Activity of the Escherichia coli mutT mutator allele in an anaerobic environment.

Mutation frequencies for an Escherichia coli mutT strain were measured in both aerobic and anaerobic environments. When cells were grown in a rich medium (L broth), mutation frequencies were similar in both aerobic and anaerobic conditions. In contrast, when grown in a minimal medium, mutT anaerobic mutation frequencies were reduced dramatically compared with aerobic values, which were similar to L broth frequencies. L broth mutT cultures treated with a commercial enzyme complex that reduces free oxygen in the medium also showed strongly reduced anaerobic mutation frequencies. These results indicate that the biological role of the MutT protein is to prevent oxidative damage from becoming mutagenic.     

A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.     

Simple and convenient method for culturing anaerobic bacteria.

A simple and convenient method for culturing anaerobic bacteria is described. Cultures can be grown in commercially available flasks normally used for preparation of sterile external solutions. A special disposable rubber flask closure maintains anaerobic conditions in the flask after autoclaving. Growth of a variety of anaerobic oral bacteria was comparable to that obtained after anaerobic incubation of broth cultures in Brewer Anaerobic Jars.     

Paper Chromatography as an Adjunct in the Identification of Anaerobic Bacteria

Modified paper chromatography procedures for the analysis of fatty acids produced by anaerobic bacteria are described. Both ethylamine and hydroxylamine derivatives of fatty acids were prepared from inoculated anaerobic culture broth. The derivatives were spotted on chromatography paper and developed with appropriate solvents. Paper chromatography is a valuable alternative to gas liquid chromatography as an ancillary procedure in the identification of anaerobic bacteria in the clinical bacteriology laboratory.     

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

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.     

Rapid detection of sulfide-producing bacteria from sulfate and thiosulfate

An original liquid medium and a field broth bottle method for the rapid detection of the most probable number of sulfide-producing bacteria (SPB) from sulfate and thiosulfate are described. The broth bottle method enables after inoculation with a sample (water, sediment) the growth of ubiquitous aerobic bacteria, causing oxygen depletion, required for the growth of the present various anaerobic and facultative anaerobic SPB. The medium regularly gives higher numbers of the SPB than the used control medium (Postgate's E) for detection of sulfate-reducing bacteria and the final results are obtained just 36 h after the medium inoculation. The method is simple and suitable for the estimation of the physiological group of SPB in fresh waters, saline waters, sediments and industrial waters.     

厌氧菌预还原琼脂平板培养方法

为简化厌氧菌分离培养方法,使其在普通实验条件下于固体培养基上形成单菌落,本研究增加庖肉培养基无氧溶液体积,用作无氧倍比稀释液,在琼脂柱下进行倍比稀释,将皿盖带有胶塞孔的厌氧琼脂平板进行预还原,注射接种倍比稀释菌液,通过厌氧指示剂监测无氧效果,初步试用于肠道厌氧菌分离培养。结果显示,该方法整个操作过程厌氧效果良好,无需专门厌氧设备即可以分离纯化培养肠道乳酸杆菌,甚至无芽胞专性厌氧菌,如双歧杆菌和韦荣球菌。     

Effect of Culture Medium and Carbon Dioxide Concentration on Growth of Anaerobic Bacteria Commonly Encountered in Clinical Specimens

Representative strains of anaerobic bacteria from human infections were used to evaluate broth media, gas mixtures, and inocula for use in developing a procedure for performing minimal inhibitory concentration antimicrobic susceptibility tests. Nine commercially available media, including two that were chemically defined, were tested. Tests were performed in atmospheres with carbon dioxide concentrations between 2.5 and 10% and also in the GasPak system (BBL) that had a disposable hydrogen-carbon dioxide generator. Growth curves on each organism grown in schaedler broth and a 5% carbon dioxide atmosphere were used to determine growth characteristics, equate time of the particular growth phases to turbidity readings, and determine the numbers of viable organisms present in the culture. Schaedler broth proved to be most advantageous in combination with an atmosphere of 5% carbon dioxide, 10% hydrogen, and 85% nitrogen. The growth curve studies yielded valuable data on the rapidity and quantity of growth under these conditions. We believe these data have provided information which can be used as the basis for developing a standardized procedure for antimicrobic susceptibility testing for anaerobic bacteria.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

Antimicrobial susceptibility tests for anaerobic bacteria. Comparison of Wilkins-Chalgren agar reference method and a microdilution method,and determination of stability of antimicrobics frozen in broth

Susceptibility results for anaerobic bacteria obtained with a microdilution method with either Wilkins-Chalgren broth or Brain Heart Infusion broth were compared to results obtained with the reference agar dilution method proposed by the National Committee for Clinical Laboratory Standards. Results were generally comparable, but the minimal inhibitory concentrations obtained with the microdilution method were slightly lower than those obtained with the agar dilution method. Antibiotics stored in these broths in microdilution trays at-20°C were stable for up to 130 days.     

A COMPARATIVE STUDY OF FOLPMERS' GLUTAMIC ACID MEDIUM FOR THE DETECTION OF BACT. COLI IN WATER

SUMMARY: Folpmers' glutamic acid medium and certain modifications of it have been compared with MacConkey's broth for the detection of organisms of the coli-aerogenes group in water. Very satisfactory results were obtained with the glutamic acid medium made up in tubes and incubated in the same way as with MacConkey's broth. Less satisfactory acid were obtained when lactose was substituted for glucose in the glutamic acid medium, although both were superior to MacConkey's broth. The anaerobic method as described by Folpmers had too many disadvantages to be practicable for the routine examination of large numbers of samples.     

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.     

Pathogen survival during anaerobic digestion: Fatty acids inhibit anaerobic growth ofEscherichia coli

Summary The fate of pathogens in anaerobic digesters has been studied in a laboratory model system in which glucose-nutrient broth cultures of genetically-defined strains ofEscherichia coli received additions of fatty acids at concentrations similar to those attained during anaerobic treatment of farm wastes. Marked concentration-dependent inhibition of growth was observed for both antibiotic resistant and sensitive strains, and the effects increased with increasing chain lengths up to C₈. Survival of enteric organisms during anaerobic digestion may be limited by fatty acid toxicity.     

Comparison of the BACTEC System with Blind Subculture for the Detection of Bacteremia

One thousand blood specimens were cultured in BACTEC vials containing modified Columbia broth in aerobic, anaerobic, and hypertonic formulations. Radiometric readings and subcultures were performed on aerobic and hypertonic vials at 24 h and 7 days, and on anaerobic vials at 48 h and 7 days. Significant numbers of false-positive BACTEC readings were obtained. Although all positive cultures were eventually detected by the BACTEC, approximately 20% of blood specimens yielding positive subcultures at 24 h did not give positive BACTEC readings until 48 h.     

Radiometric Detection of Some Food-Borne Bacteria

Studies on detection of bacteria by radiometric techniques have been concerned primarily with aerobic species in clinical specimens. The data presented here are related to detection of aerobic and anaerobic species that are of significance in foods, by measurement of (14)CO(2) evolved from the metabolism of (14)C-glucose. Salmonella typhimurium and Staphylococcus aureus were inoculated into tryptic soy broth containing 0.0139 muCi of (14)C glucose/ml of medium. Detection times ranged from 10 to 3 hr for inocula of 10(0) to 10(4) cells/ml of broth. Heat-shocked spores of Clostridium sporogenes or C. botulinum were incubated in tryptic soy broth supplemented with Thiotone and NaHCO(3). The medium was rendered anaerobic with N(2). Spores were detected when 0.0833 muCi of labeled glucose was available/ml of medium but not when 0.0139 muCi of glucose was present/ml. The spores required 3 to 4 hr longer for detection than did comparable numbers of aerobic vegetative cells. The results demonstrate the importance of availability of sufficient label in the media and the potential of the application of this technique for sterility testing of foods.     

Modified microdilution antimicrobial susceptibility test for anaerobic bacteria

A modified microdilution antimicrobial susceptibility test is described for anaerobic bacteria. The microdilution procedure at 24 and 48 h of incubation was compared with agar dilution using Wilkins-Chalgren (WC) broth and agar respectively. Results showed a total of 12.2% minor and major discrepancies with the microdilution test at 24 h incubation and 7.8% at 48 h. If anaerobic isolates are to be tested for antimicrobial susceptibility, then the 24-h microdilution procedure is an acceptable alternative to agar dilution.     

Detection of Bacteremia with Liquid Media Containing Sodium Polyanetholsulfonate

Two liquid blood culture media, Tryptic soy broth (TSB) and Thiol broth, containing sodium polyanetholsulfonate were compared in 8,654 cultures. Pseudomonas and Corynebacterium (including Propionibacterium) were isolated significantly more frequently (P < 0.001) from TSB than from Thiol. Escherichia coli, Haemophilus, and Bacteroidaceae were isolated more frequently in TSB; however, the differences were not statistically significant. In no instance was Thiol superior to TSB in detecting bacteremia. In an additional 2,977 cultures, aerobic and anaerobic Vacutainer culture tubes with supplemented peptone broth were inoculated in parallel with TSB and Thiol. Significantly greater rates of detection (P < 0.01) in TSB or Thiol were noted with Pseudomonas, E. coli, Enterobacter, viridans, and group A streptococci, Bacteroidaceae, and staphylococci.     

Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.     

United States National Hospital Survey of anaerobic culture and susceptibility methods, II

To assess the status of clinical anaerobic bacteriology in the United States, we surveyed (by means of a questionnaire) 120 hospitals selected at random with bed capacities of 200-1000, and we received responses from 78 (65%), all of which performed some degree of clinical anaerobic microbiology. Separate anaerobic blood culture bottles were used by 73 labs (94%) (median, 450 specimens/mo): 56% used Bactec 7, 27 or 37; 15% used 'BacT-Alert'; 11% used Columbia broth; 5% used thioglycolate and 'lytic'; 3% each used, Dupont Isolator, Supplemented peptone or other media. Selective media was used for primary anaerobe isolation by 89% labs which included: LKV, 76%; PEA, 53%; BBE, 31%; CNA, 28%; 'CDC', 12%. Sixty labs (78%) stored anaerobes after isolation (median 7 days), most using blood agar plates (31%), chopped meat (26%) or thioglycolate broth (27%) either for further identification (30 out of 78) or susceptibility testing (33 out of 78), if clinically indicated. Only 23% performed routine anaerobic susceptibility testing of clinical isolates. Of the 77% that do not perform susceptibility studies, 59% would not even perform them upon physician request; 30% relied on published surveys; 68% did not publish results of anaerobic susceptibility in annual summaries. When susceptibility testing was performed, the test agents selected were related to availability on a commercial system (21), NCCLS recommendation (20), hospital formulary (15) or hospital committee input (20). Nine of 78 labs (12%) had discussed stopping or decreasing the performance of both anaerobic bacteriology and susceptibility testing. Despite educational and published guidelines, clinical anaerobic bacteriology is not uniformly practiced and could be improved. In addition, an educational effort must be made in order to stress the relevance and increase performance of anaerobic bacteriology.