Viable Count Method Research Articles

Note. Growth of Vibrio parahaemolyticus in lobster homogenates at different temperatures / Nota. Crecimiento de Vibrio parahaemolyticus en langosta a diferentes temperaturas

Due to the illnesses associated with the presence of Vibrio parahaemolyticus in seafoods, its survival in lobster was studied. Lobster homogenates were prepared, sterilized, inoculated with the bacteria, and incubated at -25 °C, 6 °C and 28 °C, for 60 days. Different viable cell counting methods were employed. The results of the McFarland nephelometric standard method and the direct count of viable bacterial cells, (DVC) were not significantly different (p = 0.5). However, the DVC method was better for detecting viable cells at low numbers for longer times, especially for cultures incubated at -25 °C. Therefore, this method, and the plate count (PC) method for counting colony forming units, were selected for monitoring the survival of V. parahaemolyticus under each temperature of incuba tion. The greatest survival of the bacterium was at ambient (28 °C) temperature. At freezer tempera ture (-25 °C), no viable cells were detected towards the end of the experiments. However, the cultures kept at 6 °C were viable until the end of the incubation period, these results being different from previously published works conducted at refrigeration temperatures.

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters.

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.

A RAPID METHOD FOR DETECTING BACTERIA IN DRINKING WATER

Abstract A rapid determination of the total bacterial count in drinking water is important to the operators of treatment plants and distribution systems. It will allow corrective measures in real‐time, such as increasing the disinfectant dose or removing water that has high bacterial numbers. The present heterotrophic plate count (HPC) analysis takes seven days and is not useful for operational intervention. The purpose of this study was to determine if a rapid adenosine triphosphate (ATP) assay would estimate the total number of bacteria in minutes. For quality control purposes and also to test the accuracy of both the ATP and HPC test, direct enumeration of the bacteria in a water sample was done using two epifluorescence methods. One was acridine orange direct count (AODC) method, which allows enumeration of both viable and nonviable bacteria. The other was direct viable count (DVC) method, which enumerates viable bacteria. Water samples originated from local, national, and international locations. The sample selection criteria were based on proximity to the laboratory, cooperating water utilities, and the travel of the authors. The results of the study show that the rapid ATP assay is highly correlated with the conventional plate count method and the DVC method, and estimates the bacterial quality of drinking water in minutes.

Immunity against Escherichia coli Infection in Chickens Assessed by Viable Bacterial Counts in Internal Organs

Septicemic strains of Escherichia coli cause systemic infection in chickens after the intra-airsac inoculation. We have investigated whether levels of immunity can be determined by the viable organism count in the internal organs of infected birds. The intra-airsac inoculation of O1:K1 strain caused acute systemic infection in 6 hr. The viable count was highest in the lung followed by the liver, spleen, and blood. The count was significantly (P < 0.05) lower in the liver or spleen of vaccinated birds at 6, 12, or 24 hr after inoculation than in controls. Vaccines containing various adjuvants were tested in this system, and three oil-based adjuvants demonstrated significant (P < 0.05) immunity, whereas an alum-precipitated vaccine or one without an adjuvant failed to do so compared with nonvaccinated controls. An oil-adjuvanted vaccine showed some deterioration in its immunogenicity after prolonged storage or heating at 100 C. The acute phase response induced by intravenous injection of killed O1: K1 cells or lipopolysaccharide purified from Salmonella typhimurium in aqueous suspension induced significant immunity against the E. coli infection. These results indicate that the method referred to as "in vivo viable count method" produces quantitative results in a reproducible manner and suggest that it may be used as an alternative method to mortality measurement.

ANTIBACTERIAL ACTIVITY OF SULFONATED STYRENE-GRAFTED POLYPROPYLENE FABRIC AND ITS METALLIC SALT

Antibacterial activities of sulfonated styrene-grafted polypropylene fabric and its metallic complexes against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by a viable cell counting method. After styrene was grafted to polypropylene fabric, a sulfonation reaction was carried out. Various metals were introduced to the sulfonated styrene-grafted polypropylene fabric to evaluate their antibacterial activities. Ag complex of sulfonated styrene-grafted polypropylene fabric (3.95 mM SO3H/g) had a strong biocidal effect to kill all bacteria within 30 minutes. From the antibacterial activity of the metallic salts of sulfonated-grafted PP fabric, it was found that Ag complexed fabric had strong biocidal effects for all bac-teria and the other metal complexed fabrics had different antibacterial activity depending on each bacterium.

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.

Enumeration and characterization of bacteria in mineral water by improved direct viable count method.

Fifteen strains from two emergent mineral waters were isolated and tentatively identified with API 20NE and BIOLOG GN systems. These strains were screened for their sensitivities to seven replication-inhibiting antibiotics of the (fluoro)quinolone group (nalidixic and pipemidic acid, flumequine, norfloxacin, ofloxacin, pefloxacin and ciprofloxacin). It was shown that the direct viable count (DVC) procedure could be improved by using certain antibiotic cocktails, which were active against the isolates. Geometric bacterial features were successfully determined with image analysis and adapted software (ICONIX, Perfect Image). Elongations were significant and allowed rapid discrimination of antibiotic inhibited and non-inhibited strains. Particular isolates in a mixed culture were characterized and enumerated after only 14 h exposure with the appropriate antibiotic cocktail. This method can also be applied to other communities, such as mixed cultures in bio-fermentors or in food with known microflora.

Antibacterial activities of acrylic acid‐grafted polypropylene fabric and its metallic salt

Acrylic acid (AAc) was grafted onto polypropylene (PP) fabric by a preirradiation method using a 60Co gamma ray. The effects of the absorbed dose, the reaction temperature, reaction time, storage time, as well as the addition effect of ferrous ion and sulphuric acid on the degree of grafting, were determined. It was shown that the synergistic effect of a strong acid with ferrous sulfate can increase the grafting yield. Antibacterial activities on metallic complexes of acrylic acid-grafted polypropylene (AAc-g-PP) fabric were evaluated by a viable cell counting method. An Ag complexed fabric had strong biocidal effect for all bacteria. Fe, Cu, and Zn complexed fabrics had different antibacterial activities depending on each bacterium. However, AAc-g-PP fabric and Ni-complexed fabric did not have bactericidal effect. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 69: 2213–2220, 1998

Bacteria in post-glacial freshwater sediments.

Prokaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12,000 years of deposition, were examined for culturability, viability and community structure. The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria. Direct count procedures revealed 10(12) cells (g dry wt sediment)-1 in the surface sediments, which declined to approximately 10(9) cells (g dry wt sediment)-1 at 6 m depth of core (Representing approximately 10,000 years of deposition). The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method. Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively. Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from 10(7) c.f.u. (g dry wt sediment)-1 to 10(1)-10(2) c.f.u. (g dry wt sediment)-1 below 3 m depth. Among culturable isolates, Gram-positives and Gram-negatives were found at all levels of the core, and spore-forming heterotrophs dominated. Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected at all depths. PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea. These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.

Postantibiotic effect of meropenem in combination with gentamicin or sparfloxacin on Gram-positive and Gram-negative organisms

Objective To determine the postantibiotic effect (PAE) on some Gram-positive and Gram-negative strains induced by meropenem in combination with sparfloxacin and gentamicin. Methods The determinations of the minimum inhibitory concentrations (MICs) and of the PAEs were carried out on two isolates each of Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and Pseudomonas aeruginosa with meropenem, gentamicin and sparfloxacin. The PAEs of meropenem plus gentamicin or sparfloxacin, at 0.5 and 5 MIC, were evaluated, after an exposure time of 1.5 h, by the viable count method. Results Combination of meropenem with gentamicin, at 5 MIC, significantly enhanced the PAE for enterococci, which reached values about 1 h greater than the sum of the PAEs of the two drugs (synergistic effect). Combination of meropenem with sparfloxacin, at 5 MIC, produced a distinct enhancement of the PAE for enterococci (roughly additive effect). Both combinations, meropenem plus gentamicin and meropenem plus sparfloxacin, at 5 MIC, gave PAE values that were a little greater than the highest value obtained with the most active antibiotic for S. aureus and P. aeruginosa , and were consistently similar to those of the most single active antibiotic for Enterobacteriaceae (indifferent effect). Conclusions The longer PAE of meropenem in combination with gentamicin or sparfloxacin, in comparison to that of singly tested antibiotics, could have some implications for the timing of doses during therapy with antimicrobial combinations.

Evaluation of the direct viable count method for temperature-stressed Campylobacter coli

Human pathogens, such as campylobacters, transform into the nonculturable state more or less rapidly in the environment. The question arises, whether these cells are still viable. The direct cell count (DVC) method has been used widely for enumeration and viability testing of different bacterial species. The influence of temperature induced bacterial injury on the applicability of the method, however, has not been examined. Two different Campylobacter coli strains were pre-incubated at low temperatures. The necessary nalidixic acid concentration was strain specific, and they became more sensitive to the inhibitory effect of the antibiotic after temperature downshift. The method was not applicable for cultures that had been pre-incubated at intermediate temperatures, although nonculturability had not yet set in at the beginning of the experiments.

Modelling the effectiveness of a natural antimicrobial on Salmonella enteritidis as a function of concentration, temperature and pH, using conductance measurements.

The growth of Salmonella enteritidis in a brain heart infusion medium was monitored using the traditional viable count method and by conductance measurements using a Rabit impedance instrument. Growth curves (log10 cfu ml-1 vs time) at three different concentrations of oleuropein (0, 0.2 and 0.8%), pH values in the range of 5-8 and incubation temperatures from 22 to 42 degrees C were modelled using the Gompertz equation. A good correlation between the maximum growth rate from the viable count method and the maximum slope of the conductance curve from the impedance instrument was established. Based on this correlation, the maximum specific growth rate of Salm. enteritidis was modelled as a function of the oleuropein concentration, initial pH values and the incubation temperature with a quadratic equation, using a new, large dataset of growth measurements by conductance. The developed model was validated by statistical comparison of predicted growth rates with growth rates determined by the viable count method, within the limits of the antimicrobial, pH and temperature domain.

Biologically active polymers: synthesis and antimicrobial activity of modified glycidyl methacrylate polymers having a quaternary ammonium and phosphonium groups

Polymers with antibacterial activity have been synthesized by chemical modification of poly(glycidyl methacrylate). The glycidyl methacrylate was polymerized by the free radical polymerization technique. The poly(glycidyl methacrylate) was hydrolyzed and was chloroacetylated using chloroacetyl chloride. The chloroacetylated product was modified to yield polymers with either quaternary ammonium or phosphonium salts. The antimicrobial activity of the modified glycidyl methacrylate polymers has been examined against a variety of test microorganisms by the cut plug and the viable cell counting methods using shake flask of ten times diluted nutrient broth medium. All three polymers obtained were inhibitory to the growth of Gram negative bacteria ( Escherichia coli, Pseudomones aeruginosa, Shigella sp. and Salmonella typhae) and Gram positive bacteria ( Bacillus subtilis and B. cereus) as well as the fungus ( Trichophytun rubrum). It was found that the growth inhibitory effect varied according to the structure of the polymer and the composition of the active group and increased with increasing the concentration of the polymer. The tested polymers showed more antimicrobial activity against Gram negative bacteria and the fungus, whereas were less active against Gram positive bacteria.

Comparison of CO2 generation (BACTEC) and viable-count methods to determine the postantibiotic effect of antimycobacterial agents against Mycobacterium avium complex.

The postantibiotic effects (PAEs) of antimycobacterial agents determined with a BACTEC TB-460 instrument (CO2 production) and by a traditional viable-count method against Mycobacterium avium complex (MAC) were not significantly different (P > 0.05). The longest PAEs following a 2-h exposure to 2x the MIC were induced by amikacin (10.3 h), rifampin (9.7 h), and rifabutin (9.5 h), while the shortest PAEs resulted from clofazimine (1.7 h) and ethambutol (1.1 h) exposure. CO2 generation is a valid and efficient means of determining in vitro PAEs against MAC.

Ecological implications of an improved direct viable count method for aquatic bacteria

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.

Laboratory methods for studies of bacterial adhesion

Prosthetic infection following total joint replacement can have catastrophic results both physically and psychologically for the patients, leading to complete failure of the arthroplasty, possible amputation, prolonged hospitalization, and even death. Bacterial adherence to biomaterial surfaces is an important step in the pathogenesis of prosthetic infection. The exact mechanism of prosthetic infection remains unclear. It is thought that certain bacteria, particularly coagulase-negative staphylococci, after adhering to biomaterial surfaces, secrete a layer polysaccharide, an extracellular substance (slime) and then form a biofilm (a biomass of bacteria and slime). The biofilm makes the embedded bacteria less accessible to the human defense system and significantly decreases antibiotic susceptibility. Before effective preventive or therapeutic measures can be achieved, the process, characteristics, or mechanism of bacterial adhesion to biomaterials have to be studied. In this review, the in-vitro experimental methods for bacterial adhesion will be discussed, which concentrates on (1) how to design a new in-vitro model of bacterial adhesion or biofilm formation, including the selection of bacteria, sample surface preparation, conducting bacterial adhesion or biofilm formation experiments, and the sample preparation for evaluation; and (2) methods for examining adhered bacteria and biofilm, which include microscopy for counting and morphological observation of adhered bacteria, viable bacterial counting methods, other direct or indirect bacterial counting methods, and the methods for evaluating biofilm.

Detection and enumeration of viable but non-culturable transconjugants of Escherichia coli during the survival of recipient cells in river water.

Viable but non-culturable transconjugant cells were detected by a modification of the direct viable count (DVC) method. This modification involved the addition of parental antimicrobial markers (kanamycin and streptomycin) to the elongation medium in order to promote selective elongation of the transconjugant cells. Presence of viable, other than culturable, transconjugants was demonstrated in matings with parental cells from TSB culture as well as with recipient cells from survival in river water (under illuminated and non-illuminated systems). In matings with a recipient strain from illuminated systems, culturable transconjugants were not detected after the third day of recipient cell survival. In spite of this, viable transconjugants were detected in numbers that exceeded 10(5) cells ml-1. These results clearly show that a fraction of non-culturable recipient cells is able to receive and express plasmids by conjugation processes and form viable but non-culturable transconjugant cells.

Synthesis of chitosan derivatives with quaternary ammonium salt and their antibacterial activity

N-alkyl chitosan derivatives were prepared by introducing alkyl groups into the amine groups of chitosan via Schiff’s base intermediates. Quaternization of the N-alkyl chitosan derivatives were carried out using methyl iodide to produce water soluble cationic polyelectrolytes, novel chitosan derivatives with quaternary ammonium salt. Their antibacterial activities against S. aureus were explored by the viable cell counting method in acetate buffer(pH 6.0). The antibacterial activities of the chitosan derivatives with quaternary ammonium salt increased with increase in the chain length of the alkyl substituent, and this increased activity could be ascribed to the contribution of the increased hydrophobic properties of the derivatives.

Postantibiotic effects with Bacteroides fragilis determined by viable counts and CO2 generation.

OBJECTIVE: To study the postantibiotic effect (PAE) for Bacteroides fragilis after exposure to common anaerobic antimicrobials with two different methods, by viable counting and by measuring CO2 generation in a BACTEC(R) blood culture system. METHODS: Four strains of B. fragilis were exposed for 1, 2 and 4 h to cefoxitin, chloramphenicol, clindamycin, imipenem or metronidazole at concentrations from 1 to 16 x MIC. The drugs were removed by dilution into BACTEC 7A(R) vials and growth determined with viability counts and CO2 production. RESULTS: The durations of the PAEs determined by the two methods correlated well (r=0.913, p<0.005). PAEs of up to 4-5 h were induced by imipenem and metronidazole with achievable concentrations and exposure durations. Chloramphenicol induced short or no PAEs, but cefoxitin and clindamycin induced PAEs up to 2 h with high AUC values. The imipenem PAEs and the short cefoxitin and clindamycin PAEs were dependent on AUC. CONCLUSIONS: Significant PAEs against B. fragilis were induced by imipenem and metronidazole. Determining PAE by measuring CO2 production is an accurate and less time-consuming alternative to the conventional method of viable counts.