Direct Viable Count Research Articles

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water.

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.

DETECTING THE VIABILITY OF PARTICLE ASSOCIATED BACTERIA AFTER UV-RADIATION: A MOLECULAR BIOLOGICAL APPROACH

Presence of wastewater particles is often cited as the reason for failure of UV-irradiation to inactivate targeted organisms to desired level. However, till now there is no method to directly assess the inactivation of particle-associated indicator organisms after UV-irradiation. In this study, we tried to establish a method to identify viable bacteria within wastewater particles. This method was then used to distinguish between particle-associated viable and non-viable bacteria after UV-irradiation. The objective was to assess the fraction of particles of different sizes that are associated with coliform bacteria and to analyze the impact that these particles may have on the residual coliform concentration after UV-radiation. We found that fluorescent in-situ hybridization (FISH) in conjugation with Direct viable count can be effectively used for identification and counting of particle associated Enterobacteriaceae. The method can also successfully distinguish viable bacteria after UV-radiation. We found that only 2 percent of the total coliforms in the supernatant from the secondary clarifier of the Shibaura water treatment plant is associated with particles. Association of coliforms were maximum for the particle size ranging from 41-µm to 80-µm. It was also found that UV-radiation can effectively penetrate through most of the waste water particles.

High Culturability of Bacteria in Commercially Available Personal Composters for Fed-batch Treatment of Household Biowaste

Different types of commercially available personal composters which were designed for repeated fed-batch treatment of garbage were operated for more than one month in order to study microbial populations at the steady state of this process. Both microscopic direct counts and viable plate counts of bacteria recorded had an order of magnitude of 1011 per g dry weight of solid waste-compost mixture (SCM), and the culturability of the resident bacteria was estimated to be 54% on average. High culturability was also demonstrated by comparative quinone profiling of the SCM itself and bacterial colonies recovered on the plates therefrom. These results suggest that conventional cultivation methods work much better for studying microbial community structure in the fed-batch composting process than in natural environments. The colony quinone profiling method may have great promise for the evaluation of culture bias and culturability of in situ microbial populations.

Rapid and sensitive enumeration of viable diluted cells of members of the family enterobacteriaceae in freshwater and drinking water.

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10(8) nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.

Geomicrobiology of deep, low organic carbon sediments in the Woodlark Basin, Pacific Ocean

Abstract The distribution of bacterial populations and activity were determined at two Ocean Drilling Program sites (1109 and 1115) in the Woodlark extensional Basin, near Papua New Guinea, Pacific Ocean. These relatively deep water (1 150-2 211 m), low organic carbon ( approximately 0.4%), low organic matter sedimentation sites, with average thermal gradients ( approximately 30 degrees C/km) are representative of large areas of ocean sediments. At a third site, 1118, with a higher thermal gradient (63 degrees C/km), only bacterial distributions and pore water acetate (bioavailable) concentrations were determined. Active bacterial populations were present to all depths at Sites 1109 and 1115, maximum depth 801 mbsf (metres below seafloor), and this represents the deepest and oldest marine sediments ( approximately 15 Ma) in which the presence of bacteria has been demonstrated using a combination of different approaches (direct microscopic and viable counts, radiotracer turnover and geochemical analysis). In addition, direct counts and geochemical data at Site 1118 provide strong evidence for significant bacterial populations to at least 842 mbsf (3.2x10(5)/cm(3)). Similar to previously studied subseafloor sediments, maximum bacterial populations and activity occurred in the upper approximately 20 m, with much lower rates (up to 10 000 times) in deeper layers. However, a depth integration of data demonstrated that the majority of bacterial cells and activity (sulfate reduction, methanogenesis, thymidine incorporation into DNA and acetate oxidation) occurred in the subsurface, below 20 m. For sulfate reduction this was dependent on the depth of sulfate penetration. Acetate was an important substrate for methanogenesis. Despite being degraded, acetate concentrations in subsurface layers remained relatively constant (<10 muM) and therefore, there must also have been deep acetate formation, which is consistent with the presence of acetogenic bacteria. These results extend the significance of deep acetate formation for the maintenance of subsurface bacterial populations to sediments representative of large areas of the ocean.

Geomicrobiology of deep, low organic carbon sediments in the Woodlark Basin, Pacific Ocean

The distribution of bacterial populations and activity were determined at two Ocean Drilling Program sites (1109 and 1115) in the Woodlark extensional Basin, near Papua New Guinea, Pacific Ocean. These relatively deep water (1 150–2 211 m), low organic carbon (∼0.4%), low organic matter sedimentation sites, with average thermal gradients (∼30°C/km) are representative of large areas of ocean sediments. At a third site, 1118, with a higher thermal gradient (63°C/km), only bacterial distributions and pore water acetate (bioavailable) concentrations were determined. Active bacterial populations were present to all depths at Sites 1109 and 1115, maximum depth 801 mbsf (metres below seafloor), and this represents the deepest and oldest marine sediments (∼15 Ma) in which the presence of bacteria has been demonstrated using a combination of different approaches (direct microscopic and viable counts, radiotracer turnover and geochemical analysis). In addition, direct counts and geochemical data at Site 1118 provide strong evidence for significant bacterial populations to at least 842 mbsf (3.2×10 5/cm 3). Similar to previously studied subseafloor sediments, maximum bacterial populations and activity occurred in the upper ∼20 m, with much lower rates (up to 10 000 times) in deeper layers. However, a depth integration of data demonstrated that the majority of bacterial cells and activity (sulfate reduction, methanogenesis, thymidine incorporation into DNA and acetate oxidation) occurred in the subsurface, below 20 m. For sulfate reduction this was dependent on the depth of sulfate penetration. Acetate was an important substrate for methanogenesis. Despite being degraded, acetate concentrations in subsurface layers remained relatively constant (<10 μM) and therefore, there must also have been deep acetate formation, which is consistent with the presence of acetogenic bacteria. These results extend the significance of deep acetate formation for the maintenance of subsurface bacterial populations to sediments representative of large areas of the ocean.

Survival of Campylobacter jejuni in Biofilms Isolated from Chicken Houses

Campylobacter jejuni is a thermophilic and microaerophilic enteric pathogen associated with poultry. Biofilms may be a source of C. jejuni in poultry house water systems since they can protect constituent microorganisms from environmental stress. In this study, the viability of C. jejuni in biofilms of gram-positive chicken house isolates (P1, Y1, and W1) and a Pseudomonas sp. was determined using a cultural method (modified brucella agar) and direct viable count (DVC). Two-day biofilms grown on polyvinyl chloride (PVC) coupons in R2A broth at 12 and 23°C were incubated with C. jejuni for a 6-h attachment period. Media were then refreshed every 24 h for 7 days to allow biofilm growth. Two-day biofilms of P1, Y1, and Pseudomonas spp. enhanced attachment (P < 0.01) of C. jejuni (4.74, 4.62, and 4.78 log cells/cm2, respectively)compared to W1 and controls without preexisting biofilm (4.31 and 4.22 log cells/cm2, respectively). On day 7, isolates P1 and Y1 and Pseudomonas biofilms covered 5.4, 7.0, and 21.5% of the surface, respectively, compared to 4.9% by W1. Viable C. jejuni on the surface decreased (P < 0.05) with time, with the greatest reduction occurring on surfaces without a preexisting biofilm. The number of viable C. jejuni determined by DVC was greater than that determined by the cultural method, indicating that C. jejuni may form a viable but nonculturable state within the biofilm. Both DVC and the cultural method indicate that biofilms enhance (P < 0.01) the survival of C. jejuni during incubation at 12 and 23°C over a 7-day period.

Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and ecological significance.

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 +/- 0.2) x 10(8) cells g(-1) and (53.1 +/- 16.0) x 10(8) cells g(-1) in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was "reactivated." Bacterial turnover rates estimated ranged from 0.01 to 0.1 day(-1) but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day(-1)). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.

Environmental and physico-chemical factors induce VBNC state in Listeria monocytogenes.

Investigations of bacterial survival in natural environments have indicated that some organisms lose culturability on appropriate media under certain conditions and yet still exhibit signs of metabolic activity and thus viability. This reproducible loss of culturability in many bacterial species led to the description of a "Viable But Non Culturable" (VBNC) state. The purpose of this article is to determine environmental and physico-chemical factors which induce the VBNC state in a food-borne pathogen that has become a public concern: Listeria monocytogenes. The factors, i.e. inoculum size, natural sunlight, temperature (4 degrees C or 20 degrees C), NaCl concentration (0% or 7%) and pH (5 or 6) were studied on 4 strains (LO28, ATCC 19115, Scott A, CNL 895807). The culturability of the starved cell suspension was determined in each condition tested by the spread plate count, and the cell activity was determined by the Direct Viable Count technique and CTC-DAPI double staining. A strain effect was found in different test conditions. For the LO 28 and ATCC 19115 strains, the VBNC state was very transient in certain conditions. For the other strains tested (Scott A, CNL 895807), the VBNC state was maintained throughout the observation period. In the dark, the incubation temperature was the main factor in the production of VBNC forms in L. monocytogenes. However, natural sunlight rapidly produced the VBNC state in L. monocytogenes cells in microcosm water. We conclude that because of its ubiquity and the factors studied which are met in the food industry, the presence of VBNC L. monocytogenes cells could pose a major public health problem since they cannot be detected by traditional culturing methods. Further investigations are needed to establish virulence before and after resuscitation of VBNC L. monocytogenes cells.

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state.

Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.

Inducible gene expression by nonculturable bacteria in milk after pasteurization.

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

Marine environmental pollution stress detection through direct viable counts of bacteria

Direct viable counts (DVC) of bacteria were quantified from polluted and relatively less/non-polluted coastal locations during different seasons to assess whether they can be routinely monitored for an understanding of environmental stress(es) that may impede the full functioning of bacterial communities in situ. Most notably, DVC were quite low during pre-monsoon (March–May) in pollution-affected locations when compared to relatively less/non-polluted ones. In contrast, their abundance was significantly higher (up to or >10%) suggesting a substantially higher microbial activity (thus, a larger turnover of organic matter) during monsoon (June–September) and post-monsoon (October–February) even in pollution-affected locations. The ease of reliably measuring DVC was useful in realising decreased metabolic functioning of bacteria during pre-monsoon, a season where dispersion of land discharges/effluents is much lower. From laboratory and field analyses of this study it is ascertained that DVC are direct indices of potential bacterial metabolic activity, reliable for sensing metabolic stress experienced by bacterial communities in situ and can be useful for evaluating risks in marine environment through human (industrial) activities.

Assessment of viability in the bacterial standing stock of the Antarctic sea from the Indian side

During the austral summer, we examined the bacterial population along the cruise track extending from 70° S and 18° E to 30° S and 35° E. During the cruise, three distinct fractions of the bacterioplankton viz. total count, total direct viable and retrievable counts were simultaneously enumerated in these waters. In the stations south of the convergent region designated as section I the population ranged within 10 8–9 L –1 whereas in the north of the region designated as section II they were one order higher and ranged from 10 9–10 L –1. The percentage of viability in the region was high corresponding to the generally high chlorophyll and primary productivity encountered in the eastern Aghulas bank. The study substantiates the hypothesis that in the Antarctic, not only the bacterial standing stock but also the active population of bacterioplankton (ca. 50 %) are almost equal in abundance to those in the other oceanic or coastal regions. The viable fraction forms a hitherto unreported significant component of these waters.

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields.

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

23] Assessment of metabolic potential of biofilm-associated bacteria

Publisher Summary This chapter discusses the assessment of metabolic potential of biofilm-associated bacteria. In the study of the microbial ecology of bacteria involved in biofilm formation, two major questions concern the metabolic status and phylogenetic identity of community members. Identification of bacteria has been revolutionized by rRNA-based phylogeny, and the development of rRNA-targeted oligonucleotide probes for fluorescent in sire hybridization (FISH) in natural microbial communities. If combined with spectroscopy or flow cytometry, some of them might also be useful tools at the population level. Most measurements of metabolic activities can be roughly grouped into methods detecting activities of specific enzymes, synthesis of macromolecules, or membrane potentials. Some of these methods, however, are not presented here, either because they have not yet been applied to environmental samples of biofilm populations or because they are not widely used in studies of bacterial metabolic activities. They include detection of (1) esterase activity by fluorescent substrates such as fluorescein diacetate or sulfofluorescein diacetate; (2) cell division by microcolony formation; (3) membrane potential by rhodamine or by propidium iodide; (4) membrane integrity by plasmolytic response to osmotic stress; or (5) DNA and RNA degradation by lysozyme treatment and 4', 6-diamidino-2-phenylindole (DAPI) staining. Thus, this chapter starts with the presentation of two of the most widely applied procedures for the detection of bacterial metabolic potentials: the cyanoditolyl tetrazolium chloride (CTC) method, and the direct viable count (DVC) method..

Rapid determination of bacteria in drinking water using an ATP assay

AbstractThe presently used heterotrophic plate count (HPC) for the evaluation of the total number of bacteria in a sample of drinking water takes 7 days of incubation. When the results are known, the water has been consumed and is ineffective for the protection of the health of the consumers. Operators of water treatment systems need to know the bacterial water quality in near real time. Contamination of the system, whether it is intentional, accidental, or due to an inadequate disinfectant residual needs to be discovered much sooner because intervention can then take place in the form of flushing low quality water and/or raising the disinfectant residual. The purpose of this study was therefore to determine if a rapid ATP assay can estimate the HPC in minutes. Two additional methods were used for some samples. The first method was the acridine orange direct count (AODC) that enumerates both viable and nonviable bacteria. The second method was the direct viable count (DVC) that enumerates only viable bacteria. Water samples were obtained 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. The significance of the ATP assay is that it can determine the bacterial quality of the drinking water in less than 5 min. © 2001 John Wiley &amp; Sons, Inc. Field Analyt Chem Technol 5: 185–189, 2001

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degrees C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degrees C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection. The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.

Evidence of Viable But Non-Culturable state in Listeria monocytogenes by direct viable count and CTC-DAPI double staining

Viable but Non-culturable (VBNC) state in bacteria was detected originally in environmental microbiology studies. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and moreover the cells remain potentially pathogenic under favourable conditions. But the VBNC state has not been yet described in Listeria monocytogenes. Production of VBNC L. monocytogenes cells was studied using four strains resuspended in microcosm water. Various strains of L. monocytogenes were resuspended in filtered sterilized water adjusted to pH 6·0, incubated at 20°C and 4°C with or without NaCl supplementation (0%, 7%), with gentle shaking at 100 rpm. The culturability of starved cells suspensions was determined by spread plate count (PCA). The cells activity was measured by a Direct Viable Count technique and by CTC-DAPI double staining. Two strains of four (CNL 895807, Scott A) exhibited a VBNC state. They lost their culturability and maintained a cellular activity, even after 10 weeks of starvation in microcosm water.

Survival and cell culturability of biocontrol Pseudomonas fluorescens CHA0 in the rhizosphere of cucumber grown in two soils of contrasting fertility status

The effect of cucumber roots on survival patterns of the biocontrol soil inoculant Pseudomonas fluorescens CHA0-Rif was assessed for 22 days in two non-sterile soils, using a combination of total immunofluorescence cell counts, Kogure's direct viable counts and colony counts on plates containing rifampicin. In Eschikon soil (high fertility status for cucumber), CHA0-Rif persisted as culturable cells in bulk soil and in the rhizosphere, but colony counts were lower than viable counts and total cell counts inside root tissues. The occurrence of viable but non-culturable (VBNC) cells inside root tissues (5 log cells g–1 root) was unlikely to have resulted from the hydrogen peroxide treatment used to disinfect the root surface, as hydrogen peroxide caused the death of CHA0-Rif cells in vitro. In Siglistorf soil (low fertility status for cucumber), the inoculant was found mostly as non-culturable cells. Colony counts and viable counts of CHA0-Rif were similar, both in bulk soil and inside root tissues, whereas in the rhizosphere viable counts exceeded colony counts at the last two samplings (giving about 7 log VBNC cells g–1). In conclusion, soil type had a significant influence on the occurrence of VBNC cells of CHA0-Rif, although these cells were found in root-associated habitats (i.e. rhizosphere and root tissues) and not in bulk soil.