Viable Plate Counts Research Articles

Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil.

Most characterized strains of the bacterium Photorhabdus luminescens are symbionts of entomopathogenic nematodes, whereas other strains have been isolated from human clinical specimens. The ability of P. luminescens strains to survive and grow in soil has received limited attention, with some studies indicating these bacteria have little or no ability to persist in soil. Survival and (or) growth of P. luminescens strains in previously sterilized soil, and examination of different soil amendments on their numbers in soil, have not been previously reported. Entomopathogenic P. luminescens (ATCC 29999) and a human clinical isolate (ATCC 43949) were introduced into a soil that had been sterilized by autoclaving, with or without different soil amendments, and bacterial numbers were estimated over time by viable plate count. In the previously sterilized soil receiving no exogenous amendments, numbers fell drastically over a week's time, followed by an increase in numbers by day 30. Treatments involving the addition of calcium carbonate and gelatin or casamino acids to soil usually resulted in higher bacterial numbers. For some sampling dates and soil treatments, there were statistically significant differences between the numbers of the two bacterial strains recovered from soil. The two strains of P. luminescens used in this study were able to survive and grow after being inoculated into previously sterilized soil.

Bioremediation Assessment of Hydrocarbon-Contaminated Soils from the High Arctic

The bioremediation potential of hydrocarbon-contaminated soils from the most northerly inhabited station in the world, Canadian Forces Station - Alert, was assessed. Microbial enumeration, by both viable plate counts and direct counts, combined with molecular analysis (polymerase chain reaction and colony hybridization) for hydrocarbon catabolic genes (alkB, ndoB, xylE), demonstrated the presence of significant numbers of cold-adapted hydrocarbon-degrading microorganisms. The degradative activity of these populations was assessed by mineralization of 14Clabeled hexadecane (C16) at 5°C in untreated and treated soils. Although very low rates of C16 mineralization were observed in the untreated soils, nutrient supplementation with a fertilizer markedly increased C16 mineralization. Highly active cold-adapted hydrocarbon-degrading consortia were prepared from soil slurries, and their degradative potentials were monitored by biomass measurements and mineralization activity. Bio augmentation of the contaminated soils with consortia containing the greatest percentages of degradative bacteria resulted in the shortest C16 mineralization acclimation period. However, treatment with the consortia plus fertilizer did not appreciably increase C16 mineralization or reduce total petroleum hydrocarbon concentrations to a greater extent than did the fertilizer treatment alone. These results indicate that the soils possessed sufficient numbers of cold-adapted degradative bacteria, and that fertilizer application alone was sufficient to obtain elevated levels of degradative activity at low ambient summer temperatures.

Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water.

An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.

Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water

An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.

Use of calcein and SYTO-13 to assess cell cycle phases and osmotic shock effects on E. coli and Staphylococcus aureus by flow cytometry

The fluorochromes SYTO-13 and calcein were tested by flow cytometry for their ability to assess the cell phases and the effects of osmotic shock in E. coli and S. aureus after exposure to NaCl and sucrose. Results were validated by viable plate count. Flow cytometry gave quicker results and more accurate information about intermediate states and population heterogeneity of S. aureus and E. coli than viable plate counts. SYTO-13 was an excellent marker for total counts and calcein can be used to assess esterase activity in S. aureus.

Enumeration, viability and heterogeneity in Staphylococcus aureus cultures by flow cytometry

Several fluorochromes (rhodamine 123, bis-oxonol, propidium iodide, SYTO-13 and calcein) were tested by flow cytometry for their ability to determine cell density, viability and heterogeneity in Staphylococcus aureus cultures exposed to heating (60–70–80°C for 2 min), formaldehyde 2% for 20 min and gramicidin-S at 2–5–10 μg/ml for 20 min. Results were validated by viable plate count and counts performed with a particle analyser. Flow cytometry gave quicker results and more accurate information about intermediate states and heterogeneity of S. aureus cultures than viable plate counts. Rhodamine 123 and oxonol were found to be efficient dyes for the assessment of bacterial viability. SYTO-13 was an excellent marker for total counts and calcein can be used to assess metabolic activity.

17‐β‐Estradiol Upregulates the Stress Response inCandida albicans: Implications for Microbial Virulence

Objective: The influence of 17‐β‐estradiol on the stress response of Candida albicans was studied.Methods: The survival of clinical isolates of C. albicans treated with 17‐β‐estradiol after heat and oxidative stress was measured by viable plate counts. Cellular proteins were analyzed via SDSPAGE.Results: The heat stress response induced by 17‐β‐estradiol in C. albicans grown at 25 ℃ protected the organisms against the lethal temperature of 48.5 ℃, as shown by viable plate counts. 17‐β‐estradiol also enhanced protection of C. albicans against oxidative stress (menadione exposure). SDS‐PAGE analysis of cytoplasmic extracts revealed proteins induced by 17‐β‐estradiol were similar to those induced by heat.Conclusion: 17‐β‐estradiol enhances survival of C. albicans under heat and oxidative stresses. The proteins induced by 17‐β‐estradiol are probably heat shock proteins. Because heat shock proteins are considered to be virulence factors, 17‐β‐estradiol may function to promote in vivo survival.

17-beta-estradiol upregulates the stress response in Candida albicans: implications for microbial virulence.

OBJECTIVE: The influence of 17-beta-estradiol on the stress response of Candida albicans was studied. METHODS: The survival of clinical isolates of C. albicans treated with 17-beta-estradiol after heat and oxidative stress was measured by viable plate counts. Cellular proteins were analyzed via SDS-PAGE. RESULTS: The heat stress response induced by 17-beta-estradiol in C. albicans grown at 25 degrees C protected the organisms against the lethal temperature of 48.5 degrees C, as shown by viable plate counts. 17-beta-estradiol also enhanced protection of C. albicans against oxidative stress (menadione exposure). SDS-PAGE analysis of cytoplasmic extracts revealed proteins induced by 17-beta-estradiol were similar to those induced by heat. CONCLUSION: 17-beta-estradiol enhances survival of C. albicans under heat and oxidative stresses. The proteins induced by 17-beta-estradiol are probably heat shock proteins. Because heat shock proteins are considered to be virulence factors, 17-beta-estradiol may function to promote in vivo survival.

Room temperature sterilization of surfaces and fabrics with a one atmosphere uniform glow discharge plasma.

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log10 CFU reduction of bacteria when 2 x 10(2) cells were seeded on filter paper. Results showed > or = 3 log10 CFU reduction when polypropylene samples seeded with E. coli (5 x 10(4)) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect > or = 6 log10 CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated > or = 5 log10 CFU reduction of commercially prepared Bacillus subtilis spores (1 x 10(5)); 7 min OAUGDP exposures were required to generate a > or = 3 log10 CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed.

EVALUATION OF PCR AND DNA HYBRIDIZATION PROTOCOLS FOR DETECTION OF VIABLE ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN IRRADIATED BEEF

The sensitivity of DNA hybridization and polymerase chain reaction (PCR), was evaluated in irradiated cooked and raw beef samples. A membrane‐based colony hybridization assay and a PCR protocol, both with specificity for the enterotoxin A gene of Clostridium perfringens, were compared with viable plate counts. The results of the colony hybridization procedure were in agreement with viable plate counts for detection and enumeration of enterotoxigenic C. perfringens. The PCR procedure combined a 4 h enrichment followed by a nucleic acid extraction step and assessed the amplification of 183 and 750 base pair enterotoxin gene targets. Detection of C. perfringens by PCR did not show a reliable correlation with viable plate counts or the colony hybridization assay. C. perfringens killed by irradiation were not detected by the plate count or colony hybridization methods; however, killed cells were detected with the PCR technique. By relying on the growth of viable cells for detection and/or enumeration, the colony hybridization and plate count methods provided a direct correlation with the presence of viable bacteria.; Accepted for Publication August 6, 1997

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.

Changes in Cellular States of the Marine Bacterium Deleya aquamarina under Starvation Conditions

In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.

Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii

Two bacterial isolates from Great Bay Estuary, New Hampshire, in co-culture carried out anaerobic dissimilation of citric acid with Fe(III) as the terminal electron acceptor. Neither isolate oxidized citrate with Fe(III) anaerobically in axenic culture. The Fe(III) reducer, Shewanella alga strain BrY, did not grow anaerobically with citrate as an energy source. The citrate utilizer, Aeromonas veronii, did not reduce iron axenically with a variety of electron donors including citrate. The onset of iron reduction by the co-culture occurred after initiation of citrate dissimilation and just prior to initiation of growth by either organism (as measured by viable plate counts). Anaerobic culture growth rates and final cell densities of each bacterial strain were greater in co-culture than in axenic cultures. By 48 h of growth, the co-culture had consumed 27 mM citrate as compared with 12 mM dissimilated by the axenic culture of A. veronii. By 48 h the co-culture produced half as much formate (6 mM) and twice as much acetate (40 mM) as did A. veronii grown axenically (12 mM and 20 mM, respectively). Formate produced from citrate by A. veronii appeared to have supported growth and Fe(III) reduction by S. alga.Although not obligatory, nutrient coupling between these two organisms illustrates that fermentative (A. veronii-type) organisms can convert organic compounds such as citrate to those used as substrates by dissimilatory Fe(III) reducers, including S. alga. This synergism broadens the range of substrates available for iron reduction, stimulates the extent and rate of organic electron donor degradation (and that of iron reduction) and enhances the growth of each participant.

Rapid identification of Escherichia coli O157:H7 in bovine feces using the antibody-direct epifluorescent filter technique (Ab-DEFT)

The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct detection of Escherichia coli O157:H7 in bovine feces. The method involved suspension of bovine feces in buffer, centrifugation for 30 s, treatment of the supernatant with trypsin and Triton X-100 at 50°C for 10 min, pre-filtration through 5 and 1.2 μm pore filters, and filtration through a 0.4 μm pore filter. The final filter was stained with fluorescein-labeled polyclonal antibody specific for the O157 antigen and examined by epifluorescence microscopy. The Ab-DEFT was correlated with viable plate counts for enumeration of the pathogen in artificially inoculated bovine feces ( r = 0.96). The limit of detection was approximately 10 4 – 10 5 CFU/g feces. The procedure provided a clean background for microscopic visualization of cells; however, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was detected in feces of an inoculated calf for more than 3 weeks post-inoculation. The Ab-DEFT may be useful for rapid screening of cattle for the presence of the E. coli O157:H7 and as an analytical method in ecological studies of the pathogen.

In Situ Dynamics of Aromatic Hydrocarbons and Bacteria Capable of AH Metabolism in a Coal Tar Waste-Contaminated Field Site

Field experiments utilizing randomized block designs were implemented to assess the mobility of both coal tar-derived aromatic hydrocarbons and bacteria capable of metabolizing these substances at a contaminated field site. Arrays of sorbent materials wrapped in fiberglass mesh fabric were inserted into organic matter-rich freshwater sediments in order to intercept mobile chemicals and bacteria carried by the prevailing hydraulic gradient. Polyurethane foam plugs served as a sorbent for aqueous-phase coal tar components while sterile sand from the site served as a substrate for colonization by bacteria. Replicate sorbents were removed from the sediments at varying intervals and assessed for organic compounds (via gas chromatography/mass spectrometry) and for numbers of aromatic hydrocarbon-degrading bacteria (via viable plate counts). Organic contaminants including naphthalene, methyl naphthalene, indenes, and substituted benzenes were detected in the foam sorbents. Contaminant concentrations reached a maximum after 15 days before diminishing. Both naphthalene- and phenanthrene-utilizing bacteria were mobile and reached peak titers of 104 and 103.3, respectively, within 11 days. Thus, comigration of both contaminants and microorganisms occurred at the study site. Furthermore, the in situ abundances of contaminants and microorganisms reflect a dynamic balance between processes causing accrual and elimination.

Occurrence of heterotrophic bacteria and fungi in cold and hot water distribution systems using water of different quality

The microbiological quality of cold and hot water samples of 67 Finnish buildings was studied. Most of the buildings were apartment buildings receiving their cold water from municipal groundwater or surface water plants. Disinfection with chlorine was applied in all the surface water plants and 33% of the groundwater plants. Water samples in buildings were taken from incoming cold water, from the hot water main just before and after the heat exchanger, and from a tap or shower in an apartment. The viable counts of mesophilic bacteria and fungi and total cell counts were higher in cold than in hot water samples. In hot water, the microbial counts were higher in samples from taps and showers than from the mains. In taps and showers, the decrease in hot water temperature probably increased the microbial numbers. Thermophilic bacteria appeared with high numbers in all the hot water samples, but thermophilic fungi were found in only one sample. Bacterial biomass and mean cell volume were greater in processed surface water than in processed groundwater samples. Disinfection with chlorine reduced the viable plate counts, although the chlorine concentration was extremely low in the water samples studied.Key words: heterotrophic bacteria, fungi, distribution system, groundwater, surface water.

Aspergillus parasiticus NRRL 2667 Growth and Aflatoxin Synthesis as Affected by Calcium Content and Initial Spore Load in Single Peanuts

Mycelial growth of Aspergillus parasiticus NRRL 2667 and aflatoxin production on Florunner peanuts grown under different calcium supplementation levels (CSL) (550, 1,100, 2,200 and 4,400 kg gypsum/ha) with initial spore loads of 102, 104 and 106 spores/g were investigated. Growth at 25°C for 0, 4, 7 and 14 days was determined by viable plate counts on Aspergillus flavus/parasiticus agar (AFPA) medium. Irrespective of the initial spore load, maximum growth of 108 to 109 CFU/g was attained after 14 days except for the 4,400 kg/ha Ca-supplemented nuts on which the maximum populations were one log less. Aflatoxins B1 and G1 concentrations measured by thin layer chromatography (TLC) ranged from 0–3460 (μg/g and 0–3740 (μg/g, respectively. Toxin production was highest in single peanuts with CSL of 2,200 kg/ha. A reduction of 50% or higher was observed as CSL was increased to 4,400 kg/ha.

Accucary of epifluorescence microscopy counts for direct estimates of bacterial numbers

The aim of this study was to determine the extent and source of inaccuracies for total counts of microorganisms by filtration followed by epifluorescent microscopy. The extent of these inaccuracies were determined by comparing the microscopic method with viable plate counts while their possible sources were investigated using flow cytometry and fluorescent microbeads. One factor responsible for these accuracies was found to be the microscope conversion factor which did not take into account the total filter surface area on which the bacteria were impacted. Approximately a two-fold underestimation was observed when the filtration area rather than the effective area over which the cells are disturbed were compared. Thus it is recommended that the latter be accurately measured when calculating the conversion factor in order to reduce the inaccuracy of direct microscopic counts.

Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice

In a modification of the direct epifluorescent filter technique (DEFT), direct fluorescent antibody staining was used for the rapid (<1 h), specific enumeration of foodbome Escherichia coli 0157:H7 by epifluorescence microscopy. Cell counts obtained by this method (Ab-DEFT) correlated well with DEFT counts obtained by acridine orange staining and with viable plate counts ranging from 108 to 101 cells per ml for pure cultures in buffer. Ab-DEFT also was effective for counting E. coli 0157:H7 cells inoculated into milk and juice; the sensitivity limit was about 103 for milk. The highly specific nature of the technique was demonstrated by enumeration of E. coli 0157:H7 cells in the presence of large numbers of indigenous spoilage microorganisms in milk. This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step.

Susceptibility of Starved Planktonic and Biofilm Listeria monocytogenes to Quaternary Ammonium Sanitizer as Determined by Direct Viable and Agar Plate Counts

Effective food plant cleaning procedures remove microbial nutrients from surfaces, which could result in contaminating bacteria being subject to a starvation microenvironment. This research investigated the effect of starvation on the susceptibility of Listeria monocytogenes to benzalkonium chloride (BAC). Cells were starved in phosphate buffer at 21°C for 4 d. Biofilm and planktonic listeriae reacted differently to starvation. When cells were grown in tryptic soy broth (TSB), starvation reduced the susceptibility of planktonic cells to BAC by 2.3- to 4.7-fold but had no effect on the susceptibility of biofilm cells. Planktonic cells grown in diluted TSB were 390 times more resistant than normal TSB-grown cells, but when these cells were starved, they lost their increased resistance. This phenomenon was not observed with biofilm cells. Increased resistance of listeriae grown in diluted TSB was associated with dilution of the salt/buffer components of the medium. Sanitizer-treated cells were enumerated by using tryptic soy agar-yeast extract pour plates and by a direct viable count method. Results indicate that some cells exposed to BAC were not detected by the plate count procedure but were still viable.