
C. rasmosum* (avg = 32.5, SD = 0.3)
C. cadaveris (avg = 32.7, SD = 1.9)
C. perfringens° (avg = 24.6, SD = 4.3)
C. bifermentans* (avg = 18.4, SD = 2.7)
35.5 23.9 14.8 44.4 32.8 20.9 26.6 32.2 32.2 44.1 17.9 23.6 31.3 46.7 32.4 47.8 32.7 31.9 24.2 20.0 PYGc a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without asterisk were calculated from abbreviated studies where only logarithmic growth was monitored. bAverage and standard deviation for C. perfringens were taken from the data in Table 1. c Plus the appropriate supplements.
Schaedler broth Thioglycolate brothc Lombard brothc
VGROWTH RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, 1977
Similarly, an analysis of variance of the gt values for B. fragilis- subsp. thetaiotaomicron, listed in Table 4, indicated that there were statistically significant differences in the growth rates among the four different media. A summary of the data used to compare the two B. fragilis subspecies on a medium-by-medium basis appears in Table 5. The results of testing indicated that there were no significant differences in the growth rates of the two subspecies in any given medium tested, even though the growth rates in different media are significantly different. Upon combining the g, data for subspecies, also noted in Table 5, an
485
analysis of variance indicated that one or more of the average gt values in the four media were significantly different from the others at the 99.0% confidence level. The averageg, value for both subspecies is shortest in Schaedler broth and longest in supplemented PYG. By deleting the data for one of the media exhibiting either the longest or shortest average gt value, the remaining media can be tested for significant differences. After deleting the data for Schaedler broth and performing an analysis of variance on the three remaining media, the results indicated that one or all ofthe averageg, values were still significantly different. Similarly,
TABLE 3. gt values of Bacteroides fragilis subsp. fragilis gt (min/doubling) in: Schaedler broth (avg = 46.6, SD = 4.4)
Isolate accession no.
Thioglycolate
(avg - 55.0, SD =
6.7)
46.5 3326-1 46.5 3468-4 3492-1 44.0 53.7 3585-1 42.2 ATCC 25285 a Plus appropriate supplements.
Lombard brotha (avg = 50.4, SD = 5.5)
42.8 49.7 58.0 48.8 52.5
51.1
45.3 58.0 59.2 61.4
PYGa (avg = 57.3, SD = 1.8) 55.0
58.3 lost 59.0 56.7
TABLE 4. gt values ofBacteroides fragilis subsp. thetaiotaomicrona g, (min/doubling) in:
Isolate accession no.
Schaedler broth (avg = 43.4, SD =
Thioglycolate5 (avg = 51.9, SD =
Lombard broth" (avg = 51.9, SD =
PYGb (avg = 59.2, SD 9.2) 92 D92
(v
3.7) 4.6) 0.8) 2957-9* 41.6 50.7 52.2 50.9 3139-1 43.1 50.2 51.8 51.7 3708-1 40.1 48.1 50.5 67.8 807-4 48.6 58.5 52.3 66.3 a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without an asterisk were calculated from abbreviated studies where only logarithmic growth was monitored. b Plus appropriate supplements.
TABLE 5. Summary of comparative statistical data for Bacteroides fragilis subsp. fragilis and B. fragilis subsp. thetaiotaomicron Subspecies
9t
Schaedler broth
Thioglycolate
Lombard broth
PYG
Bacteroides fragilis subsp. fra-
43.3
51.9
51.7
59.1
fragilis subsp. thetaiotaomi-
46.6
55.0
50.4
57.3
gilis
B.
cron
Significant differences between None None None None subspeciesa Average gib for both subspecies 45.1 53.6 51.0 58.2 combined SD 4.2 5.7 4.2 6.2 a Determined by Student's t test. b Average g, values were computed from all the entries for each medium listed in Tables 3 and 4.
486
after deleting the data for supplemented PYG and repeating the analysis in the first three media, the results indicated that one or all of the average gt values were significantly different. This suggests that both the average gt in Schaedler broth and average gt in supplemented PYG were significantly different from the two intermediate values. Testing the difference between average gt in supplemented thioglycolate broth and the average gt of both subspecies in supplemented Lombard-Dowell broth combined indicated that there was no significant difference between these two. Thus, it would appear from the data that the significant differences at the 95% confidence level between media for both subspecies of B. fragilis combined were as follows: (i) Schaedler broth, generation time = 45.1 min (standard 4.2 min); (ii) thioglycolate deviation [SD] and Lombard-Dowell broth, generation time = 52.3 min (SD = 5.0 min); and (iii) PYG, generation time = 58.2 min (SD = 6.2 min). An analysis of variance of the gt data for the Fusobacterium species, listed in Table 6, indicated no significant differences in average gt values among any of the four media. However, we believe that, because of the small sample size and wide apparent ranges in gt values, our conclusions about this genus must remain tentative. Isolates 360-1, 3872-2, and F-2 were all F. nucleatum, whereas isolate F-1 was F. necrophorum. The standard inoculum in all of the =
growth studies
was
about 5
x
103 CFU/ml,
which may have been too low for the fusobacteria to rapidly initiate growth. In three cases, the isolates simply did not grow in some of the test media during the time samples were being taken. Examples of the turbidity and viable count data plotted against time for C. perfringens (1058-1), C. ramosum, and B. fragilis subsp. thetaiotaomicron (2957-9) appear in Fig. 1, 2, and 3, respectively. In Fig. 1, it is apparent that turbidity ofC. perfringens does not appreciably increase until the number of cells reaches ap-
Isolate accession no.
a
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SOTTILE AND ZABRANSKY
proximately 107 to 108 CFU/ml, which is about the end of logarithmic growth for this organism. Furthermore, the stationary-phase density is about 108 to 109 CFU/ml, and appears rather stable over a 10- to 12-h period. The turbidity and viable count curves behave differently for C. ramosum (Fig. 2). Again, turbidity does not increase appreciably until the number of cells is about 106 to 107 CFU/ml, which occurs toward the end ofthe logarithmic growth. However, in this case, the viable cell count appears to drop slightly within the range of 108 to 109
CFU/ml, whereas the turbidity tends to inat a more gradual rate than is evident during logarithmic growth. In the third example, B. fragilis subsp. thetaiotaomicron (Fig. 3), the turbidity in three of the media does not appreciably increase until the cell population has reached a stationary phase of growth with approximately 109 CFU/ml. In contrast, the turbidity in Schaedler broth began to increase in the mid-logarithmic phase of growth and did not begin to taper off until the stationary phase of growth was established. In all three examples presented, the viable counts were more predictable in terms of growth in different media than the comparable turbidity measurecrease
ments.
DISCUSSION The standardization of an antimicrobial susceptibility testing procedure for anaerobic bacteria will depend ultimately upon an understanding of the physiological requirements and responses of these organisms. It is recognized that any given procedure will not be satisfactory for all anaerobic organisms, just as the aerobic disk diffusion technique is not suitable for all aerobic and facultative organisms. For this reason, we chose to study the least fastidious organisms with rapid rates of growth, which are also the organisms most commonly isolated from clinical specimens. Our objective has been to compare four commonly used ananerobic culture media in terms of their ability
TABLE 6. gt values of Fusobacterium spp. gt (min/doubling) in: Lombard brotha Thioglycolate Schaedler broth (avg = 57.3, SD = (avg = 54.9, SD = (avg 51.7, SD = 3.1)
360-1 53.9 3872-2 54.6 F-1 48.3 F-2 49.8 Plus appropriate supplements.
5.4) 57.2 58.8
lost 48.7
7.9) 62.5 60.6 48.0 lost
PGYa (avg = 60.1, SD 12.3) 67.0 67.5 45.9
lost
VGROWTH RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, 1977
487
10
9
t8
E
0
1 5'
,s 143
7 o-
13
a11 .6
Z 124 , -j
ve
C .c
11
1
4
3
2
.1
H OURS
FIG. 1. Growth curves ofClostridium perfringens in four culture media. Open symbols represent CFUlml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, 0), supplemented thioglycolate broth (A, A), supplemented Lombard-Dowell broth (0, U), supplemented PYG broth (C), *).
to support rapid growth, and to determine which single medium, if any, would support the most rapid growth. The only species for which we could detect significant differences in gt values in different media were the two subspecies of B. fragilis. All species tested grew in Schaedler broth as well, if not better than, in the other media
tested. Stalons et al. (14) have published a study similar to ours using a wider variety of media and organisms; however, their criterion for comparison was the "percent of change in turbidity calculated after a 24-h incubation pe-
riod." From the three examples presented above (Fig. 1-3), it can be seen that an organism may grow at approximately the same rate and have the same number of organisms in different media, but produce strikingly different turbidometric responses. The examples above demonstrate that turbidity may or may not increase during logarithmic growth and, consequently, does not accurately reflect the number of organisms in the four different media. Although turbidity is a function of cell concentration, it can be influenced by size, shape, and presence or absence of cellular inclusions. Casual observation of some isolates
488
SOTrIE AND ZABRANSKY
ANTIMICROB. AGENTS CHZMOTIiZR.
10o 9
8 E
10 75 50
.c
z
6N
I.-J le
5
4
3
2
2
4
6
8
10
12
14
16
18 20 22 24 26 28 30
HOURS
FIG. 2. Growth curves of Clostridium ramosum in four culture media. Open symbols represent CFU/ml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, *), supplemented thioglycolate broth (A, A), supplemented Lombard-Dowell broth (0, N), supplemented PYG broth ( 0>} *).
over the growth cycle disclosed some change in overall size and Gram-staining characteristics, but none of these effects appeared to be particularly characteristic of any given medium. We, therefore, do not think that turbidity is a satisfactory basis on which to compare bacterial growth, unless the relationship between t bty and CFU per mililliter is actually known. Nevertheless, despite the differences in approach to the problem of comparative growth rates, our results are in general agreement with those of Stalons et al.
Ericsson and Sherris (7) cite results obtained with different aerobic cultures. Their data indicate the cultures in stationary-growth stages may show differences of up to 1,000-fold in viable counts within individual species. In contrast, we observed a rather narrow range for different species. Early-stationary-phase counts for most species tested routinely fell between 1 x 108 and 5 x 108 CFU/ml, regardless of the growth and medium used. We propose that an appropriate dilution of an early-stationary-phase culture grown in Schae-
RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, ,GROWTH 1977
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o10
9
8 0E 1
7
0-
.6
C
1n m
if
C.3
.2
.1
2
4
6
8
10
12
14
16
8 20 22
24 26 28
30
HOURS
FIG. 3.-Growth curves ofBacteroides fragilis subsp. thetaiotaomicron in four culture media. Open symbols represent CFUlml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, 0), supplemented thiogylcolate broth (A, A), supplemented Lombard-Dowell broth (0, U), supplemented PYG broth(, *).
dler broth would provide a more satisfactory inoculum for anaerobic susceptibility testing than would adjustment of turbidity to agree with a turbidity standard. The use of an "overnight" culture as an inoculum source has been used with acceptable results by other investigators (4, 5, 12, 14, 17, 18).
ACKNOWLEDGMENT The technical assistance of Kenneth Hauser is greatly
appreciated. LITERATURE CITED 1. Barry, A. L., and C. D. Fay. 1974. Evaluation of four disc diffusion methods for antimicrobic susceptiblity
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tests with anaerobic gram negative bacilli. Clin. Pathol 61:592-598. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. Blair, J. E., E. H. Lennette, and J. P. Traunt (ed). 1975. Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Bethesda, Md. Blazevic, D. J. 1975. Evaluation of the modified brothdisk method for determining antibiotic susceptibilities of anaerobic bacteria. Antimicrob. Agents Chemother. 7:721-723. Dornbusch, K., C. E. Nord, and B. Olsson. 1975. Antibiotic susceptibility testing of anaerobic bacteria by standardized disc diffusion method with special reference to Bacteroides fragilis. Scand. J. Infect. Dis. 7:59-66. Dowell, V. R., and T. M. Hawkins (ed.). 1968. Laboratory methods in anaerobic bacteriology. Center for Disease Control, Atlanta, Ga. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta. Path. Microbiol. Scand. Sect. B 217(Suppl.). Hauser, K. J., J. A. Johnston, and R. J. Zabransky. 1975. Economical agar dilution techniques for susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 7:7-12. Holdeman, L. V., and W. E. C. Moore (ed.). 1973. Anaerobe laboratory manual, 2nd ed. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, Va. Overman, S. V., D. W. Lambe, and J. V. Bennett. 1974. Proposed standardized method for testing and interpreting susceptibility of Bacteroides fragilis to tetracycline. Antimicrob. Agents Chemother. 5:357-361.
ANTIMICROB. AGENTS CHEMOTHER. 11. Rahimi, A., W. J. Martin, and J. A. Washington. 1974. Effects of medium and inoculum on antimicrobial
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susceptibility of anaerobic bacteria. Am. J. Clin. Pathol. 62:425-427. Rotilie, C. A., R. J. Fass, R. B. Prior, and R. L. Perkins. 1975. Microbilution technique for antimicrobial susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 7:311-315. Schoutens, E., and E. Yourassowsky. 1974. Speed of bactericidal action of penicillin G, ampicillin, and carbenicillin on Bacteroides fragilis. Antimicrob. Agents Chemother. 6:227-231. Stalons, D. R., C. Thornsberry, and V. R. Dowell. 1974. The effect of culture medium and carbon dioxide concentration on the growth of anerobic bacteria commonly encountered in clinical specimens. Appl. Microbiol. 27:1098-1104. Sutter, V. L., Y-Y. Kwok, and S. M. Finegold. 1972. Standardized antimicrobial disc susceptibility testing of anaerobic bacteria I. Susceptibility of Bacteroides fragilis to tetracycline. Appl. Microbiol. 23:268-275. Sutter, V. L., Y-Y. Kwok, and S. M. Finegold. 1973. Susceptibility of Bacteroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing. Antimicrob. Agents Chemother. 3:188-193. Wilkins, T. D., L. V. Holdemann, I. J. Abramson, and W. E. C. Moore. 1972. Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 1:451-
459. 18. Wilkins, T. D., and T. Thiel. 1973. Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 3:350-356. 19. Zabransky, R. J. 1970. Isolation of anaerobic bacteria from clinical specimens. Mayo Clin. Proc. 45:256-264.
Vol. 11, No. 3 Printed in U.S.A.
Comparative Growth Rates of Selected Anaerobic Species in Four Commonly Used Broth Media WILLIAM SOTTILE II* Apm R. J. ZABRANSKY Microbiology Research Laboratory, Department of Pathology, Mount Sinai Medical Center, Milwaukee, Wisconsin 53201 Received for publication 1 June 1976
Generations times (gd of selected anaerobic species were compared in four culture media commonly used for anaerobic bacteria to evaluate these media for growing inocula for anaerobic antibiotic susceptibility determinations. Twentytwo clinical isolates of Bacteroides fragilis, Fusobacterium spp., Clostridium perfringens, and Clostridium spp. were tested in Schaedler broth, supplemented Lombard-Dowell broth, supplemented thioglycolate broth, and supplemented peptone-yeast extract-glucose broth. Growth curves were performed in an anaerobic chamber; changes in cell density were followed by viable count procedures and, in some cases, turbidometric procedures. The clostridia did not appear to have significantly different gt values in different media. The gt values for the five clostridial species tested ranged from 18 to 45 min. The fusobacteria also showed no apparent differences in gt values in the four media; values ranged from 51 to 60 min. The two subspecies of B. fragilis tested showed no significant differences in gt values when compared with each other in the same media. The combined data, however, indicated that the growth rate in Schaedler broth was faster than in the other media (averagegt, 45.1 min), whereas the growth rate in peptone-yeast extract-glucose was slower (average gt, 58.2 min). The growth rates in supplemented thioglycolate and supplemented Lombard-Dowell broth (average gt, 53.6 min and average gt, 51.0 min, respectively) were not significantly different.
Considerable advances have been made in routine isolation and identification of clinically significant anaerobic bacteria from clinical specimens. After isolation and identification, there are three primary techniques that may be used for antimicrobial susceptibility testing of anaerobic bacteria: (i) broth dilution, (ii) agar dilution, and (iii) agar diffusion. Each technique has numerous modifications and proponents (e.g., references 11, 16, 18, 19). Unfortunately, none of these techniques is nationally standardized as is the agar diffusion technique for aerobic susceptibility testing (National Committee for Clinical Laboratory Standards, Approved standard: ASM-2. Performance standards for antimicrobial disk susceptibility tests, Villanova, Pa. 1975). Regardless of the susceptibility testing technique used, it is important to standardize the initial inoculum because the in vitro antimicrobial susceptibility of an organism can be significantly affected by organism concentration (2, 7, 16). Currently used procedures for anaerobic susceptibility testing use a
variety of growth media and inoculum standardization methods; some investigators (1, 8, 11, 15, 16) use the no. 1 MacFarland nephelometric standard or a 50% dilution of the same standard (10) as in the aerobic disk diffusion method, whereas other investigators (4, 5, 12, 13, 17, 18) use various dilutions for 18- to 24-h broth cultures. In using a nephelometric standard to standardize inocula, one must assume that different cultures with the same turbidity have approximately the same number of colony-forming units (CFU) per milliliter. Use of a diluted 18- to 24-h culture for an inoculum source presumes that all cultures will have approximately the same CFU-per-milliliter density at the time the sample is taken. Neither possibility has been adequately documented for anaerobic bacteria. We thought, therefore, that it would be appropriate to investigate the effects of various broth culture media on the growth of anaerobic isolates routinely recovered in a clinical laboratory. It was the objective of this study to compare four commonly used broth media designed for anaerobes that could be used to grow the inoculum for subsequent antimicrobial suscep-
' Present address: Department of Pathology, Chicago Osteopathic Hosptial, Chicago, IL 60615. 482
VOL. 11, 1977
GROWTH RATES OF ANAEROBES IN BROTH MEDIA
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Mich.) with an atmosphere of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. Anaerobic conditions were maintained with a palladium-coated catalyst (Englehard Industries, East Newark, N.J.). All equipment was kept in the glove box for the duration ofthe study. Turbidity measurements were MATERIALS AND METHODS made within the anaerobic chamber with a KlettOrganisms. All bacterial cultures were obtained Summerson colorimeter (Klett Manufacturing Co., from the clinical microbiology laboratory at Mount New York) using a no. 66 red filter (Klett ManufacSinai Medical Center, Milwaukee, Wis., through turing Co.). In the resultant turbidity measureroutine isolation procedures for clinical specimens ment, 1 Klett unit equals 0.002 optical density units. (3, 19) except one isolate, Bacteroides fragilis subsp. Broth cultures and inoculated plates were incubated fragilis ATCC 25285, which was provided by T. Wil- at 37°C in an incubator (Thelco model Z) within the kins. Each isolate was identified by the procedures anaerobic chamber. Growth curve studies. The inoculum for each outlined by Dowell and Hawkins (6) and was assigned an accession number representing the clini- study was grown for 18 to 24 h in Schaedler broth cal specimen from which the isolate was derived. within the glove box and then diluted in saline so The organisms used in the study were as follows: that the inoculated test media would contain a 5 x Bacteroides fragilis subsp. fragilis, 5 isolates; Bacte- 10-4 dilution of the inoculum broth. In practice, the roides fragilis subsp. thetaiotaomicron, 4 isolates; inoculated test media contained about 5 x 103 CFU/ Fusobacterium species, 4 isolates; Clostridium per- ml in the late-log or early-stationary phase of fringens, 5 isolates; C. bifermentans, 1 isolate; C. growth. Two kinds of growth curve studies were undercadaveris, 1 isolate; C. ramosum, 1 isolate; and C. innoculum, 1 isolate. These particular organisms taken. For in-depth studies, representative isolates were chosen because they represent the more fre- were selected for each group, and their growth cyquently encountered, rapidly growing isolates seen cles were followed by both turbidometric and viable count procedures in each of the four broth media, in our laboratory. Media. Four media were used for comparative (these strains are indicated by an asterisk in Tables growth curve studies. They were as follows: (i) 1, 2, and 4). Turbidity measurements (Klett units) Schaedler broth (BBL) without modification; (ii) thi- in side arm flasks and samples for viable count oglycolate broth without indicator (Difco) supple- within the flasks were taken at 2-h intervals during mented with 0.5% horse serum (GIBCO) and 1% the lag, logarithmic, and early-stationary phases of vitamin K,-hemin (VK-H) solution (9); (iii) Lom- growth; longer sampling intervals were used therebard-Dowell broth supplemented with 0.5% glucose after. The in-depth growth studies were repeated (G. L. Lombard and J. C. McLaughlin, Abstr. within 1 week. The reproducibility of g, values for a Annu. Meet. Am. Soc. Microbiol. 1974, M334, p. given strain in a given medium was within 10%. Abbreviated studies were done in screw-cap 122); and (iv) peptone-yeast extract-glucose (PYG) broth supplemented with 1% VK-H solution (9). As- tubes. Only viable count procedures were used and cites fluid (16) or horse serum (19) is frequently only during the logarithmic phase of growth. In added to thioglycolate broth as a supplement to these cases, samples were taken at the appropriate enhance the growth of fastidious organisms, and we time intervals suggested by the preliminary and believed it appropriate to include such a supplement more detailed studies. in this study. VK-H solution was added to thioglySamples for determinations of CFU per milliter colate broth so that all media would contain compa- were taken with a Selecta-pipette (Clay-Adams, rable amounts of these growth supplements. Glu- New Jersey) and diluted in 0.85% saline. Blood agar cose was added to Lombard-Dowell broth so that all plates were inoculated in duplicate with 0.1 ml of a media would have comparable amounts of the same given dilution and spread over the agar surface with fermentable carbohydrate. Two-hundred milliliters a glass spreader. The time involved in sampling, ofeach broth medium was dispensed into 300-ml side diluting, and spreading was less than 7 min for each arm flasks or, alternatively, 15 ml was dispensed sample. Preliminary studies indicated that this prointo screw-cap tubes (16 by 150 mm). All media were cedure had no significant effect on the number of autoclaved for 15 min at 1210C at 15 lb/in2; after viable cells over a 7-min time period in an anaerobic cooling to 40 to 500C, all liquid media were promptly atmosphere. Calculation of gi values. Linear regression of placed in an anaerobic chamber until used. Viable counts were done on Columbia agar (BBL) sampling time (t_) on log10 CFU per mililliter obplates supplemented with 0.5% yeast extract (Difco), served for that sample was used to construct a 1% VK-H solution (9), and 5% defibrinated sheep regression line, from which the gt could be calcublood (GIBCO). After hardening, plates were lated. This was done to minimize random error due promptly placed in an anaerobic chamber until to any given CFU-per-mililliter determination. Data points for the early- and or late-logarithmic used. Both broth and plate media were allowed to become reduced for 24 h before use. phases of growth were included in the analysis as Anaerobic system and instrumentation. All pro- long as the correlation coefficient for the regression cedures (inoculation, incubation, dilution, spread remained above 0.950. The g,, defined as minutes plating, and reincubation) were done in an anaero- per generation, was calculated on the basis of the bic glove box (Coy Manufacturing, Ann Arbor, formula: gt = (tf - t1 +. (log10 CFUflml - log10
tibility testing. The criteria for comparison of each medium to support rapid growth was the generation time (g,) for selected anaerobes isolated from clinical specimens.
484
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cies appear in Table 2. Except for C. perfringens, the other clostridia were tested only once in each medium, and it cannot be stated whether the observed differences in gt values for a given organism are significantly different in different media. However, when the average gt values observed for one organism are compared with those of another organism, some significant differences can be found. Specifically, the mean gt values for C. ramosum and C. cadaveris, 32 and 33 min, respectively, are not significantly different, whereas the mean gt value for C. innoculum, 46 min, is significantly different from those observed for C. ramosum or C. catests. daveris. Furthermore, the meangt value for C. perfringens, 25 min, appears to be significantly RESULTS less than that of C. ramosum and significantly The gt values for five isolates of C. perfrin- more than that of C. bifermentans, 18 min. In contrast to C. perfringens, the two subspegens on each of the four media are shown in Table 1. Each isolate is denoted by its accession cies of B. fragilis that were studied show statisnumber in the left-hand column. Although it tically significant differences in gt values in would appear that the averagegt was longest in different media. Analysis of variance of the supplemented thioglycolate broth, the analysis observed gt values in different media. Analysis ofvariance indicates that there is no significant of variance of the observed gt values for B. difference in growth rate of C. perfringens in fragilis subsp. fragilis, listed in Table 3, indicated that one or more of the average gt values any of the four media at the 95% confidence level. Values for gt of four other clostridial spe- were significantly different from the others.
CFUJ/ml) log,02], where CFU,4ml and CFUJIml are the final and initial CFU per milliliter, respectively, and tf and tf are the times at which the final and initial samples were taken. Statistical analysis. One-way analysis of variance or Student's t test was used, where appropriate, to determine whether observed differences in mean gt values were statistically significant. The one-way analysis of variance was used to test the significance of differences between three or more sample means. Unless otherwise stated, the 95% confidence level (a = 0.05) was chosen as the threshold for determination of significant differences between mean values. The reader is referred to standard texts on statistics for details concerning the performance of these +
TABLE 1. gt values of Clostridcium perfringensa gt
Isolate accession no.
Schaedler broth (avg = 23.9, SD = 4.2) 23.6 22.1 31.2 22.4 20.4
(min/doubling) in:
Thioglycolateb (avg = 26.6, SD = 7.1)
Lombard brothb (avg = 23.6, SD = 3.8) 19.0 26.9 20.6
PGYb (avg = 24.2, SD = 0.4)
24.2 31.0 1058-1* 24.7 21.2 3708-6 23.6 36.9 3544-6 24.3 27.5 23.6 4640-1 24.1 20.5 24.1 4839-1 a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without asterisk were calculated from abbreviated studies in which only logarithmic growth was monitored.
b Plus appropriate supplements.
TABLE 2. Comparison of gt values for five Clostridium speciesa gt (min/doubling) for:
Medium
C. innoccum (avg = 45.8, SD = 1.8)
C. rasmosum* (avg = 32.5, SD = 0.3)
C. cadaveris (avg = 32.7, SD = 1.9)
C. perfringens° (avg = 24.6, SD = 4.3)
C. bifermentans* (avg = 18.4, SD = 2.7)
35.5 23.9 14.8 44.4 32.8 20.9 26.6 32.2 32.2 44.1 17.9 23.6 31.3 46.7 32.4 47.8 32.7 31.9 24.2 20.0 PYGc a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without asterisk were calculated from abbreviated studies where only logarithmic growth was monitored. bAverage and standard deviation for C. perfringens were taken from the data in Table 1. c Plus the appropriate supplements.
Schaedler broth Thioglycolate brothc Lombard brothc
VGROWTH RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, 1977
Similarly, an analysis of variance of the gt values for B. fragilis- subsp. thetaiotaomicron, listed in Table 4, indicated that there were statistically significant differences in the growth rates among the four different media. A summary of the data used to compare the two B. fragilis subspecies on a medium-by-medium basis appears in Table 5. The results of testing indicated that there were no significant differences in the growth rates of the two subspecies in any given medium tested, even though the growth rates in different media are significantly different. Upon combining the g, data for subspecies, also noted in Table 5, an
485
analysis of variance indicated that one or more of the average gt values in the four media were significantly different from the others at the 99.0% confidence level. The averageg, value for both subspecies is shortest in Schaedler broth and longest in supplemented PYG. By deleting the data for one of the media exhibiting either the longest or shortest average gt value, the remaining media can be tested for significant differences. After deleting the data for Schaedler broth and performing an analysis of variance on the three remaining media, the results indicated that one or all ofthe averageg, values were still significantly different. Similarly,
TABLE 3. gt values of Bacteroides fragilis subsp. fragilis gt (min/doubling) in: Schaedler broth (avg = 46.6, SD = 4.4)
Isolate accession no.
Thioglycolate
(avg - 55.0, SD =
6.7)
46.5 3326-1 46.5 3468-4 3492-1 44.0 53.7 3585-1 42.2 ATCC 25285 a Plus appropriate supplements.
Lombard brotha (avg = 50.4, SD = 5.5)
42.8 49.7 58.0 48.8 52.5
51.1
45.3 58.0 59.2 61.4
PYGa (avg = 57.3, SD = 1.8) 55.0
58.3 lost 59.0 56.7
TABLE 4. gt values ofBacteroides fragilis subsp. thetaiotaomicrona g, (min/doubling) in:
Isolate accession no.
Schaedler broth (avg = 43.4, SD =
Thioglycolate5 (avg = 51.9, SD =
Lombard broth" (avg = 51.9, SD =
PYGb (avg = 59.2, SD 9.2) 92 D92
(v
3.7) 4.6) 0.8) 2957-9* 41.6 50.7 52.2 50.9 3139-1 43.1 50.2 51.8 51.7 3708-1 40.1 48.1 50.5 67.8 807-4 48.6 58.5 52.3 66.3 a Asterisk indicates that generation times for this isolate were calculated from data taken during "indepth" studies where all phases of growth were monitored. Generation times for isolates without an asterisk were calculated from abbreviated studies where only logarithmic growth was monitored. b Plus appropriate supplements.
TABLE 5. Summary of comparative statistical data for Bacteroides fragilis subsp. fragilis and B. fragilis subsp. thetaiotaomicron Subspecies
9t
Schaedler broth
Thioglycolate
Lombard broth
PYG
Bacteroides fragilis subsp. fra-
43.3
51.9
51.7
59.1
fragilis subsp. thetaiotaomi-
46.6
55.0
50.4
57.3
gilis
B.
cron
Significant differences between None None None None subspeciesa Average gib for both subspecies 45.1 53.6 51.0 58.2 combined SD 4.2 5.7 4.2 6.2 a Determined by Student's t test. b Average g, values were computed from all the entries for each medium listed in Tables 3 and 4.
486
after deleting the data for supplemented PYG and repeating the analysis in the first three media, the results indicated that one or all of the average gt values were significantly different. This suggests that both the average gt in Schaedler broth and average gt in supplemented PYG were significantly different from the two intermediate values. Testing the difference between average gt in supplemented thioglycolate broth and the average gt of both subspecies in supplemented Lombard-Dowell broth combined indicated that there was no significant difference between these two. Thus, it would appear from the data that the significant differences at the 95% confidence level between media for both subspecies of B. fragilis combined were as follows: (i) Schaedler broth, generation time = 45.1 min (standard 4.2 min); (ii) thioglycolate deviation [SD] and Lombard-Dowell broth, generation time = 52.3 min (SD = 5.0 min); and (iii) PYG, generation time = 58.2 min (SD = 6.2 min). An analysis of variance of the gt data for the Fusobacterium species, listed in Table 6, indicated no significant differences in average gt values among any of the four media. However, we believe that, because of the small sample size and wide apparent ranges in gt values, our conclusions about this genus must remain tentative. Isolates 360-1, 3872-2, and F-2 were all F. nucleatum, whereas isolate F-1 was F. necrophorum. The standard inoculum in all of the =
growth studies
was
about 5
x
103 CFU/ml,
which may have been too low for the fusobacteria to rapidly initiate growth. In three cases, the isolates simply did not grow in some of the test media during the time samples were being taken. Examples of the turbidity and viable count data plotted against time for C. perfringens (1058-1), C. ramosum, and B. fragilis subsp. thetaiotaomicron (2957-9) appear in Fig. 1, 2, and 3, respectively. In Fig. 1, it is apparent that turbidity ofC. perfringens does not appreciably increase until the number of cells reaches ap-
Isolate accession no.
a
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SOTTILE AND ZABRANSKY
proximately 107 to 108 CFU/ml, which is about the end of logarithmic growth for this organism. Furthermore, the stationary-phase density is about 108 to 109 CFU/ml, and appears rather stable over a 10- to 12-h period. The turbidity and viable count curves behave differently for C. ramosum (Fig. 2). Again, turbidity does not increase appreciably until the number of cells is about 106 to 107 CFU/ml, which occurs toward the end ofthe logarithmic growth. However, in this case, the viable cell count appears to drop slightly within the range of 108 to 109
CFU/ml, whereas the turbidity tends to inat a more gradual rate than is evident during logarithmic growth. In the third example, B. fragilis subsp. thetaiotaomicron (Fig. 3), the turbidity in three of the media does not appreciably increase until the cell population has reached a stationary phase of growth with approximately 109 CFU/ml. In contrast, the turbidity in Schaedler broth began to increase in the mid-logarithmic phase of growth and did not begin to taper off until the stationary phase of growth was established. In all three examples presented, the viable counts were more predictable in terms of growth in different media than the comparable turbidity measurecrease
ments.
DISCUSSION The standardization of an antimicrobial susceptibility testing procedure for anaerobic bacteria will depend ultimately upon an understanding of the physiological requirements and responses of these organisms. It is recognized that any given procedure will not be satisfactory for all anaerobic organisms, just as the aerobic disk diffusion technique is not suitable for all aerobic and facultative organisms. For this reason, we chose to study the least fastidious organisms with rapid rates of growth, which are also the organisms most commonly isolated from clinical specimens. Our objective has been to compare four commonly used ananerobic culture media in terms of their ability
TABLE 6. gt values of Fusobacterium spp. gt (min/doubling) in: Lombard brotha Thioglycolate Schaedler broth (avg = 57.3, SD = (avg = 54.9, SD = (avg 51.7, SD = 3.1)
360-1 53.9 3872-2 54.6 F-1 48.3 F-2 49.8 Plus appropriate supplements.
5.4) 57.2 58.8
lost 48.7
7.9) 62.5 60.6 48.0 lost
PGYa (avg = 60.1, SD 12.3) 67.0 67.5 45.9
lost
VGROWTH RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, 1977
487
10
9
t8
E
0
1 5'
,s 143
7 o-
13
a11 .6
Z 124 , -j
ve
C .c
11
1
4
3
2
.1
H OURS
FIG. 1. Growth curves ofClostridium perfringens in four culture media. Open symbols represent CFUlml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, 0), supplemented thioglycolate broth (A, A), supplemented Lombard-Dowell broth (0, U), supplemented PYG broth (C), *).
to support rapid growth, and to determine which single medium, if any, would support the most rapid growth. The only species for which we could detect significant differences in gt values in different media were the two subspecies of B. fragilis. All species tested grew in Schaedler broth as well, if not better than, in the other media
tested. Stalons et al. (14) have published a study similar to ours using a wider variety of media and organisms; however, their criterion for comparison was the "percent of change in turbidity calculated after a 24-h incubation pe-
riod." From the three examples presented above (Fig. 1-3), it can be seen that an organism may grow at approximately the same rate and have the same number of organisms in different media, but produce strikingly different turbidometric responses. The examples above demonstrate that turbidity may or may not increase during logarithmic growth and, consequently, does not accurately reflect the number of organisms in the four different media. Although turbidity is a function of cell concentration, it can be influenced by size, shape, and presence or absence of cellular inclusions. Casual observation of some isolates
488
SOTrIE AND ZABRANSKY
ANTIMICROB. AGENTS CHZMOTIiZR.
10o 9
8 E
10 75 50
.c
z
6N
I.-J le
5
4
3
2
2
4
6
8
10
12
14
16
18 20 22 24 26 28 30
HOURS
FIG. 2. Growth curves of Clostridium ramosum in four culture media. Open symbols represent CFU/ml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, *), supplemented thioglycolate broth (A, A), supplemented Lombard-Dowell broth (0, N), supplemented PYG broth ( 0>} *).
over the growth cycle disclosed some change in overall size and Gram-staining characteristics, but none of these effects appeared to be particularly characteristic of any given medium. We, therefore, do not think that turbidity is a satisfactory basis on which to compare bacterial growth, unless the relationship between t bty and CFU per mililliter is actually known. Nevertheless, despite the differences in approach to the problem of comparative growth rates, our results are in general agreement with those of Stalons et al.
Ericsson and Sherris (7) cite results obtained with different aerobic cultures. Their data indicate the cultures in stationary-growth stages may show differences of up to 1,000-fold in viable counts within individual species. In contrast, we observed a rather narrow range for different species. Early-stationary-phase counts for most species tested routinely fell between 1 x 108 and 5 x 108 CFU/ml, regardless of the growth and medium used. We propose that an appropriate dilution of an early-stationary-phase culture grown in Schae-
RATES OF ANAEROBES IN BROTH MEDIA VOL. 11, ,GROWTH 1977
489
o10
9
8 0E 1
7
0-
.6
C
1n m
if
C.3
.2
.1
2
4
6
8
10
12
14
16
8 20 22
24 26 28
30
HOURS
FIG. 3.-Growth curves ofBacteroides fragilis subsp. thetaiotaomicron in four culture media. Open symbols represent CFUlml; closed symbols represent Klett turbidity values. Symbols: Schaedler broth (0, 0), supplemented thiogylcolate broth (A, A), supplemented Lombard-Dowell broth (0, U), supplemented PYG broth(, *).
dler broth would provide a more satisfactory inoculum for anaerobic susceptibility testing than would adjustment of turbidity to agree with a turbidity standard. The use of an "overnight" culture as an inoculum source has been used with acceptable results by other investigators (4, 5, 12, 14, 17, 18).
ACKNOWLEDGMENT The technical assistance of Kenneth Hauser is greatly
appreciated. LITERATURE CITED 1. Barry, A. L., and C. D. Fay. 1974. Evaluation of four disc diffusion methods for antimicrobic susceptiblity
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459. 18. Wilkins, T. D., and T. Thiel. 1973. Modified broth-disk method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. 3:350-356. 19. Zabransky, R. J. 1970. Isolation of anaerobic bacteria from clinical specimens. Mayo Clin. Proc. 45:256-264.
