Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of isopropyl cinodine with nalidixic acid in the direct viable count

Isopropyl cinodine and nalidixic acid were compared in the direct viable count. With raw water and biofilms, elongated cells were seen in the presence of isopropyl cinodine. Increased incubation time led to an increased direct viable count. Individual bacteria responded differently to isopropyl cinodine. Five organisms grew in the presence of 0.01 μg ml^-1 of isopropyl cinodine but were inhibited by 0.1 μg ml^-1. These values for a sixth organism were 0.1 μg ml^-1 and 1.0 μg ml^-1 respectively. The direct viable count was done with inocula taken when the cells were in either lag, log or stationary phases of growth. No differences were seen in the percentage of elongated cells within an experiment but there was variation between experiments. The effect of nalidixic acid and isopropyl cinodine appeared to be additive with respect to inhibition of growth, but little or no additive effect was seen upon the percent of nutrient responsive cells.     

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae.

The direct viable count method, used to detect viable but nonculturable bacteria in aquatic systems, was modified to examine epiphytic populations of Pseudomonas syringae. Viable-population sizes determined from the number of cells that elongated when incubated with yeast extract and nalidixic acid were compared with those determined by the conventional plate count method. The plate count method accurately determined the number of viable cells in epiphytic P. syringae populations in a state of active growth under conditions of high relative humidity. The plate count method also accurately determined the number of viable cells in P. syringae inoculum, or a growing P. syringae population, subject to desiccation stress under conditions of low relative humidity. In epiphytic populations of P. syringae older than 80 h, however, the plate count underestimated the viable-population size by about two- to fourfold, suggesting that up to 75% of the P. syringae population was nonculturable. These nonculturable cells may have entered a starvation-survival state, induced by low nutrient availability in the phyllosphere environment. Epiphytic P. syringae populations undergoing rapid size changes due to growth and death under fluctuating environmental conditions in the field should be accurately enumerated by the plate count method. However, the possible underestimation of viable-population size under some circumstances should be considered in epidemiological studies of phytopathogenic bacteria and when genetically engineered microorganisms in terrestrial ecosystems are monitored.     

Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae.

The direct viable count method, used to detect viable but nonculturable bacteria in aquatic systems, was modified to examine epiphytic populations of Pseudomonas syringae. Viable-population sizes determined from the number of cells that elongated when incubated with yeast extract and nalidixic acid were compared with those determined by the conventional plate count method. The plate count method accurately determined the number of viable cells in epiphytic P. syringae populations in a state of active growth under conditions of high relative humidity. The plate count method also accurately determined the number of viable cells in P. syringae inoculum, or a growing P. syringae population, subject to desiccation stress under conditions of low relative humidity. In epiphytic populations of P. syringae older than 80 h, however, the plate count underestimated the viable-population size by about two- to fourfold, suggesting that up to 75% of the P. syringae population was nonculturable. These nonculturable cells may have entered a starvation-survival state, induced by low nutrient availability in the phyllosphere environment. Epiphytic P. syringae populations undergoing rapid size changes due to growth and death under fluctuating environmental conditions in the field should be accurately enumerated by the plate count method. However, the possible underestimation of viable-population size under some circumstances should be considered in epidemiological studies of phytopathogenic bacteria and when genetically engineered microorganisms in terrestrial ecosystems are monitored.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.     

Use of substrate responsive-direct viable counts to visualize naphthalene degrading bacteria in a coal tar-contaminated groundwater microbial community

A microscopy-based method was developed to distinguish naphthalene-degrading bacteria within the microbial community of a coal tar-contaminated groundwater system. Pure cultures of Pseudomonas putida NCIB 9816-4 were used to develop the substrate responsive-direct viable count (SR-DVC) method. Cells were concentrated on membrane filters, placed on agar plates of Stanier's minimal basal salts media containing antibiotics (nalidixic acid, piromidic acid, pipemidic acid, and cephalexin), and exposed to vapors of naphthalene. Following brief incubation, samples were fixed in 2% formaldehyde and examined by epifluorescent microscopy. Pure cultures displayed the expected cell elongation response to the SR-DVC assay and required a minimum incubation time of 9 h for differentiation of elongated cells. When applied to groundwater samples from the study site, naphthalene responsive cells in the groundwater community were easily distinguished from unresponsive cells and debris (350+/-180 substrate responsive cells/ml, relative to negative controls with no added growth substrate). In an attempt to reduce background counts of elongated bacteria and fungi, the SR-DVC procedure was modified by adding a wash step prior to incubation and a fungal inhibitor, cyclohexamide, to the plates. When groundwater samples were subjected to the modified procedure, only cells in washed samples showed a significant response to naphthalene (150+/-25 cells/ml), indicating the presence of inhibitory substances in the groundwater. Variations in response of the groundwater microbial community to the two SR-DVC procedures suggest that subsurface conditions (microbial and chemical composition) vary temporally. SR-DVC allows the phenotypes of individual naturally occurring cells to be assessed.     

Metabolic activity of bacterial cells enumerated by direct viable count.

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Metabolic activity of bacterial cells enumerated by direct viable count

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Metabolic activity of Flavobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat

Abstract: The physiological state of introduced Flavobacterium strain P25 cells was determined in starvation cultures, in bulk soil, and in the rhizosphere of wheat using direct viable counts (DVC; based on cell elongation after use of nalidixic acid and substrate addition, resulting in a potential activity measurement) and the redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC; based on respiration without substrate additions, resulting in an in situ activity measurement). Both methods clearly demonstrated that the metabolic activity of Flavobacterium P25 cells decreased during starvation, followed by increased activity after amendment with substrate. This confirmed the applicability of DVC and CTC methods to Flavobacterium P25. Both DVC and CTC methods showed that the percentage of active cells in an introduced Flavobacterium P25 population in rhizosphere soil was lower than that in bulk soil in the first 1–2 weeks after planting wheat seedlings. After two weeks, the percentage of metabolically active cells in the P25 population in rhizosphere soil was higher than in bulk soil. Since different aspects of cellular physiology are measured when applying DVC and CTC, the impact of variations in environmental factors on the metabolic state of introduced strains may be monitored closely by these methods.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Physiological responses of bacteria in biofilms to disinfection.

In situ enumeration methods using fluorescent probes and a radioisotope labelling technique were applied to evaluate physiological changes of Klebsiella pneumoniae within biofilms after disinfection treatment. Chlorine (0.25 mg of free chlorine per liter [pH 7.2]) and monochloramine (1 mg/liter [pH 9.0]) were employed as disinfectants in the study. Two fluorgenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride and rhodamine 123, and tritiated uridine incorporation were chosen for assessment of physiological activities. Results obtained by these methods were compared with those from the plate count and direct viable count methods. 5-Cyano-2,3-ditolyl tetrazolium chloride is an indicator of bacterial respiratory activity, rhodamine 123 is incorporated into bacteria in response to transmembrane potential, and the incorporation of uridine represents the global RNA turnover rate. The results acquired by these methods following disinfection exposure showed a range of responses and suggested different physiological reactions in biofilms exposed to chlorine and monochloramine. The direct viable count response and respiratory activity were affected more by disinfection than were the transmembrane potential and RNA turnover rate on the basis of comparable efficiency as evaluated by plate count enumeration. Information revealed by these approaches can provide different physiological insights that may be used in evaluating the efficacy of biofilm disinfection.     

Large-Scale Production of Rhizobium meliloti on Whey

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey, the number of cells of Rhizobium meliloti Balsac reached 5 × 10⁹ CFU/ml in 48 h of incubation. This is comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia. In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells reached 10¹⁰ CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for the large-scale production of fast-growing rhizobia.     

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.     

Basal medium for the selective enumeration of rumen bacteria utilizing specific energy sources.

A 40% rumen fluid basal medium has been developed that without added substrate will support growth of about 10% or less of the total colony count obtained with 40% rumen fluid-glucose-cellobiose-starch-agar medium (RGCSA). The basal medium is prepared by anaerobic incubation of all ingredients in RGCSA medium except the carbohydrates, Na2CO3, and cysteine for 7 days at 38 degrees C. After incubation, substrate(s), Na2CO3 and cysteine are added and the medium is tubed and sterilized as in normal medium preparation. When xylose was included with glucose, cellobiose, and starch as added carbohydrates in the incubated medium, colony counts were comparable to those obtained with RGCSA medium. The addition of specific carbohydrates or other substrates as energy sources to the basal medium suggested that the percentage of the bacterial population capable of utilizing these energy sources was influenced by the ration of the animal; however, considerable animal variation and day-to-day variation in a given animal was observed. Comparison of the population in animals fed either orchardgrass hay or 60% corn-40% orchardgrass (60-40) indicated little or no difference for the percentage of bacteria utilizing glucose, pectin, xylan, or mannitol. Increases in the percentages of xylose-, cellobiose-, Glycerol-, and lactate-utilizing bacteria occurred with the orchardgrass hay ration, whereas the percentage of starch-digesting bacteria was increased significantly (P less than 0.01) in the animals fed the 60-40 ration. A limited number of bacterial strains were isolated from the basal medium without added substrate, most of which were atypical with respect to the predominant rumen bacteria. Growth of these strains, even in complex media, was very slow and limited. Based on these data with isolated strains and colony counts obtained in roll tube medium containing only minerals, resazurin, agar, Na2CO3, and cysteine, the selective medium overestimated the percentage of bacteria able to use a specific energy source. This overestimate was 6 to 7% of the total culturable count.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. 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 the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 degrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water.

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.