Enumeration of bifidobacteria in gastrointestinal samples from piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

A new selective medium for Bifidobacterium spp.

A new selective antibiotic-free medium for Bifidobacterium spp. is defined. This medium has lactulose as the main carbon source and includes methylene blue, propionic acid, and lithium chloride as inhibitors of some related bacterial species. The low pH of the medium contributes to the inhibition of the growth of Enterobacteriaceae. This new selective medium has a simple composition, and the level of recovery it yields is similar to those yielded by nonselective media for Bifidobacterium strains. It could thus be used for routine analysis in environmental or food microbiology.     

An assessment of three selective media for bifidobacteria in faeces

Three previously described media enumerating Bifidobacterium spp. in faeces were compared with respect to their selectivity and quantitative recovery. The results of this study indicate that of the three media studied, Beerens'agar is the most suitable medium for isolation and enumeration of Bifidobacterium spp. from the gut microflora.     

New, simple medium for selective, differential recovery of Klebsiella spp

A highly selective, differential medium for the enumeration and isolation of Klebsiella spp. was developed. With pure cultures, 100% recovery of Klebsiella spp. was observed. Recovery of Klebsiella spp. on MacConkey-inositol-potassium tellurite (MCIK) agar was as good as or better than on MacConkey-inositol-carbenicillin agar either with pure cultures or environmental samples. Recovery and percent colony confirmation with MCIK agar were greater and easier to obtain than for other proposed Klebsiella selective media.     

New, simple medium for selective, differential recovery of Klebsiella spp.

A highly selective, differential medium for the enumeration and isolation of Klebsiella spp. was developed. With pure cultures, 100% recovery of Klebsiella spp. was observed. Recovery of Klebsiella spp. on MacConkey-inositol-potassium tellurite (MCIK) agar was as good as or better than on MacConkey-inositol-carbenicillin agar either with pure cultures or environmental samples. Recovery and percent colony confirmation with MCIK agar were greater and easier to obtain than for other proposed Klebsiella selective media.     

Selective Media for the Enumeration of Chromobacterium spp. in Soil and Water

The development of a selective medium for counting Chromobacterium spp. in river water is described. A mixture of sodium deoxycholate and colistin as inhibitors favoured growth and recovery of chromobacteria. Addition of cycloheximide to this medium controlled the growth of fungi when soil samples were examined.     

Bifidobacterium-selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium

AIMS: To determine the efficacy and selectivity of an acidified, antibiotic-selective, oligosaccharide-containing media for enumerating Bifidobacterium spp. from chicken caeca samples. METHODS AND RESULTS: Transoligosaccharide propionate agar medium (TOS) modified by addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v), did not inhibit the growth of bifidobacteria compared with the control media yet inhibited the growth of Lactobacillus acidophilus, Lactobacillus gallinarum, Lactobacillus helveticus and Streptococcus gordonii. CONCLUSIONS: Addition of mupirocin (50 microg ml-1) and glacial acetic acid (1%, v/v) to TOS (TOS-AM50), is an effective selective medium for isolation and enumeration of Bifidobacterium spp. from chicken caeca samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of an intestinal bifidobacteria-selective media contributes to the study of probiotics and prebiotics in poultry and potentially other species.     

Distribution, diversity, and activity of sulfate-reducing bacteria in the water column in Gek-Gel Lake, Azerbaijan

The distribution and activity of sulfate-reducing bacteria (SRB) in the water column of the alpine meromictic Gek-Gel lake were studied. Apart from traditional microbiological methods based on cultivation and on measuring the process rates with radioactive labels, in situ fluorescent hybridization (FISH) was used, which enables identification and quantification without cultivating organisms. The peak rate of sulfate reduction, 0.486 microg S/(l day), was found in the chemocline at 33 m. The peak SRB number of 2.5 x 106 cells/ml, as determined by the end-point dilutions method on selective media, was found at the same depth. The phylogenetic position of the SRB, as determined by FISH, revealed the predominance of the Desulfovibrio spp., Desulfobulbus spp., and Desulfoarculus spp./Desulfomonile spp. groups. The numbers of spore-forming Desulfotomaculum spp. increased with depth. The low measured rates of sulfate reduction accompanied with high SRB numbers and the predominance of the groups capable of reducing a wide range of substrates permit us to propose utilization of electron acceptors other than sulfate as the main activity of the SRB in the water column.     

Listeria monocytogenes: diagnostic problems

The first isolation methods for the detection of Listeria spp. were generally based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult. In time, new techniques were developed, based on a variety of selective and elective agents in isolation and enrichment media, which gained better and quicker results. Current reference methods allow the recovery of L. monocytogenes from a variety of foods with relative ease. However, more comparative studies are needed to select one horizontal method. It is suggested that the procedure of the International Organization for Standardization is a good base for such comparisons.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Efficiency of various selective media in determining Pythium population in soil

Of 15 selective media recommended for isolation and enumeration ofPythium spp. directly from soil, corn meal agar (CMA) supplemented with agar, sucrose, minor elements, thiamine, rose bengal, pimaricin, pentachloronitrobenzene and vancomycin (MPVM) was the most efficient. Streptomycin (30–50 ppm) and rose bengal (33–60 ppm) as used in certain tested media effectively suppressed development of bacteria and actinomycetes. However, these chemicals adversely affected germination of spores and mycelial growth and thereby the recovery ofPythium spp. from soil. Media containing pimaricin (5 to 100 ppm) were more effective than those with nystatin (40 ppm) in suppressing development of nonphycomycetous fungi on isolation plate. MPVM with pimaricin at 5 ppm was more efficient than that with 10 ppm of the antibiotic in recoveryingPythium from soil. However, there was no difference in recovery ofPythium by this medium containing rose bengal at 5 ppm or at 10 ppm, butPythium colonies were more dense and better delineated when the medium contained 10 ppm of rose bengal. CMA containing pimaricin (5–100 ppm) and vancomycin (200 ppm) permitted occasionally development of a large number ofMortierella and bacterial colonies from certain soils, that interfered with accurate determination of colonies of certainPythium spp. on the plates. Vancomycin at 300 ppm, as used in MPVM, substantially reduced development of bacterial colonies compared to 200 ppm of the antibiotic. Surface-soil dilution-plate was more effective than the soil-dilution-plate method in reducing bacteria andMortierella colonies on isolation plates without affecting recovery ofPythium. The importance of basal medium, complement of antimicrobial agents, and isolation methods for efficiency of selective medium in recovery ofPythium spp. directly from soil is discussed.     

A multipathogen selective enrichment broth for simultaneous growth of Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae

A selective enrichment broth (SVV) was formulated to allow concurrent growth of Salmonella spp., V. parahaemolyticus, and V. cholerae. Potassium tellurite and sodium citrate were added as the inhibitors, while glucose, mannitol, anhydrous sodium sulfite and sodium pyruvate were employed as the growth-promoters. When mixed in equal or varied proportions, the target pathogens in SVV had a great accumulation (10(5)-10(8) CFU/ml) and effectively inhibited the growth of competitive microflora. In the artificially contaminated samples, a high recovery of these 3 target pathogens was obtained in SVV. Finally, Salmonella spp., V. parahaemolyticus, and V. cholerae were detected from 608 suspicious food samples by SVV with real-time PCR, and no false-positive or -negative results were reported. In summary, SVV has been shown to be a suitable broth for the simultaneous detection of the 3 pathogens by multipathogen detection on a single-assay platform.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.     

Distribution, diversity, and activity of sulfate-reducing bacteria in the water column in Gek-Gel lake, Azerbaijan

The distribution and activity of sulfate-reducing bacteria (SRB) in the water column of the alpine meromictic Gek-Gel lake were studied. Apart from traditional microbiological methods based on cultivation and on measuring the process rates with radioactive labels, in situ fluorescent hybridization (FISH) was used, which enables identification and quantification without cultivating organisms. The peak rate of sulfate reduction, 0.486 μg S 1⁻¹ day⁻¹, was found in the chemocline at 33 m. The peak SRB number of 2.5×10⁶ cells/ml, as determined by the most probable number method on selective media, was found at the same depth. The phylogenetic affiliation of the SRB, as determined by FISH, revealed the predominance of the Desulfovibrio spp., Desulfobulbus spp., and Desulfoarculus spp./Desulfomonile spp. groups. The numbers of spore-forming Desulfotomaculum spp. increased with depth. The low measured rates of sulfate reduction accompanied by high SRB numbers and the predominance of the groups capable of reducing a wide range of substrates permit us to assume utilization of electron acceptors other than sulfate as the main activity of the SRB in the water column. Original Russian Text ? O.V. Karnachuk, N.V. Pimenov, S.K. Yusupov, Yu.A. Frank, Ya.A. Puhakka, M.V. Ivanov, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 1, pp. 101–109.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43 degrees C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37 degrees C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43°C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37°C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.