Effect of eight growth media upon fermentation profiles of ten anaerobic bacteria

A study was made of the influence of eight growth media upon the fermentation end-product patterns obtained with ten species of anaerobes. Acidic and neutral end-products of fermentation were analysed by gas chromatography and the results were examined statistically by computer. Differences in end-product profile were at least as great between different media used to grow a single anaerobic species, as between different anaerobes grown in a single type of medium. When individual fermentation end-products produced by a single anaerobe species were examined, statistically significant differences were found between different media for individual end-products. It is concluded that standardisation of growth medium is essential in diagnostic laboratories concerned with identification of anaerobes with the aid of gas chromatography.     

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.     

Development of media to accelerate the isolation of indigo-reducing bacteria,which are difficult to isolate using conventional media

Indigo-reducing bacteria perform natural fermentation in indigo fermentation fluid. Owing to the stochastic nature of the process, the constituent in indigo fermentation fluid differ depending on the prepared batch and fermentation period. To identify new indigo-reducing bacteria, isolation of the bacteria is indispensable. However, isolation of indigo-reducing bacteria is difficult because conventional media are often unsuitable to isolate these slow-growing bacteria that also exist in low numbers. Hydrolysates of polysaccharides and mixtures of plant base constituents are candidates to accelerate the isolation of indigo-reducing bacteria that cannot be isolated using conventional media. In this current study, wheat bran hydrolysate and composted indigo leaves (sukumo) were used as ingredients in the fermentation fluid in the selective medium for indigo-reducing bacteria in anaerobic culture. The results suggested that obligate and oxygen-non-metabolizing facultative anaerobes are difficult to isolate using conventional media, whereas oxygen-metabolizing facultative anaerobes, relatively rapid-growing and major bacterial strains are relatively easy to isolate. Media containing sukumo hydrolysate facilitated the isolation of novel species of Bacillus pseudofirmus-related strains, whereas media containing wheat bran hydrolysate facilitated the isolation of Amphibacillus spp. (including new species). Seven species (including two new species) of indigo-reducing bacteria were isolated using wheat bran hydrolysate-containing media, whereas six species (including three new species) of indigo-reducing bacteria were isolated using media containing both wheat bran and sukumo hydrolysates. These newly developed culture media will facilitate the isolation of unknown bacteria in indigo fermentation and in environments similar to indigo fermentation fluid.     

APPLICATIONS FOR BACTERIAL MEMBRANE FRACTIONS IN STERILE PHARMACEUTICAL QUALITY CONTROL

The commercially available bacterial membrane preparation Oxyrase was examined for use in quality control laboratory procedures for culturing anaerobic microorganisms, and as a media additive for parenteral product filling line validation by media fill to detect anaerobes. Comparison studies between Oxyrase products and conventional anaerobic culturing methods were performed. The results from the studies showed Oxyrase for Broth to be effective in promoting the growth of anaerobes in nonprereduced media not normally used for anaerobic cultivation.     

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.     

PCR-based diagnostics for anaerobic infections

Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.     

Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era

The isolation of anaerobes from patients has declined in recent years, whereas their detection by molecular techniques has increased. In the present work, we analyzed the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing to routine identification of anaerobes in clinical microbiology laboratory. We identified 544 isolates of 79 species by routine culture from deep samples in our hospital. MALDI-TOF MS allowed identification of 332 isolates (61%). The remaining 212 (39%) were identified by 16S rRNA gene sequencing, allowing identification of 202 at the species level. The most common anaerobes were Propionibacterium spp. (12%), Finegoldia magna (4%), Fusobacterium spp. (6%) and Bacteroides spp. (6%). However, among the 79 identified species, seven were new species or genera, including two Prevotella conceptionensis, a species previously detected by our team by amplification and sequencing, five Anaerococcus sp. and one Prevotella sp. Beyond the identification of these new species, we also identified several uncommon or previously not described associations between species and specific pathologic conditions. MALDI-TOF MS-based identification, which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology.     

Anaerobes isolated from clinical material over three years

Data on anaerobic bacteria isolated from clinical specimens at the bacteriology department within the 3-year period (1992-1994) were analysed. Anaerobic cultivation was carried out in all aspirates and swabs were transferred in transport media in syringes or blood cultures. Established growth occurred in all samples cultivated in thioglycollate broth after 4 days of incubation. Cultivation methods included enrichment media, GasPak jar, and API (BioMerieux) for final identification. A sulfite-reduction test using the Wilson-Blair medium and the Ellner-Smith sporulation medium was also used for the isolation of Clostridium perfringens. Anaerobes were diagnosed in 899 samples. Wound swabs (266 samples) and aspirates (106 samples) were the most common clinical material used. In total, 964 anaerobes were isolated: Peptostreptococcus species (299 strains), Eubacterium species (188 strains), Propionibacterium species (153 strains), Bacteroides fragilis(149 strains), Bacteroides species (95 strains) and Clostridium perfringens(80 strains).     

General and specialized media routinely employed for primary isolation of bacterial pathogens of fishes

There are a number of significant diseases among cultured and free-ranging freshwater fishes that have a bacterial etiology; these represent a variety of gram-negative and gram-positive genera. Confirmatory diagnosis of these diseases involves primary isolation of the causative bacterium on bacteriologic media. Frequently used "general" bacteriologic media simply provide the essential nutrients for growth. For most of the major pathogens, however, there are differential and/or selective media that facilitate primary recovery. Some specialized media are available as "ready-to-use" from suppliers, while others must be prepared. Differential media employ various types of indicator systems, such as pH indicators, that allow diagnosticians to observe assimilation of selected substrates. An advantage to the use of differential media for primary isolation is that they hasten bacterial characterization by yielding the appropriate positive or negative result for a particular substrate, often leading to a presumptive identification. Selective media also incorporate agent(s) that inhibit the growth of contaminants typically encountered with samples from aquatic environments. Media that incorporate differential and/or selective components are ideally based on characters that are unique to the targeted bacterium, and their use can reduce the time associated with diagnosis and facilitate early intervention in affected fish populations. In this review, the concepts of general and differential/selective bacteriologic media and their use and development for fish pathogens are discussed. The media routinely employed for primary isolation of the significant bacterial pathogens of fishes are presented.     

Rapid preliminary diagnosis of anaerobic bacterial infections. I. Natural fluorescence of pus, purulent fluids and dressing under UV radiation]

For evaluation of usefulness of natural fluorescence of clinical materials in UV radiation as rapid diagnostic method of infections with anaerobes, 405 samples of pus, bloody-purulent fluids, blood, wound secretions, dressings and other materials were investigated. Occurrence of red-brick UV fluorescence of clinical materials was compared with results of culture aimed at isolation of non-sporeforming anaerobes from "B. melaninogenicus group (P. melaninogenica, P. intermedia and P. saccharolytics). Significant correlation red-brick fluorescence of clinical materials resulting from UV irradiation with presence in these materials of anaerobes such as P. melaninogenica, P. intermedia and P. asaccharolytics was detected. Investigation of clinical materials with application of fluorescence in UV radiation lasts only 1-2 minutes and together with preparation and microscopical inspection which is Gram-stained--only 15-20 min. Positive results of this test may constitute a basis for rapid, preliminary identification of the etiologic factor and for direction of chemotherapy in the early period of infection.     

Putative anaerobic activity in aerated composts

It has been suggested that anaerobic microenvironments develop in aerobic composts, regardless of the aeration system used, and that anaerobic activity is responsible for odor generation and nitrogen losses. This study was designed to measure levels of microorganisms capable of anaerobic growth in two aerated composts: municipal solid waste, a relatively nutrient-rich compost, and pulp and paper-mill solid waste, which is relatively nutrient-poor. Anaerobic microorganisms were isolated from both composts at mesophilic and thermophilic temperatures. The majority of the anaerobic mesophiles were facultative anaerobes, whereas facultative, anaerobic thermophiles varied from 0 to 100%. Serially-diluted samples were spot-plated onto various media to preserve microbial consortia. Levels of aerobic and anaerobic exoenzyme production on spot-plates were similar on cell-wall, starch, and casein media. Although microbial levels on spread plates indicate that aerobes are present in much higher numbers than anaerobes (in 47 of 56 subsamples, 90% of the population were aerobes), microbial growth levels and exoenzyme production on spot-plates indicate that anaerobes may be responsible for a large portion (greater than or equal to 72%) of the metabolic activity in anaerobic microenvironments of aerobic composts.     

一种嗜热厌氧纤维素降解细菌的分离纯化方法

根据纤维素降解细菌对不溶性纤维素底部的粘附作用,利用Ｈｕｎｇａｔｅ厌氧操作技术直接以不溶性纤维素粉为基质进行滚管,分离和纯化获得嗜热厌氧纤维素降解细菌。     

Some Properties of the Nonsporing Anaerobes from Poultry Caeca

S ummary : A study has been made of the ability of 48 strains of anaerobic bacteria, representing 20 different groups of Gram negative and Gram positive nonsporing rods and peptostreptococci, isolated from poultry caeca, to attack certain substrates that may be of ecological importance. The organisms were predominantly saccharolytic; only the strains of Propionibacterium acnes showed proteolytic activity. None of the strains hydrolysed amorphous cellulose or xylan. A few were weakly pectolytic. Starch was hydrolysed by all the strains of Bacteroides fragilis and by one strain of Eubacterium. Two groups of Gram negative bacteria (Bacteroidaceae) and one of the peptostreptococci attacked uric acid. The resistance of the anaerobes to inhibitory substances (bacitracin, virginiamycin, kanamycin, neomycin, vancomycin and nalidixic acid) was determined. Selective media for the isolation of certain of the groups of Gram negative anaerobes have been developed.     

A biochemical protocol for the isolation and identification of current species of Vibrio in seafood

AIMS: We report a biochemical method for the isolation and identification of the current species of vibrios using just one operative protocol. METHODS AND RESULTS: The method involves an enrichment phase with incubation at 30 degrees C for 8-24 h in alkaline peptone water and an isolation phase on thiosulphate-citrate-salt sucrose agar plates incubating at 30 degrees C for 24 h. Four biochemical tests and Alsina's scheme were performed for genus and species identification, respectively. All biochemical tests were optimized as regards conditions of temperature, time of incubation and media composition. The whole standardized protocol was always able to give a correct identification when applied to 25 reference strains of Vibrio and 134 field isolates. CONCLUSIONS: The data demonstrated that the assay method allows an efficient recovery, isolation and identification of current species of Vibrio in seafood obtaining results within 2-7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This method based on biochemical tests could be applicable even in basic microbiology laboratories, and can be used simultaneously to isolate and discriminate all clinically relevant species of Vibrio.     

Oxolinic acid as a selective agent for the isolation of non-sporing anaerobes from clinical material

The use of oxolinic acid as a selective agent for the isolation of non-sporing anaerobes from clinical material was investigated. At a concentration of 5 mg/l it compared favourably with nalidixic acid for this purpose and had the advantage of inhibiting the growth of staphylococci.     

A COMPARISON STUDY BETWEEN OXYRASE ANAEROBIC AGAR PLATES AND CONVENTIONAL ANAEROBIC CHAMBER FOR THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM CLINICAL INFECTIONS

Misplaced confidence in the broad-spectrum antibiotics, increased resistance among previously predictable anaerobic antibiograms, and the push to maximize productivity of available space and downsizing trends has created a need for a simplified cost-effective, and superior method for the isolation and identification of anaerobic bacteria. In this study, the Oxyrase anaerobic plate system which requires no extraneous apparatus to create an anaerobic environment was compared to an anaerobic chamber in the isolation of anaerobic bacteria from 212 consecutive wound specimens. Brucella blood agar and KVL agar plates were used in this comparison study. RapID ANA II, AP120A, special potency disks, and GLC were used for identification. Of the 212 specimens cultured, 87 yielded anaerobic bacteria comprising 182 strains. Thirty-nine strains failed to grow in the anaerobic chamber but grew on the OxyPlates^(TM). These strains were predominantly Peptostreptococcus species (28%), Eubacterium species (20%), and Propionibacterium species (20%). Fourteen strains failed to grow on the OxyPlates, but grew in the anaerobic chamber. No trend was noted and all organisms in this category grew on the OxyPlates from other specimens. In conclusion, the Oxyrases anaerobic plate system appears to be an excellent alternative to the conventional anaerobic chamber in the isolation and identification of clinically significant anaerobes found in human samples, obviating the need for separate anaerobic-aerobic workstations, expensive anaerobic apparatus, and additional incubator space.     

厌氧微生物培养分离：过去、现在和未来

厌氧菌是地球上数量最多、物种最丰富的微生物,也是分类上报道最少的微生物。它们对氧气敏感、生长条件苛刻,不容易培养分离。本文简要总结了厌氧微生物的研究历史,分析了限制厌氧微生物培养分离的主要因素,讨论了厌氧微生物培养分离的策略和方法,回顾了国内外厌氧微生物的系统分类学现状,并展望了厌氧微生物培养分离的发展趋势。     

Bacteria of the genus Acinetobacter. Methods for their isolation and primary identification

A liquid accumulation medium and a solid isolation medium for primary identification of bacteria of the genus Acinetobacter have been proposed. Both media have identical composition and contain ethanol as the only source of carbon and energy and sodium ammonium phosphate as the source of nitrogen. Besides, the modification of Baumann's medium with sodium acetate as the source of carbon and potassium nitrate as the source of nitrogen has been developed. This modified medium has simplified composition and is not inferior to the above-mentioned media in selectivity, though its use gives less satisfactory results. The two proposed media, both liquid and solid, are recommended for wide use.     

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen? AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen? AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen? AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen? AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.     

Simplified Approach to Identification of Aerobic Actinomycetes by Thin-Layer Chromatography

A system has been developed for the identification of aerobic actinomycetes in the clinical laboratory based on analysis of whole cells for diaminopimelic acid and carbohydrates and on the ability of the organism to decompose casein, tyrosine, and xanthine media. The whole-cell analyses were performed by a simple thin-layer chromatographic procedure that is described. Eighteen reference cultures were correctly identified and, subsequently, 35 isolates from clinical material were grouped by using this system. The method is well suited for use in routine clinical laboratories.