Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Butyl Rubber Stoppers Increase the Shelf Life of Prereduced, Anaerobically Sterilized Media

Butyl rubber stoppers as compared with neoprene or black rubber stoppers significantly increased the shelf life of prereduced, anaerobically sterilized media for growth of obligately anaerobic bacteria.     

Bacteremia After Tooth Extractions Studied with the Aid of Prereduced Anaerobically Sterilized Culture Media

Both prereduced molten agar and broth and aerobic molten agar and broth were inoculated with blood samples collected from patients with periodontitis, but in otherwise good health, both before and after extraction of two or more teeth. Postoperative blood samples from 23 of 25 patients sampled yielded anaerobic and facultative species. Colony counts from nine samples yielded from less than 1 to over 100 colonies per ml of blood. Organisms detected were species belonging to the genera Bacteroides, Fusobacterium, Peptostreptococcus, Leptotrichia, Propionibacterium, Peptococcus, Veillonella, plus Streptococcus mitis, S. salivarius, vibrio forms, and strains resembling S. mutans. The data indicate that prereduced anaerobically sterilized culture medium with polyanethol sulfonate is effective for detecting anaerobic species in bacteremia and that anaerobic species can be prevalent in bacteremias immediately after tooth extraction in patients with periodontitis.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Isolation of anaerobic oxalate-degrading bacteria from freshwater lake sediments

Enrichment cultures that anaerobically degraded oxalate were obtained from lake sediment inocula. From these, 5 pure cultures of anaerobic oxalate-degrading bacteria were isolated and partially characterized. The isolates were Gram-negative, non-sporeforming, non-motile, obligate anaerobes. Oxalate was required for growth and was stoichiometrically converted to formate; ¹⁴CO₂ was also recovered when ¹⁴C-oxalate was added. Maximal growth occurred when the oxalate concentration was 50 mM. Acetate stimulated growth in the presence of oxalate, however, ¹⁴C-experiments indicated that acetate was only utilized for cell carbon.The isolates were either spiral-shaped or rod-shaped organisms. The first morphotype grew much more slowly than the second and exhibited 13-fold lower cell yields. These isolates represent a new strain of oxalate-degrading bacteria. The second morphotype was similar to the anaerobic oxalate-degrading bacteria previously found in rumen. This report extends the known habitats in which anaerobic oxalate-degrading organisms have been found to include aquatic sediments.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

"Phoenix phenomenon" in the growth of Clostridium perfringens.

The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III). The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied. This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar). When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon. The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed. The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited. This indicated that phases I and II were an injury-recovery process.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 x 10 cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH(3) was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by CO(2) evolution from [2-C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut x h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.     

New Selective Media for Enumeration and Recovery of Fluorescent Pseudomonads from Various Habitats

New media (S1 and S2) were formulated that provide a high degree of selectivity and detection of fluorescent pseudomonads on initial plating. The selectivity of the S-type media was based on a detergent, sodium lauroyl sarcosine, and an antibiotic, trimethoprim. A total of five soils from different geographical locations and one sewage sludge sample were examined. On S1 medium, isolates from two soils with low fluorescent pseudomonad populations exhibited a high frequency of arginine dihydrolase (78%) and oxidase-positive (95%) phenotypes, but no fermentative isolates were recovered. Medium S2 was more defined and selective than S1, but lower numbers of fluorescent pseudomonads were recovered on S2. In soils in which fluorescent pseudomonads represent a small proportion of the total population, S1 medium consistently recovered high percentages of fluorescent phenotypes (82.5%).     

Effect of high-fiber and high-oil diets on the fecal flora of swine.

Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48:964-969, 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Medium Containing Trypan Blue and Antibiotics for the Detection of Cryptococcus neoformans in Clinical Samples

A medium containing trypan blue, gentamicin, and chloramphenicol is introduced for the detection of Cryptococcus neoformans and Cryptococcus species from clinical samples. Ten recently isolated strains of Cryptococcus species as well as several clinical isolates of C. neoformans incorporated trypan blue and produced dark blue colonies on this mycological medium, whereas other common yeasts were light blue. The laboratory diagnosis of two cases of cryptococcosis was accomplished by the isolation of C. neoformans on the antibiotic-dye-containing medium. Compared to conventional media supporting large numbers of Pseudomonas aeruginosa and other gram-negative bacilli, the new medium was selective for yeasts. In one instance, the colonization of the respiratory tract by C. neoformans which led to fungemia was traced by the use of the antibiotic-dye medium. The antibiotic mixture, utilized herein, was more effective in suppressing bacteria contained in samples from patients than a medium containing cycloheximide and chloramphenicol.     

Effect of high-fiber and high-oil diets on the fecal flora of swine

Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48:964-969, 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of hexavalent chromium-reducing Cr-tolerant facultative anaerobes from tannery effluent

Several facultative anaerobes tolerant to high levels of chromate (>400 mg/ml) were isolated from tannery effluents. These isolates displayed varying degrees of Cr(VI) reduction under aerobic and anaerobic conditions at room temperature (24+/-2 degrees C). Interestingly, eight isolates were efficient in reducing 70% Cr(VI) anaerobically. This includes 5 isolates of genus Aerococcus, two isolates of Micrococcus and single isolate of genus Aeromonas. These isolates were subjected to further characterization for possible use in Cr(VI) detoxification of industrial wastes. This is the first report of Aerococcus sp. capable of Cr(VI) reduction >70% anaerobically. These bacteria were further checked for tolerance to a variety of other heavy metals. Our study indicates the possible use of these bacteria in environmental clean up.     

Methods for Detecting Indole Production by Gram-Negative Nonsporeforming Anaerobes

To help select the most appropriate method for detecting indole production with anaerobic gram-negative bacilli, several recommended methods were compared. Indole was measured both quantitatively and qualitatively after varying periods of incubation. Studies evaluated the results obtained in different media, the effect of adding glucose and/or tryptophan, the requirement for strict anaerobiasis, and the effects of reducing the total volume of broth. A 1-ml amount of thioglycolate broth without glucose but with 0.02% tryptophan gave optimal results after 2 to 7 days of incubation in anaerobic (Gas-Pak) jars. The majority of clinical isolates will give strong positive tests after 1 to 2 days but a few require 3 to 7 days of incubation. Prolonged incubation was required more frequently with conventional methods.     

Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation.

Eight strains of rumen bacteria capable of degrading phloroglucinol (1,3,5-trihydroxybenzene) under anaerobic conditions were isolated from enrichment cultures of the bovine rumen microflora established in a prereduced medium containing 0.02 M phloroglucinol. Five of the strains were facultatively anaerobic Gram-positive streptococci which were identified as Streptococcus bovis. Three strains of obligately anaerobic Gram-positive cocci were assigned to the genus Coprococcus. Anaerobic cultures of the Streptococcus bovis strains in a 40% rumen fluid medium initially containing 0.02 M phloroglucinol degraded 50-80% of the substrate within 2 days, whereas cultures of the Coprococcus strains degraded more than 80% of the substrate under the same conditions. The Streptococcus bovis strains were incapable of degrading phloroglucinol in brain heart infusion or in the medium of de Man, Rogosa, and Sharpe (MRS broth) incubated aerobically.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery.

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.