Showing papers in "Letters in Applied Microbiology in 1995"
TL;DR: Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.
Abstract: The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum, a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium, was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum. Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.
278 citations
TL;DR: The microplate method was developed and compared to a standard method for assays of auxin compounds produced by bacteria, similar to the standard method in accuracy of determination, required less chemical reagents, and considerably reduced the time required for analyses.
Abstract: Rapid and efficient methods for determining the ability of soil and rhizosphere bacteria to produce key metabolites which are useful in growth promotion or suppression of plant growth are needed. A microplate method was developed and compared to a standard method for assays of auxin compounds produced by bacteria. The microplate method was similar to the standard method in accuracy of determination, required less chemical reagents, and considerably reduced the time required for analyses.
267 citations
TL;DR: During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus and B. thuringiensis were isolated from four individuals and spice.
Abstract: During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus. An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus, all showed characteristic cytotoxic patterns.
207 citations
TL;DR: This study supports the use of tea‐tree oil in the treatment of acne, and demonstrates that terpinen‐4‐ol is not the sole active constituent of the oil.
Abstract: Major components of two tea-tree oil samples were identified using thin layer and gas-liquid chromatography (TLC and GLC). Using a TLC-bioautographic technique, the tea-tree oils, terpinen-4-ol, alpha-terpineol and alpha-pinene were found to be active against Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes whereas cineole was inactive against these organisms. The MIC values of the three active compounds increased in the order alpha-terpineol < terpinen-4-ol < alpha-pinene for all three micro-organisms. MIC values of the tea-tree oils and terpinen-4-ol were lower for P. acnes than for the two staphylococci. This study supports the use of tea-tree oil in the treatment of acne, and demonstrates that terpinen-4-ol is not the sole active constituent of the oil.
202 citations
TL;DR: A logistic regression model is proposed which enables one to model the boundary between growth and no growth for bacterial strains in the presence of one or more growth controlling factors such as temperature, pH and additives such as salt and sodium nitrite.
Abstract: A logistic regression model is proposed which enables one to model the boundary between growth and no growth for bacterial strains in the presence of one or more growth controlling factors such as temperature, pH and additives such as salt and sodium nitrite. The form of the expression containing the growth limiting factors may be suggested by a kinetic model, while the response at a given combination of factors may either be presence/absence (i.e. growth/no growth) or probabilistic (i.e. r successes in n trials). The approach described represents an integration of the probability and kinetic aspects of predictive microbiology, and a unification of predictive microbiology and the hurdle concept. The model is illustrated using data for Shigella flexneri.
188 citations
TL;DR: A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed and fingerprints were achieved without the need to isolate genomic DNA.
Abstract: A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.
169 citations
TL;DR: Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains and with some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%.
Abstract: Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G + C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient (r) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.
160 citations
TL;DR: In this article, the effect of different water activities (aw, 0.968 and temperature) and temperature (25 degrees C and 30 degrees C) on colonization and production of fumonisin B1 (FB1) and B2 (FB2) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks.
Abstract: The effect of different water activities (aw, 0.968, 0.956, 0.944, 0.925) and temperature (25 degrees C and 30 degrees C) on colonization and production of fumonisin B1 (FB1) and B2 (FB2) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing aw and best at 30 degrees C. All three isolates produced more FB1 than FB2 regardless of aw or temperature. Very little FB1 and FB2 were produced at 0.925 aw, with maximum produced at 0.956 and 0.968 aw at both temperatures tested. Most FB1 and FB2 were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all aw levels and both temperatures there was an increase in FB1 and FB2 concentration with time. Statistical analyses of aw, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB1 and FB2 production.
130 citations
TL;DR: A gamma ray‐induced mutant of Pseudomonas aeruginosa strain S8, capable of hyperproduction of biosurfactant from hydrocarbons, was isolated and named as EBN‐8 and showed 3–4 times more hydrocarbon emulsification/conversion as compared to the parent when grown on Khaskheli crude oil in minimal medium.
Abstract: A gamma ray-induced mutant of Pseudomonas aeruginosa strain S8, capable of hyperproduction of biosurfactant from hydrocarbons, was isolated and named as EBN-8. The mutant showed 3-4 times more hydrocarbon emulsification/conversion as compared to the parent when grown on Khaskheli crude oil in minimal medium. Enhanced biosurfactant production and hydrocarbon utilization by the mutant was also observed during growth on heptadecane in minimal medium as indicated by emulsion index and surface tension of cell-free culture broth. Using heptadecane as carbon and energy source, time course for the growth (cfu ml-1) and biosurfactant production were compared for both parent and mutant. These studies were carried out for 24 d at 30 +/- 2 degrees C and for 20 d at 37 degrees C. Growth of EBN-8 was much faster compared to the parent as well as being 2-3 times more hyperproductive.
122 citations
TL;DR: Several strains of Lact.
Abstract: Seventy-three strains of the Lactobacillus acidophilus group and a Lact. reuteri isolated from human faeces were examined for production of antimicrobial agents against 16 strains of six species of food-borne enteric pathogenic bacteria. Several strains of Lact. gasseri showed wide inhibitory activity against the tested bacteria. Gassericin A produced by Lact. gasseri LA39 was one of the most widely active bacteriocins. It was bactericidal without causing cell lysis.
115 citations
TL;DR: Aqueous garlic extract and concentrated garlic oil along with various commercial garlic supplements and pharmaceutical prescriptions were used in an in‐vitro study and AGE and especially CGO were found to have antifungal activity.
Abstract: Otomycosis due to saprophytic keratolytic fungi represents a small percentage of clinical external otitis. Although there are certain antibacterial and antifungal agents available, they usually are very caustic, potentially ototoxic and cannot be used if the ear drum is perforated. Garlic is utilized as a folk medicine in many countries for its antimicrobial and other beneficial properties. In response to a lack of otic preparations, the authors studied the efficacy of garlic extracts against the fungi belonging to the genus Aspergillus which are the most common cause of this infection. Aqueous garlic extract (AGE) and concentrated garlic oil (CGO) along with various commercial garlic supplements and pharmaceutical prescriptions were used in an in-vitro study. AGE and especially CGO were found to have antifungal activity. These agents showed similar or better inhibitory effects than the pharmaceutical preparations and demonstrated similar minimum inhibitory concentrations.
TL;DR: It was demonstrated that the ability of the strain to adhere and colonize the intestinal cell in vivo or the cultured intestinal cells in intro was similar.
Abstract: The validity of the in vitro adhesion tests performed with cultured cell lines, was determined in this study by comparison with results obtained in vivo, in a previous study. To make this experiment the in vitro adhesion tests were performed during a long period by utilization of an appropriate medium, to determine the capacity of the adhered strain to colonize the intestinal tract. It was demonstrated that the ability of the strain to adhere and colonize the intestinal cell in vivo or the cultured intestinal cells in intro was similar.
TL;DR: It is concluded that growing bifidobacteria cells are able to remove cholesterol both by precipitation and assimilation.
Abstract: To determine the validity of the hypothesis of assimilation or precipitation of cholesterol by Bifidobacterium species, resting cell assays and cultures were undertaken in TPY medium containing oxgall With resting cell assays (pH 5), cholesterol was precipitated and redissolved in phosphate buffer (pH 7) At the end of the cultures, only part of the removed cholesterol from the culture medium was found in the phosphate buffer, while the missing cholesterol was in cell extracts It appeared that removal of cholesterol during culturing was not solely due to its precipitation It is concluded that growing bifidobacteria cells are able to remove cholesterol both by precipitation and assimilation
TL;DR: Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis, and recommended dosages of glyphosate did not affect growth rates.
Abstract: A. SANTOS AND M. FLORES. 1995. The effect of the herbicide glyphosate (N-(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha -1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O 2 h -1 g -1 dry weight for A. chroococcum and 12.09 mmol h -1 g -1 for A. vinelandii. When 20 kg ha -1 was used with A. vinelandii, respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha -1 . Nitrogen fixation dropped drastically 80% with 20 kg ha -1 in A. vinelandii and 98% with 28 kg ha -1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.
TL;DR: The very sensitive response of lux‐marked terrestrial bacteria to PTEs identified the potential for a rapid and flexible ecotoxicity assay for assessing the pollution of soil or fresh water environments.
Abstract: The bioluminescence response of a genetically modified (lux-marked) bacterium to potentially toxic elements (PTEs) was monitored using an in vitro assay. Washed cells of Pseudomonas fiuorescens were added to solutions containing various concentrations of metal salts. Bioluminescence, involving either plasmid or chromosomally encoded lux genes, declined as the metal concentration increased. The plasmid marked construct was significantly more sensitive to all metals except Cr. The order of metal sensitivity was found to be Cu = Zn > Cd > Cr > Ni for the chromosomally marked construct and Cu = Zn > Cd > Ni > Cr for the plasmid marked construct. The very sensitive response of lux-marked terrestrial bacteria to PTEs identified the potential for a rapid and flexible ecotoxicity assay for assessing the pollution of soil or fresh water environments.
TL;DR: Both Salmonella sp.
Abstract: Growth and survival of enteric bacteria in freshwater sediments should be of concern to public health officials because of potential contributions of bacteria to the water column. Bacteria densities were measured in sediment and water using direct counts (DC), direct viable counts (DVC) and standard plate counts (PC). Both Salmonella sp. and Escherichia coli survived in microcosms containing autoclaved water and sediment for at least 28 d, as measured by all three methods of enumeration. Moreover, when bacteria were enumerated from sediment-containing microcosms, the DC, DVC and PC values were equivalent. Plate counts showed that both organisms were present at high densities on day 56. In addition, parallel platings on MacConkey agar of E. coli from microcosms containing sediment gave results identical to PC values, indicating that E. coli was not stressed in these microcosms. In microcosms containing water only, E. coli densities declined gradually over 2 weeks by all three measures of enumeration. By day 14, only 58% of total DC was viable as measured by DVC. Due to the protective nature of sediments, the use of standard media may be adequate to enumerate E. coli from some freshwater sediments.
TL;DR: Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.
Abstract: Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.
TL;DR: Environmental factors such as temperature, pH and nutrient level affect enterobacterial acid sensitivity, as well as the presence of phosphate and Na+ and the extent of aeration, and the involvement of phoE, fur and atp in acid tolerance is investigated.
Abstract: Environmental factors such as temperature, pH and nutrient level affect enterobacterial acid sensitivity, as do the presence of phosphate and Na+ and the extent of aeration. The mechanisms governing these effects are partially understood and the involvement of phoE, fur and atp in acid tolerance, of phoE, envZ, tonB, (p)ppGpp and cAMP in salt-induced acid sensitivity and of rpoS in stationary-phase acid tolerance are of particular interest. It should be noted that surface attachment enhances acid resistance.
TL;DR: The isolated bacterium was identified to be a Pseudomonas pickettii strain and was found to be capable of readily metabolizing other organic compounds such as phenol, benzoic acid, hydroxybenzoic acids and hydroquinone, suggesting that the bacterium degrades monochlorophenols through a chlorocatechol pathway.
Abstract: A Gram-negative aerobic bacterium capable of using 2-chlorophenol (2-CP), 3-chlorophenol (3-CP) and 4-chlorophenol (4-CP) as sole carbon sources was isolated and characterized. The bacterium, designated LD1, was identified to be a Pseudomonas pickettii strain. LD1 was able to totally degrade and dechlorinate 2-CP (initial concentration: 1.51 mmol l-1), 3-CP (initial concentration: 0.57 mmol l-1) and 4-CP (initial concentration: 0.75 mmol l-1) within 30, 30 and 40 h of incubation, respectively, under growing-cell batch conditions. LD1 was also found to be able to metabolize chlorocatechols in growing- and resting-cell conditions. This suggests that the bacterium degrades monochlorophenols through a chlorocatechol pathway. In addition, LD1 was found to be capable of readily metabolizing other organic compounds such as phenol, benzoic acid, hydroxybenzoic acids and hydroquinone. Because of the broad spectrum of monochlorophenols and organic compounds that LD1 can degrade, this bacterium appears to have the potential for being successfully used in the biotreatment of wastewaters and in soil decontamination.
TL;DR: The inhibitory effect of commercial ‘pure’ oleuropein was tested against Salmonella enteritidis in a coliform broth and in reconstituted milk and it was found that the inhibition of this organism in the broth was influenced by the initial inoculum size, the pH of the medium and the concentration of additive.
Abstract: The inhibitory effect of commercial 'pure' oleuropein was tested against Salmonella enteritidis in a coliform broth and in reconstituted milk (model food system). It was found that the inhibition of this organism in the broth was influenced by the initial inoculum size, the pH of the medium and the concentration of additive. The inhibition was more pronounced in samples with low pH and low inoculum size. No such inhibition was evident in the model food system.
TL;DR: Re recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA‐1 and lactococcin A, which could only be achieved by placing it under control of a lactococcal promoter.
Abstract: Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter. Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.
TL;DR: Experiments on maize grain at different water activities have demonstrated the influence of aw on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels, and found that at a threshold aw of 0.85–0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumolisin production.
Abstract: The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities (aw) (1, 0.95, 0.90, 0.85) have demonstrated the influence of aw on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when aw was lowered by 5%, but growth rate was unchanged. A 10% reduction in aw from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold aw of 0.85-0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.
TL;DR: The authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes‐like bacteria and report the results of a comparative sequence analysis, and propose two new ActInomyces species.
Abstract: In a previous study the authors reported the characterization of some facultatively anaerobic, Gram-positive, non-sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes-like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169T and DSM 9168T, respectively.
TL;DR: A membrane filter dissolution method was used to recover Cryptosporidium oocysts from spiked water and the average recovery rate was 70.5%.
Abstract: A membrane filter dissolution method was used to recover Cryptosporidium oocysts from spiked water. The average recovery rate was 70.5%.
TL;DR: The antibacterial activity of ibuprofen was affected by pH, being more effective at values below pH 7.5, and may have an ancillary benefit in topical application, in controlling bacteria.
Abstract: The effect of ibuprofen on growth in vitro of six bacterial species was tested. Ibuprofen inhibited growth of the Gram-positive species, but the two Gram-negative species were unaffected. Growth of Staphylococcus aureus was suppressed by ibuprofen concentrations greater than 150 micrograms ml-1 at initial pH 7. At pH 6, such concentrations prevented growth. The antibacterial activity of ibuprofen was affected by pH, being more effective at values below pH 7. Ibuprofen may have an ancillary benefit in topical application, in controlling bacteria.
TL;DR: The addition of selected lactic acid bacteria strains had a remarkable inhibitory effect on the growth dynamics of microflora associated with ready‐to‐use vegetables, during refrigerated storage.
Abstract: The addition of selected lactic acid bacteria strains had a remarkable inhibitory effect on the growth dynamics of microflora associated with ready-to-use vegetables, during refrigerated storage. In particular, coliforms and enterococci were strongly reduced or eliminated from the products from the third day of storage. Lactobacillus casei strains proved more effective than pediococci. The use of lactic cultures able to produce bacteriocins and to grow at low temperatures could be a useful tool to preserve fresh vegetables and to ensure their microbiological safety.
TL;DR: A high incidence of biogenic amine‐producing bacterial strains associated with poultry is highlighted, with three bacterial genera tested apparent as well as amongst strains of the same genus.
Abstract: The production of biogenic amines by 50 poultry-associated bacterial strains (25 Pseudomonas, 13 Salmonella and 12 Listeria) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy-four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine-producing bacterial strains associated with poultry.
TL;DR: A 200‐mer fragment of the fla A gene from Listeria monocytogenes was amplified using the polymerase chain reaction (PCR) incorporating biotin‐ and fluorescein amadite (FAM‐)labelled primers, finding advantages in the specificity and speed of this approach compared to standard agarose gel electrophoresis.
Abstract: A 200-mer fragment of the fla A gene from Listeria monocytogenes was amplified using the polymerase chain reaction (PCR) incorporating biotin- and fluorescein amadite (FAM-)labelled primers. Methods are described for isolating the single stranded FAM-labelled 200-mer. A central portion of this 200-mer was successfully hybridized onto a complementary sequence coated onto a fibre optic biosensor. Advantages in the specificity and speed of this approach compared to standard agarose gel electrophoresis and probing methods are discussed.
TL;DR: The bacteriocins nisin and enterocin 4 are rapidly adsorbed on polypropylene disposables (pipette tips, microfuge tubes) and on glassware and this effect may lead to considerable errors in determination of bacteriOCin activity.
Abstract: H.M.L.J. JOOSTEN AND M. NUNEZ. 1995. The bacteriocins nisin and enterocin 4 are rapidly adsorbed on polypropylene disposables (pipette tips, microfuge tubes) and on glassware. Losses were estimated to exceed 50% if small volumes were transferred to microfuge tubes. This effect may lead to considerable errors in determination of bacteriocin activity. Addition of Tween 80 prevents adsorption.
TL;DR: The incorporation of a resuscitation period on Trypticase Soy Agar before overlay with MSMA was found to significantly improve recovery of heat‐stressed cells and this recovery protocol was shown not to result in the loss of the major known virulence factors of E. coli O157: H7.
Abstract: The use of Sorbitol MacConkey Agar supplemented with 4-methylumbelliferyl beta-D-glucuronide (MSMA), which is commonly used in the isolation of Escherichia coli O157:H7, has been shown to perform poorly when stressed cells of the pathogen are present. The incorporation of a resuscitation period (2 h at 25 degrees C) on Trypticase Soy Agar (TSA) before overlay with MSMA was found to significantly (P < or = 0.01) improve recovery of heat-stressed (52 degrees C/60 min) cells. Maximal recovery was, however, obtained by adding catalase (1000 U) to the TSA before overlaying with MSMA. This recovery protocol was shown not to result in the loss of the major known virulence factors of E. coli O157:H7 (genes encoding eae, VT1 and VT2).