Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Evaluation of factors affecting the membrane filter technique for testing drinking water.

The following studies were done in response to questions regarding the adoption and use of the membrane filter (MF) technique for testing drinking water for the total coliform indicator group. A comparison with the most-probable-number technique showed that MF procedures with m-Endo agar LES were somewhat superior to the most-probable-number methods in terms of numbers of coliform organims recovered. Medium preparation and storage studies indicated that rehydration of m-Endo agar LES should be done with boiling water for less than 15 min, that m-Endo agar LES should not be exposed to light for more than 4 to 6 h, and that m-Endo agar LES plates may be used for up to 4 weeks and broth verification media for up to 3 weeks under given storage conditions. MF culture colonies were commonly found which did not produce sheen as expected for coliforms and yet were verified as coliforms. The occurrence and morphology of these atypical colonies were studied. Parallel inoculation of both lauryl tryptose (LT) and brilliant green bile (BGB) broth was found to be a better colony verification approach than recommended LT preenrichment before transfer to BGB. Comparison of parallel verification results indicated very little justification for the use of LT medium in MF verification procedures. In the case of overgrown or confluent cultures, the best coliform recoveries resulted from swabbing the MF plate and directly inoculating BGB medium with the swab. The occurrence of overgrowth was defined and evidence was collected suggesting that overgrowth is a function of sample holding time. Evaluation of routine test data and bacterial population reductions as a function of time indicated that nonquantitative recovery of coliforms may not be significantly affected for at least a 72-h sample holding time.     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.     

Protozoan grazing and its impact upon population dynamics in biofilm communities

AIMS: To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community. METHODS AND RESULTS: Grazing by Acanthamoeba castellanii and the ciliate Colpoda maupasi upon biofilm and planktonic communities, composed of Klebsiella pneumoniae, Pseudomonas fluorescens and Staphylococcus epidermidis was investigated. Biofilms were formed using glass coverslips, held in a carousel device, as substrata for biofilm formation or in glass flow cells. The predatory effects of the amoeba were generally confined to the biofilm, where grazing rates corresponded to losses from the biofilm equivalent to ca 30,000 biofilm cells cm(-2) h(-1), with the amoeba becoming an integral part of the community. C. maupasi reduced the thickness of mature multispecies biofilms at steady-state from 500 to <200 microm. CONCLUSIONS: We report that the presence of the protozoa A. castellanii and C. maupasi markedly influence population dynamics within defined biofilm communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study dispels the popular opinion that biofilms are protected against predation by protozoa. A. castellanii clearly has the capacity to graze mixed biofilm communities and to become integrally associated with them, whereas the ciliate C. maupasi reduced biofilm thickness by up to 60%.     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

Construction and analysis of fractional multifactorial designs to study attachment strength and transfer of Listeria monocytogenes from pure or mixed biofilms after contact with a solid model food

The aim of this study was to establish which of seven factors influence the adhesion strength and hence bacterial transfer between biofilms containing Listeria monocytogenes (pure and two-species biofilms) and tryptone soya agar (TSA) as a solid organic surface. The two-species biofilms were made of L. monocytogenes and one of the following species of bacteria: the nonpathogenic organisms Kocuria varians, Pseudomonas fluorescens, and Staphylococcus sciuri and CCL 63, an unidentified gram-negative bacterium isolated from the processing plant environment. We used biofilms prepared under conditions simulating open surfaces in meat-processing sites. The biofilm's adhesion strength and population were evaluated by making 12 contacts on a given whole biofilm (4.5 cm(2)), using a new slice of a sterilized TSA cylinder for each contact, and plotting the logarithm CFU . cm(-2) detached by each contact against the contact number. Three types of detachment kinetics were observed: biphasic kinetics, where the first slope may be either positive or negative, and monophasic kinetics. The bacteria that resisted a chlorinated alkaline product and a glutaraldehyde- and quaternary ammonium-based disinfectant had greater adhesion strengths than those determined for untreated biofilms. One of the four non-Listeria strains studied, Kocuria varians CCL 56, favored both the attachment and detachment of L. monocytogenes. The stainless steel had smaller bacterial populations than polymer materials, and non-Listeria bacteria adhered to it less strongly. Our results helped to evaluate measures aimed at controlling the immediate risk, linked to the presence of a large number of CFU in a foodstuff, and the delayed risk, linked to the persistence of L. monocytogenes and the occurrence of slightly contaminated foods that may become dangerous if L. monocytogenes multiplies during storage. Cleaning and disinfection reduce the immediate risk, while reducing the delayed risk should be achieved by lowering the adhesion strength, which the sanitizers used here cannot do at low concentrations.     

Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii

AIMS: To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. METHODS AND RESULTS: The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17.65 g l(-1) and the other 8.84 g l(-1) sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16.8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5.6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17.65 g l(-1) sucrose and having a C : N : P ratio of 334 : 28 : 5.6 resulted in significantly (P < 0.05) higher counts of attached cells for both single-species biofilms at 7.73 log(10) CFU g(-1) GAC and 9.3 log(10)CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8.2 log(10) CFU g(-1) GAC and 6.34 log(10) CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5.6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. CONCLUSIONS: The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5.6 ratio showed significantly (P < 0.05) higher counts of attached cells for both single-species and binary species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Biofilm reduction by a new burn gel that targets nociception

AIMS: To compare the ability of an amorphous first aid topical gel containing vinegar, citric acid and EDTA (RescuDerm(TM); RESC) and various derivative formulations to eradicate Pseudomonas aeruginosa (PSEUD) and Staphylococcus epidermidis (STAPH) biofilms. METHODS AND RESULTS: 24-h biofilms prepared using the Minimum Biofilm Elimination Concentration (MBEC) Assay System were exposed for 4 or 24 h to the different gel formulations. Citric acid-free, acetic acid-free or acetic acid-free/sodium acetate-supplemented RESC gels reduced PSEUD and STAPH biofilm formation as effectively as RESC. Substituting the weak organic acids with equivalent concentrations of glacial acetic acid reduced the effectiveness of gel against PSEUD and STAPH biofilms by half, but viable bacterial counts still remained below 4 log(10) CFU/peg. Removal of gelling agent and/or EDTA enhanced efficacy against PSEUD but not STAPH biofilms. An acidified placebo gel formulation generated an only marginal bactericidal effect compared to that of RESC. CONCLUSIONS: RESC is a promising new antimicrobial agent. Its weak organic acid content, rather than merely acidic pH, mediates its considerable in vitro bactericidal efficacy against bacterial biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: These data, taken together with the observation that RescuDerm possesses broad in vitro bactericidal activity against other pathogen species, suggest the potential usefulness of this product for controlling biofilm formation on a variety of cutaneous traumatic and surgical wounds.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

A glimpse under the rim – the composition of microbial biofilm communities in domestic toilets

Aim: To determine the microbial composition of biofilms in domestic toilets by molecular means. Methods and Results: Genomic DNA was extracted from six biofilm samples originating from households around Düsseldorf, Germany. While no archaeal 16S rRNA or fungal ITS genes were detected by PCR, fingerprinting of bacterial 16S rRNA genes revealed a diverse community in all samples. These communities also differed considerably between the six biofilms. Using the Ribosomal Database Project (RDP) classifier tool, 275 cloned 16S rRNA gene sequences were assigned to 11 bacterial phyla and 104 bacterial genera. Only 15 genera (representing 121 sequences affiliated with Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes and Proteobacteria) occurred in at least half of the samples or contributed at least 10% of the sequences in a single biofilm. These sequences were defined as ‘typical’ for toilet biofilms, and they were examined in more detail. On a 97% sequence similarity level, these sequences represented 56 species. Twelve of these were closely related to well‐described bacterial species, and only two of them were categorized as belonging to risk group 2. No 16S rRNA genes of typical faecal bacteria were detected in any sample. Virtually all ‘typical’ clones were found to be closely related to bacteria or to sequences obtained from environmental sources, implicating that the flushing water is the main source of recruitment. Conclusion: In view of the great diversity of mostly yet‐uncultured bacteria and the considerable differences between individual toilets, very general strategies appear to be most suited for the removal and prevention of toilet biofilms. Significance and Impact of the Study: For the first time, a molecular fingerprinting and cloning approach was used to monitor the species composition in biofilm samples taken from domestic toilets. Knowledge about the microbial composition of biofilms in domestic toilets is a prerequisite for developing and evaluating strategies for their removal and prevention.     

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.     

Differential biofilm formation and chemical disinfection resistance of sessile cells of listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.     

Photomechanical wave-assisted molecular delivery in oral biofilms

Photomechanical waves (PW), the product of an intense light beam interaction with a target material, enhance molecular delivery across biological membranes and skin. The ability to deliver methylene blue (MB), a fluorescent probe and photosensitizer, into bacterial biofilms was demonstrated by applying PW on saliva-derived multi-species biofilms that were developed on agar surfaces in 24-well plates. PW were generated with a Q-switched Nd:YAG laser and were directed into the biofilms in the presence of 25 μg/ml MB. The biofilms were then irradiated with red light at 665 nm. After illumination, adherent bacteria were scraped and spread over the surface of blood agar plates. Survival fractions were calculated by counting bacterial colonies. Microbial analysis was performed via a colony lift method and a DNA checkerboard assay using whole genomic probes to 40 oral microorganisms. Visual analysis by confocal scanning laser microscopy demonstrated that the application of PW enhanced the penetration depth of MB in biofilms. Exposure to MB, PW and light led to a significant reduction of the mean levels of log₁₀ CFU counts compared with the group that received MB and light (P = 0.006). The DNA checkerboard assay showed some benefit from PW-assisted phototargeting in 25 biofilm microorganisms relative to phototreatment alone. Our data provide a basis for further exploration and optimization of PW parameters for complete eradication of microorganisms in oral microcosm biofilms.     

The assessment of airborne bacterial contamination in three composting plants revealed site-related biological hazard and seasonal variations

AIMS: The purpose of this study was to evaluate the degree of bacterial contamination generated by three Italian composting plants (1, 2 and 3) in two different seasons and to assess the health risk for the employees. METHODS AND RESULTS: Aerosols samples were collected with an agar impact sampler. Several plant sites and external upwind and downwind controls were examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to isolate pathogenic bacteria. The levels of total mesophilic and thermophilic micro-organisms ranged between 33 and >40,000 CFU m(-3) in plant 1, 39 and 18,700 CFU m(-3) in plant 2 and 261 and 6278 CFU m(-3) in plant 3. Strains of Escherichia coli, Staphylococcus aureus and Clostridium perfringens were also found. CONCLUSIONS: The plants monitored in this study have proved to be sources of aerosolized bacteria. The activities involving mechanical movement of the composting mass and the indoor activities were of greatest potential risk. In all the studied plants, a statistically significant dependence was found between the bacterial contamination and the season for some or almost all the analysed parameters, but a clear seasonal trend could not be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides broad evidence of bacterial aerosol dispersion and site-related biological hazards that may be useful to the regional government to implement regulations on worker safety in composting plants.     

Mathematical model of the interactions between Micrococcus spp. and Pseudomonas aeruginosa on agar surface

C. CHAPUIS AND J.-P. FLANDROIS. 1994. The interactions between six different Micrococcus species and two strains of Pseudomonas aeruginosa were studied on an agar surface. This type of interaction on solid surface could act as a model of situations occurring either in the environment, in food or in man. The hypothesis of an amensalistic relationship between a Micrococcus spp. and Pseudomonas aeruginosa , due essentially to Ps. aeruginosa bacteriolytic enzymes, is retained as the basis for a mathematical model of the variations of the colony surface of Micrococcus spp. ( S ) with respect to the distance ( d ) from Ps. aeruginosa cells. The diffusion of the bacteriolytic substance in agar explains the limitation of the growth of the Micrococcus spp. This model S = S _max (1 - e^-md***2) is shown to be adapted to all the interactions studied.     

A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms.

Recovery of total coliforms and Escherichia coli on a new membrane filtration (MF) medium was evaluated with 25 water samples from seven states. Testing of the new medium, m-ColiBlue24 broth, was conducted according to a U.S. Environmental Protection Agency protocol. For comparison, this same protocol was used to measure recovery of total coliforms and E. coli with two standard MF media, m-Endo broth and mTEC broth. E. coli recovery on the new medium was also compared to recovery on nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Comparison of specificity, sensitivity, false positive error, undetected target error, and overall agreement indicated E. coli recovery on m-ColiBlue24 was superior to recovery on mTEC for all five parameters. Recovery of total coliforms on the new medium was comparable to recovery on m-Endo.     

Biostimulation of Estuarine Microbiota on Substrate Coated Agar Slides: A Novel Approach to Study Diversity of Autochthonous Bdellovibrio- and Like Organisms

Characterization of Bdellovibrio- and like organisms (BALOs) from environmental samples involves growing them in the presence of Gram-negative prey bacteria and isolation of BALO plaques. This labor-intensive enrichment and isolation procedure may impede the detection and phylogenetic characterization of uncultivable BALOs. In this article, we describe a simple slide biofilm assay to improve detection and characterization of BALO microbiota. Agar spiked with biostimulants such as yeast extract (YE), casamino acids (CA), or concentrated cells of Vibrio parahaemolyticus P5 (most widely used prey bacteria for isolation of halophilic BALOs) was plated onto buffed glass slides and exposed to water samples collected from Apalachicola Bay, Florida. After incubating for a week, diversity of the biofilm bacterial community was studied by culture-dependent and culture-independent molecular methods. The results revealed that most probable numbers (MPNs) of BALOs and total culturable bacteria recovered from YE agar slide were significantly higher than the numbers on CA- or P5-spiked agar slides. Polymerase chain reaction–restriction fragment length polymorphism followed by 16S rDNA sequencing of clones from different biostimulants resulted in identification of a plethora of Gram-negative bacteria predominantly from the alpha, gamma, delta-proteobacteria, and the Cytophaga–Flavobacterium–Bacteroides group. Corresponding to the higher biomass on the YE agar slide, the BALO clone library from YE was most diverse, consisting of Bacteriovorax spp. and a novel clade representing Peredibacter spp. Microbiota from all three biostimulated biofilms were exclusively Gram-negative, and each bacterial guild represented potential prey for BALOs. We propose the use of this simple yet novel slide biofilm assay to study oligotrophic aquatic bacterial diversity which could also potentially be utilized to isolate marine bacteria with novel traits.