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)     

Physiological state of epiphytic bacteria on submerged stems of the reed Phragmites australis compared with planktonic bacteria in gravel-pit ponds

Suspensions of epiphytic bacteria from submerged stems of Phragmites australis , collected from a gravel-pit pond, were prepared by treatment in a stomacher and by brushing. The two procedures were equally successful in dislodging bacteria. These epiphytic bacteria were compared with planktonic bacteria in water samples from within the reed bed. Colony-forming units (cfu) as a percentage of acridine-orange direct counts (AODCs), percentage of cells capable of intracellular reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, percentage of cells able to form microcolonies, and cell length were all greater for epiphytic bacteria. Epiphytic bacteria of P. australis from six further gravel-pit ponds were also compared with bacterioplankton; cfu as a percentage of AODCs, and percentage of cells capable of INT reduction were again greater for epiphytic bacteria. Because the epiphytic bacteria in these non-organically-enriched, gravel-pit ponds were physiologically different from the planktonic bacteria it is suggested that there was not continual casual exchange, by largely identical bacterial cells, between the epiphytic and planktonic mode. Instead, epiphytic and planktonic populations were independent of each other and/or if there was exchange then bacteria were more successful while in the attached mode, perhaps because of greater organic-nutrient availability at the stem surface.     

Comparison of methods for detection and enumeration of airborne microorganisms collected by liquid impingement.

Bacterial agents and cell components can be spread as bioaerosols, producing infections and asthmatic problems. This study compares four methods for the detection and enumeration of aerosolized bacteria collected in an AGI-30 impinger. Changes in the total and viable concentrations of Pseudomonas fluorescens in the collection fluid with respect to time of impingement were determined. Two direct microscopic methods (acridine orange and BacLight) and aerodynamic aerosol-size spectrometry (Aerosizer) were employed to measure the total bacterial cell concentrations in the impinger collection fluid and the air, respectively. These data were compared with plate counts on selective (MacConkey agar) and nonselective (Trypticase soy agar) media, and the percentages of culturable cells in the collection fluid and the bacterial injury response to the impingement process were determined'. The bacterial collection rate was found to be relatively unchanged during 60 min of impingement. The aerosol measurements indicated an increased amount of cell fragments upstream of the impinger due to continuous bacterial nebulization. Some of the bacterial clusters, present in the air upstream of the impinger, deagglomerated during impingement, thus increasing the total bacterial count by both direct microscopic methods. The BacLight staining technique was also used to determine the changes in viable bacterial concentration during the impingement process. The percentage of viable bacteria, determined as a ratio of BacLight live to total counts was only 20% after 60 min of sampling. High counts on Trypticase soy agar indicated that most of the injured cells could recover. On the other hand, the counts from the MacConkey agar were very low, indicating that most of the cells were structurally damaged in the impinger. The comparison of data on the percentage of injured bacteria obtained by the traditional plate count with the data on percentage of nonviable bacteria obtained by the BacLight method showed good agreement.     

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)     

Recovery of Viable Bacteria from Probiotic Products that Target Oral Health

Probiotic therapy has predominantly been directed toward promoting and maintaining intestinal health. In recent years, however, probiotic regimens that target oral health have appeared on the market. These regimens are often delivered in the form of lozenges. Despite the oral health claims made by the manufacturers of these products, there is little independent evidence in the literature to support such claims. In theory, probiotic organisms can be beneficial by several different means including direct inhibition of pathogens and boosting of the host immune response, with the underlying assumption that these mechanisms require a critical number of viable organisms. In this study, five brands of probiotics marketed for oral health were tested for the recovery of viable bacteria. For only one brand could viable bacteria be recovered within one log of the manufacturer’s stated starting amount of bacteria. Nearly, a billion viable bacteria could be recovered from a lozenge of this brand. The other brands claimed similar starting amounts of bacteria at the time of manufacture but at least a three-log drop-off was observed in the amount of viable bacteria recovered from those products. Refrigeration of the probiotics significantly improved the recovery for one brand, but recoveries for all but one brand remained below the recommended daily dosage for probiotic regimens. It is concluded that probiotic brands differ significantly in the quantities of bacteria that remain viable with most failing to meet recommended dosage targets.     

Generalized relationship between numbers of bacteria and their viability in biofilms

Bacterial biofilms are confined communities that are encapsulated in protective layers of extracellular polymeric substances. Microscopic evaluation of biofilms of diverse bacterial strains on various substrata reveals that, in general, the percentage of viable bacteria decreases with the total number of bacteria in a biofilm.     

Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]

Strict anaerobic culture techniques were used to quantitate heterotrophic bacteria present in hindguts of Reticulitermes flavipes. The grand mean number of viable cells per hindgut was 0.4 X 10(5) (first-instar larvae), 1.3 X 10(5) (third-instar larvae), 3.5 X 10(5) (workers), and 1.5 X 10(5) (soldiers). Of a total of 344 isolates, 66.3% were streptococci that were always obtained regardless of the origin of termites, their developmental stage or caste, or their length of captivity. Most of the remaining isolates were strains of Bacteroides and Enterobacteriaceae. A small percentage were strains of Lactobacillus, Fusobacterium, and unidentified anaerobic gram-positive rods. Recovery of bacteria from worker hindguts was 13.0% of the direct microscopic count. Isolations performed aerobically failed to reveal strict aerobes. Attempts to isolate cellulolytic bacteria were uniformly unsuccessful. Of 145 streptococcal strains isolated from freshly collected termites, almost all were Streptococcus lactis and S. cremoris. Enterobacteriaceae isolates from the same termite specimens were indole-positive Citrobacter, citrate-negative Citrobacter, and Enterobacter cloacae. The possibility of in situ interspecies lactate transfer, between lactate producers (e.g., streptococci) and lactate fermenters (Bacteroides), is discussed.     

The Microflora and Concentrations of Volatile Fatty Acids in The Rumen of Cattle Fed on Single Component Rations

The composition of the rumen microflora and the volatile fatty acids were examined in cattle free-grazing on grass or stall-fed on hay, grass pellets, oats or dried beet pulp with molasses. Total and viable counts of anaerobic bacteria were highest on the grass feeding, but viable counts as a percentage of total counts were highest when oats or beet pulp with molasses were fed. Counts of cellulolytic bacteria were lowest on these latter 2 diets, and highest on grass or grass pellet diets. Studies of the anaerobic flora showed that the composition in animals fed on grass pellets resembled more that found in animals free-grazing on grass than in those fed on hay. Counts of aerotolerant bacteria were only a small percentage of the total count, but were highest on the hay diet. On this latter diet and on grass-feeding the streptococci (identified as Streptococcus bovis) were predominant, but contrary to expectation, streptococci were found only in small numbers on the oats diet, where coryneform rods were the major type present. Although a period of 4–6 weeks was allowed for the animals to adapt to the feeds, the 2 periods of feeding on oats and dried beet pulp with molasses markedly affected the composition of the rumen flora in the subsequent periods of feeding grass pellets and hay. Ruinen volatile fatty acid analysis showed a propionogenic effect of oats and the highest percentage of butyric acid when beet pulp with molasses was fed. The expected propionogenic effect of grass pellets was not observed.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms

Dental plaque bacteria form complex and robust cell aggregates which cannot be counted accurately using epifluorescence microscopy. This causes a significant problem for quantifying their viability. The aim of the investigation was to develop a fluorescence assay to quantify the viable biomass of dental plaque biofilms. Using an artificial mouth system, microcosm plaques were grown under a range of fluoride and mineralizing conditions, and were treated with the oral antiseptics chlorhexidine (CHX) and Listerine. Plaques were harvested, made into suspension and stained in microtitre plates with a di-chromatic fluorescent stain (Live/Dead BacLight). The percentage of viable biomass was calculated from the regression data generated from a viability standard. The standard was constructed using different proportions of viable (green fluorescence) and non-viable (red fluorescence) plaque bacteria, and growth conditions for optimizing green fluorescence were investigated. The results from the assay showed that fluoride at 1000 and 3000 ppm promoted plaque viability by at least 15%, from approximately 45 to 60%, and at 5000 ppm to approximately 87% (P<0.05). Plaques treated with Listerine and CHX from d 0 yielded insufficient biomass to be tested for viability, however 14 d post-treatment, viability was comparable to untreated plaques (approximately 55%, P>0.05). Treatment with Listerine and CHX from d 3 reduced biomass but not viability. Development of this assay enabled viability of plaque bacteria which cannot be resolved with epifluorescence microscopy to be evaluated. It offers a rapid alternative to epifluorescence microscopy and could be applied to nonoral bacteria.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

The Influence of Immunization with Live or Killed Staphylococcus aureus Vaccines on the Early Development of Opsonizing and Bactericidal Factors in Lymph and Blood of Sheep

In order to obtain sensitive measurements on the synthesis of opsonins following immunization with live or killed S. aureus vaccines, lymph was collected from the efferent popliteal lymphatic duct of sheep during the early phase of the immune response. Lymph and blood serum were assayed for opsonizing capacity using ³H-labeled S. aureus. Within 1 hr after vaccination there was a rapid, transitory decrease in uptake by neutrophils of bacteria opsonized with lymph from sheep given the killed vaccine (Group 2). These results were in contrast to the relatively constant uptake rates of bacteria opsonized with lymph from sheep given the live vaccine (Group 1) and non-vaccinated controls (Group 3) at this time. At 72, 96, and 120 hr post-injection mean uptake values for bacteria opsonized with lymph from either vaccinated group were significantly greater than comparable values for controls. Mean uptakes for organisms opsonized with blood serum from Group 1 at 72 and 96 hr post-injection were significantly greater than comparable values for the control group. The percentage of viable neutrophil-associated bacteria decreased when lymph collected from animals in Group 2 in the first hour post-injection was used to opsonize the organisms. Percentages of viable, neutrophil-associated S. aureus for assays in which blood serum was used to opsonize remained relatively constant at around 45% for Groups 2 and 3. In contrast, however, values of viable neutrophil-associated bacteria for Group 1 decreased during the 120 hr after immunization.     

The Effect of Rumen and Post-Rumen Feeding of Carbohydrates on the Caecal Microflora of Sheep

S ummary . When sheep were bottle-fed with solutions of glucose, sucrose or maltose, so avoiding fermentation in the rumen, there was a marked increase in the number of caecal bacteria, with a higher percentage of viable bacteria, compared with caecal counts from sheep fed conventionally on carbohydrates. Bottle-feeding also resulted in a redistribution of bacterial types, 40–46% of the flora comprising Gram positive rods and a significant increase in butyric acid and decrease in propionic acid in the caecal contents. Feeding cellulose suspensions by bottle or conventionally failed to produce significant differences in counts of cellulolytic caecal bacteria. When sucrose was infused in increasing amounts into the abomasum, Streptococcus bovis was the principal organism, isolated from faeces.     

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.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.     

Inability to sustain intraphagolysosomal killing of Staphylococcus aureus predisposes to bacterial persistence in macrophages

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate‐limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis‐associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.     

Post-processing microflora and the shelf stability of gari marketed in Port Harcourt

Gari was examined for its post-processing microbial content. Aerobic mesophilic bacteria and fungi were isolated from all samples. The total viable bacterial counts ranged from 2.0 X 10(2) to 8.0 X 10(4) cfu/g. Fungal counts ranged from 1.0 X 10(2) to 1.5 X 10(4) cfu/g. The total viable counts of fresh samples were much lower than those of market and packaged samples. Bacillus, Micrococcus and Proteus spp. were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp. the fungi. Food borne parasites and pathogens such as Staph. aureus and Clostridium perfringens were not found. The gari samples were quite stable, having a shelf life of 3-6 months. The water activities of the samples ranged from 0.52 to 0.68. Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 10(4) cfu/g dry sample.