Most Probable Number Technique Research Articles

Environmental and Anthropogenic Influences on Coliform Concentrations in the Octopus insularis Production Chain in the Veracruz Reef System, Gulf of Mexico.

Coliforms are relatively common in aquatic environments, but their concentrations can be increased by environmental changes and anthropogenic activities, thus impacting fisheries resources. To determine the microbiological quality in the octopus production chain (capture, post-capture, processing and commercialization), total (TC) and fecal (FC) coliforms were quantified in sea water, fresh octopus, fresh water, ice and octopus in two presentations: packed in ice and boiled. Samples came from fishing zones Enmedio, Chopa and La Gallega at the Veracruz Reef System (VRS) during dry, rainy and windy seasons. The coliforms were determined using the most probable number technique (MPN). The most relevant results indicated that octopus packed in ice coming from the commercialization stage had FC levels >540 MPN/100 g, which exceeded the permissible limits (230 MPN/100 g). Therefore, these products present a risk for human consumption. Differences in FC were observed in octopuses between the three fishing zones (H = 8.697; p = 0.0129) and among the three climatic seasons, increasing during the rainy season, highlighting La Gallega with 203.33 ± 63 MPN (H = 7.200; p = 0.0273). The results provide evidence of the environmental and anthropogenic influences on coliform concentrations and the urgent need to implement an efficient cold chain throughout octopus production stages with adequate handling practices to reverse this situation.

Bacterial contamination of water sources for domestic use in JOS and environs

Purpose: The purpose of the study was to assess and examine water bodies in Jos, Plateau State for bacterial contamination. Methods: A total of ninety four (90) water samples were collected aseptically from different sources (stream, well, borehole, tap, dam, storage tanks and water vendors) from different locations and investigated for their bacterial contaminants using the Most Probable Number Technique (MPN). Structured questionnaires were used to obtain information on the location, depth, sanitary condition and closeness of the water sources to septic tanks. The bacterial isolates present in the water samples were identified based on their morphology and biochemical characteristics. Total bacterial count was determined using the pour plate technique and total coliform count was by the Most Probable Number (MPN) method. Results: From the T-Test result of the mean coliform count of all the seven different water sources investigated, storage tanks (ST) were found to be the least contaminated followed by Tap water (TW) while stream (STR) water had the highest rate of contamination with MPN index of 300/100 ml. However, borehole water 6 (BW6) was the least contaminated among all the samples collected with MPN index of 7/10 ml. The MPN index range from 7/100 ml to 300/100 ml in BH6 and STR7 respectively while the Total viable count ranged from 1.0 × 102 to 3.3 × 103. A total of seven different isolates were characterized and identified as Pseudomonas aeroginosa, Klebsiella pneumonia, Escherichia coli, Salmonella typhi, Proteus vulgaris, Staphylococcus aureus and Proteus mirabilis. The results obtained were analysed using SPSS Version 21. Conclusion: This study reveals that all the different water samples collected and investigated were contaminated with bacteria.

Drinking water quality monitoring of centralized water storage reservoirs in various zones of the National University

Physico-chemical and microbiological pollutants may compromise quality of drinking water. This study aims to highlight various physico-chemical and microbial parameters of drinking water samples from centralized water storage reservoirs of National University. Major water quality parameters which were examined in this study includes pH, Electrical conductivity (EC), Turbidity, Total suspended solids (TSS), Total dissolved solids (TDS), Dissolved oxygen (DO), Residual chlorine, Hardness, Alkalinity. Moreover, microbial analysis of water samples was also carried out through Most Probable Number technique (MPN). Results show that all the physico-chemical parameters were within prescribed limits of World Health Organization and Pakistan Drinking Water Quality Standards (WHO/PDWQS) in both underground and overhead water storage reservoirs except DO ( 9.05-9.2 and 9.05-9.1 mg/L ), TSS (10-20 and 3.3-10 mg/L) and residual chlorine (0.08-0.14 and 0.19-0.28 mg/L) respectively. The MPN index values for both underground and overhead water storage reservoirs ranges between 16- >23 and 1.1-12 respectively and it shows high microbial contamination in water due to low detection of residual chlorine. The results highlight that water from centralized water storage reservoirs of National University is unfit for drinking purposes. Statistical analysis such as paired t-test also reveals that water quality parameters from underground and overhead storage reservoirs are not significantly different from each other except EC and TDS having P values <0.05. It is necessary to safeguard water contamination in water storage reservoirs by monitoring regular inspections and chemical cleaning of water storage reservoirs otherwise it will cause serious threats to well-being of the community.
 Key words: water storage reservoirs, drinking water, microbial quality of water, physico-chemical parameters, paired t-test

Cultivable nitrifying bacteria of the limnetic zone of Tota lake, Boyacá, Colombia

Fish production activities in Lake Tota, Boyaca are increasing concentrations of ammonium and nitrate, nutrients that are accelerating eutrophication processes. Some of the microbial communities that regulate the entry of allochthonous nitrogen are nitrifying bacteria, and the determination of their abundance in ecosystems is a measure of pollution bioindication. The objective of the study was to quantify and biochemically characterize the cultivable nitrifying bacteria of the limnetic zone of Lake Tota. The most probable number technique (MPN) was used to measure abundance. The samples were taken in the Lago Chico and Lago Grande sectors, in limnetic areas with fish activity and without intervention. Physical and chemical variables were measured in situ and the concentration of nutrients in the laboratory was determined. The highest quantifications of nitrifying bacteria were recorded in the area of influence of fish farming. The group of ammonium oxidizing bacteria (BOA) and nitrite oxidants (BON) was smaller in the central area of Lago Grande compared to fish farms in Hato Laguna and El Tunel. 14 isolates of nitrifying bacteria were obtained, genera Nitrosomonas spp. and Nitrobacter spp. Changes in BOA abundance were related to alkalinity, while BON values correlated with electrical conductivity and dissolved inorganic nitrogen. A greater abundance of nitrifying bacteria was recorded in the lake’s limnetic areas affected by fish farming activity, associated with the increase in nutrients dissolved in the water column.

Coexistence of sulfate reducers with the other oil bacterial groups in Diyarbakır oil fields

The existence of sulfate-reducing bacteria (SRB) is a major concern in oil industry due to the detrimental effects of SRB in oil technology. SRB are co-habited with diverse microbial populations in oil fields. The presence of other bacterial groups in oil fields may alter SRB activity in different ways. Therefore, understanding this coexistence may provide insights into problems induced by SRB activity and possible solutions to these problems. To investigate this aspect, not only the presence and abundance of SRB but also bacterial population that coexists with SRB in sulfate-reducing enrichment cultures obtained from the Diyarbakır oil fields in southeast of Turkey was determined by using cultivation- and molecular-based approaches. The most probable number technique (MPN) was used to determine the number of sulfidogenic bacteria in the enrichments. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rRNA gene fragments was performed to examine the bacterial diversity of the enrichments. The results demonstrated that the number of sulfidogenic bacteria in the enrichments was low (<103 cells/mL). The DGGE analysis indicated that community members belonging to the Firmicutes were more abundant than those of other phyla. Members belonging to SRB mainly consisted of the genera Desulfosporosinus, Desulfovibrio, Thermodesulfovibrio, and Desulfotomaculum. Fermentative bacteria, acetogens, nitrate reducers, and sulfur reducers were also detected in the enrichments. The results of this study not only provide information regarding the diversity of the cultivable portion of the bacterial community that coexists with cultivable SRB, but they also offer insights into the interactions of bacteria in complex microbial communities that inhabit natural environments.

Microbial Quality Assessment of Processed Meat Product (Tsire) Sold Within Wudil Town, Wudil Local Government Area, Kano State, Nigeria

Microbial quality assessment of Tsire meat products sold within Wudil town was conducted between February-April 2018. A total of fifteen (15) samples were purchased from five different spots and analyzed microbiologically using the pour plate method. The total aerobic plate count on nutrient agar ranged from 1.4x104cfu/ml to 2.95x105cfu/ml. The coliform count using most probable number technique (MPN) ranged from 3cfu/g-240cfu/g. Total fungal count (yeast and mold) on Sabaroud dextrose agar with Chloramphenicol (as control) ranged from 1x103cfu/ml-8x103cfu/ml. The bacteria isolated include Staphylococcus aureus, Escherichia coli, Shigella spp, and Salmonella spp. The fungal isolates obtained from this study are Penicillium spp, and Aspergillus niger.

Evaluación de Microorganismos de Algunos Suelos Procedentes de la Región de Azuero

La cuenta viable de microorganismos del suelo puede realizarse por la técnica de cuenta en placa o la técnica del número más probable (NMP). Los principios fundamentales de estas técnicas son dispersión de una muestra en un diluyente apropiado, distribución en un medio apropiado de crecimiento, incubación bajo condiciones óptimas, y conteo de las colonias desarrolladas. La producción microbiológica de CO2 en el suelo puede ser determinada incubando el suelo en jarras, el objetivo de este proyecto consistió en Caracterizar los microorganismos existentes de tres tipos de suelos: Agrícola, Ganadero y Forestal, a través de múltiples técnicas de microbiología. Se seleccionará un área preferiblemente rectangular de aproximadamente 10x5 metros, sobre la cual se empleara un muestreo en forma zigzag en donde se tomaran 5 submuestras de aproximadamente 10g de cada una (que se depositan en bolsas plásticas de 1 lb), a 20 cm de profundidad, para luego homogenizarla en una sola muestra. Una vez realizada el procedimiento las muestras son llevadas a la Unidad de investigación del Centro Regional Universitario de Azuero. Al finalizar este proyecto de investigación hemos llegado a la conclusión de que la Hipótesis de investigación fue aceptada ya que se presenta una mayor ocurrencia y diversidad de microorganismos en uno de los tres suelos evaluados.&#x0D; &#x0D;

Simultaneous and individual quantitative estimation of Salmonella, Shigella and Listeria monocytogenes on inoculated Roma tomatoes (Lycopersicon esculentum var. Pyriforme) and Serrano peppers (Capsicum annuum) using an MPN technique

Simultaneous and individual enumeration of Salmonella, Shigella and Listeria monocytogenes was compared on inoculated Roma tomatoes and Serrano peppers using an Most Probable Number (MPN) technique. Samples consisting of tomatoes (4 units) or peppers (8 units) were individually inoculated with a cocktail of three strains of Salmonella, Shigella or L. monocytogenes, or by simultaneous inoculation of three strains of each pathogen, at low (1.2–1.7 log CFU/sample) and high (2.2–2.7 log CFU/sample) inocula. Samples were analyzed by an MPN technique using universal pre-enrichment (UP) broth at 35 °C for 24 ± 2 h. The UP tubes from each MPN series were transferred to enrichment and plating media following adequate conventional methods for isolating each pathogen. Data were analyzed using multifactorial analysis of variance (p < 0.05) and LSD multiple rang test. There were differences (p < 0.05) in recovery of simultaneous and individual bacteria inoculated (individual > simultaneous), type of bacteria (Salmonella > Shigella and L. monocytogenes), type of sample (UP broth > pepper and tomato), and inoculum level (high > low). The MPN technique was effective for Salmonella on both commodities. Shigella counts were higher on tomatoes compared to peppers, (p < 0.05), and for L. monocytogenes on peppers (p < 0.05).

Defined and undefined commercial probiotics cultures in the prevention of Salmonella Enteritidis in broilers

ABSTRACT: This study aimed to evaluate the efficacy of probiotics from different formations, defined and undefined cultures, applied in the control of Salmonella Enteritidis in broilers, identifying the compositions and states for which the probiotics are more effective. For that, 390 broilers were inoculated orally with 1.00 ml of Salmonella Enteritidis at a concentration of 1.2x109 CFU (Colony Forming Units). The experimental design used was randomized blocks with 5 treatments and 6 replications, totaling 30 boxes with 13 birds/box (13 birds/m2). The treatments were provided via drinking water 1 hour after inoculation, keeping a daily treatment of 12 hours with probiotics, for 3 consecutive days (birds at 1, 2 and 3 days of age). In general, the five treatments conducted were: T1 - Control without probiotic, T2 - Probiotic A (defined culture - lyophilized form, strain 7), T3 - Probiotic B (defined culture - lyophilized form, strain 11), T4 - Probiotic C (undefined culture liquid form), T5 - Probiotic D (undefined culture - liquid form). After treatments, performance was evaluated through average body weight, feed conversion and mortality counting. Microbiological analysis and Salmonella isolation were performed using MPN (Most Probable Number) and selective enrichment technique methods, respectively. Samples of ileum and liver pool, cecal tonsils, cecum, heart and spleen pool were collected at 5 and 31 days of age. No differences were observed on growth performance and isolation of Salmonella Enteritidis (p≥0.05). All probiotics applied were effective on reducing Salmonella Enteritidis colonization in the ileum, cecal tonsils, and cecum at 5 days of life. Probiotics T2 and T5 has shown effectiveness in reducing colonization at 31 days, being considered the most efficient on Salmonella Enteritidis control, for the intestines segments evaluated. It was not possible to affirm which probiotics formation, defined or undefined, is more efficient for Salmonella Enteritidis control.

Novel approach to enumerate clostridial endospores in milk

Clostridial contamination of raw milk causes late-blowing, a severe quality defect in cheese. Consequently, milk containing high numbers of cheese-damaging clostridial spores should not be used for the production of certain types of hard and semi-hard cheese. Currently, there is no officially standardised method available to monitor clostridial spore levels in milk, and major drawbacks like long analysis time, labour intensity, uncertainty of results and insufficient selectivity for clostridia exist for usually used conventional MPN (most probable number) techniques. Therefore, an optimised medium in combination with a semi-automated application for the enumeration of clostridia in milk was developed. The aim of this study was to evaluate this new methodology in comparison with a conventional method (using Bryant and Burkey broth) based on the analysis of 84 milk samples. Method inclusivity was further tested using pure clostridial cultures, and selectivity was assessed by molecular identification of isolates obtained from the new assay. The novel approach proved to be suitable for the detection of clostridia in both suppliers’ and processed milk, also indicating that it is superior in selectivity, sensitivity and analysis time compared to conventional techniques.

Estimation of Microbial Concentration in Food Products from Qualitative, Microbiological Test Data with the MPN Technique.

Microbial concentration in samples of a food product lot has been generally assumed to follow the log-normal distribution in food sampling, but this distribution cannot accommodate the concentration of zero. In the present study, first, a probabilistic study with the most probable number (MPN) technique was done for a target microbe present at a low (or zero) concentration in food products. Namely, based on the number of target pathogen-positive samples in the total samples of a product found by a qualitative, microbiological examination, the concentration of the pathogen in the product was estimated by means of the MPN technique. The effects of the sample size and the total sample number of a product were then examined. Second, operating characteristic (OC) curves for the concentration of a target microbe in a product lot were generated on the assumption that the concentration of a target microbe could be expressed with the Poisson distribution. OC curves for Salmonella and Cronobacter sakazakii in powdered formulae for infants and young children were successfully generated. The present study suggested that the MPN technique and the Poisson distribution would be useful for qualitative microbiological test data analysis for a target microbe whose concentration in a lot is expected to be low.

Importance of on-farm management practices on lactate-fermenting Clostridium spp. spore contamination of total mixed ration of Manchega ewe feeding. Determination of risk factors and characterization of Clostridium population

This work studies the on-farm management practices that increase the risk of developing high lactate-fermenting Clostridium spp. spore counts in Total Mixed Ration (TMR) of Manchega sheep. Moreover the ecology of the Clostridium population was studied in relation to those management practices. A total of 136 TMR samples belonging of 23 Manchega sheep flocks were analyzed for lactate-fermenting Clostridium spores by the Most Probable Number technique (MPN). Information about the feeding characteristics was also collected from the 23 flocks. A logistic regression analysis indicated silages and wet breweŕs grains used for feeding as the on-farm management risk factors that lead to an increase of Clostridium spp. spore counts. A total of 155 Clostridium isolates from TMR samples were typed using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The 56 different genotypes obtained were subsequently identified by restriction analysis of 16S-rRNA gene (16S-ARDRA), so three populations were observed: 92.90%, 5.81% and 1.29% corresponding to Clostridium (C.) sporogenes, Clostridium beijerinkii and Clostridium butyricum respectively. The risks factors of high Clostridium spp. spore counts also increased Clostridium population diversity and favoured the presence of C. butyricum. The results confirmed that these risk factors should be taken into account in developing strategies in the control of Clostridium spp. spores contamination in TMR.

A most-probable number technique for methanotrophic bacteria determination in biological reactors using methane as an electron donor for denitrification

The most-probable number (MPN) technique along with methane uptake determinations were used to estimate the density of methanotrophic organisms in the biological reactors used for wastewater treatment. The experimental technique was conducted using serum bottles seeded with an inoculum taken from an aerobic sequencing batch reactor that used methane as the sole carbon source. To verify the presence of methanotrophic organisms in the support media, biomass samples were subjected to molecular cloning and sequencing techniques. When compared with the sequences published in databanks, the nucleotide sequences obtained showed a phylogenetic similarity of 98% to Methylomonas sp. (access number AF150792) and a phylogenetic similarity of 96% to Chryseobacterium sp. (access number AB264124), which are type I methanotrophs and denitrifiers, respectively.

Enteroccoccus in Water, Sediment and Clams in a Tropical Environment,Maracaibo Lake, Venezuela

The genus Enterococcus has widespread extra enteric sources and reservoirs. It has been suggested that coastal and Great Lakes States adopt enterococci as an alternative indicator for the monitoring of recreational water quality. Limited information, however, is available about the presence of enterococci in Lake Maracaibo, which is an important estuary in Venezuela, with the income and interchange of the Caribbean Sea, and is used by the people for recreational purposes and the culture of marine organisms. In this study, the density and species composition of enterococci in sediment, clams and water were examined at Lake Maracaibo.Enterococci was enumerated by the Most Probable Number Technique (MPN), and isolated by standard methods. Results obtained by MPN analyses indicated that enterococci were present in all samples, and their densities were generally higher in clams than sediment and water with means of 1.0x106 MPN/100 g, 2.1x103 MPN/100 g, and 6.0x101 MPN/100 ml, respectively. Dominant Enterococcus species were E. faecalis (65%), E. casseliflavus (20%), E. sanguinicola (5%), E. faecium (5%), and unidentified strains (5%). Results suggest that some enterococci are able to persist in Lake Maracaibo, especially in clams and sediment, for a prolonged amount of time after being introduced.

A simple medium modification for isolation, growth and enumeration of Acidithiobacillus thiooxidans (syn. Thiobacillus thiooxidans) from water samples

High concentrations of H2S in groundwater are commonly removed using Biological Trickling Filter (BTF) that contains high numbers of biofilm immobilized sulfur oxidizing bacteria (mainly Thiobacillus thiooxidans). BTF performance requires continuous monitoring of these bacteria at several sampling points. The Most Probable Number (MPN) technique is at the moment the method of choice to enumerate viable T. thiooxidan cells under the above conditions. However, this method is extremely time-consuming (7–10days) and not always suitable for environmental monitoring. In the present study, Thiobacillus agar recommended for isolation and cultivation of Thiobacillus species by Spread plate method was modified by addition of bromocresol green (BCG) in order obtain a clear-cut resolution of the growing colonies resulting in similar or higher numbers compared to other methods. Visual emergence of bacterial colonies on the 3rd and 4th days, from the initial plating, was associated with sulfuric acid production, resulting in an unambiguous color change from blue to yellow, around each colony. This study revealed that BCG modified Thiobacillus agar is substantially time saving and much easier to infer compared to MPN technique.

BIOGENIC PRODUCTION OF SULFIDES IN WATER-OIL SAMPLES AND ITS CORRELATION WITH THE DETERIORATION OF STORAGE TANKS

Production of H2S is one of the main problems in the petroleum industry and one of the leading causes of corrosion in storage tanks. The microbial species found in marine/oil environments and tanks include the general anaerobic heterotrophic bacteria (GAHB) and the sulfate-reducing bacteria (SRB). This research implemented a method based on the semi-continuous production of sulfide, over 15 days, in an attempt to make correlations between sulfide production and microbial quantification. Observation of the most suitable conditions for biogenic-sulfide production indicated that an increase in salinity and temperature produce a significant decrease in the semi-continuous biogenic production of sulfide. Dilution of the culture medium promoted a decrease in cell growth and production of sulfides. The quantification of SRB and GAHB was evaluated using the MPN (Most Probable Number) technique according to FDA regulations (Food and Drug Administration). The use of this procedure contributed to the increase in reliability coefficient used in the quantification data.

Evaluation of Microbiological Safety of Herbal Concoctions Sold in Some Markets in Gabasawa and Nassarawa Local Government Areas in Kano State

A study was carried out on the microbial safety of herbs sourced from some markets in Gabasawa Local Government and Nassarawa Local Government Areas in Kano state. A total of 12 different sample of herbal concoctions were collected from vendors in 2 market from the two Local Government Areas. Enumeration of coliform count, and aerobic bacterial count were carried out using MPN (Most Probable Number) and pour-plate techniques respectively. Enumeration of fungi was also carried out using pour-plate technique. Specific pathogens were also isolated and characterized using standard procedures. The result showed that coliform counts ranged between &lt;3 and &gt;2, 4000 MPN/ML. the mean aerobic viable bacterial count/ml ranged between &lt;3 x 102 cfu/ml and TNC (too numerous to count) cfu/ml, while that of fungal counts ranged from &lt;1x101 to TNC cfu/ml. the occurrence of Escherichia coli is 58.33%, yeast (75%), Aspergillus spps. (41.67%). Penicillium spps. (16.67%), Mucor spps. (33.33%), Rhizopus spps. (41.67%). The findings showed a high microbial contamination among the varieties of concoctions sampled. Adoption of hygiene and good manufacturing practices are therefore recommended as they can improve safety and acceptability of herbal preparations.

Methods for fitting a parametric probability distribution to most probable number data

Every year hundreds of thousands, if not millions, of samples are collected and analyzed to assess microbial contamination in food and water. The concentration of pathogenic organisms at the end of the production process is low for most commodities, so a highly sensitive screening test is used to determine whether the organism of interest is present in a sample. In some applications, samples that test positive are subjected to quantitation. The most probable number (MPN) technique is a common method to quantify the level of contamination in a sample because it is able to provide estimates at low concentrations. This technique uses a series of dilution count experiments to derive estimates of the concentration of the microorganism of interest. An application for these data is food-safety risk assessment, where the MPN concentration estimates can be fitted to a parametric distribution to summarize the range of potential exposures to the contaminant. Many different methods (e.g., substitution methods, maximum likelihood and regression on order statistics) have been proposed to fit microbial contamination data to a distribution, but the development of these methods rarely considers how the MPN technique influences the choice of distribution function and fitting method. An often overlooked aspect when applying these methods is whether the data represent actual measurements of the average concentration of microorganism per milliliter or the data are real-valued estimates of the average concentration, as is the case with MPN data. In this study, we propose two methods for fitting MPN data to a probability distribution. The first method uses a maximum likelihood estimator that takes average concentration values as the data inputs. The second is a Bayesian latent variable method that uses the counts of the number of positive tubes at each dilution to estimate the parameters of the contamination distribution. The performance of the two fitting methods is compared for two data sets that represent Salmonella and Campylobacter concentrations on chicken carcasses. The results demonstrate a bias in the maximum likelihood estimator that increases with reductions in average concentration. The Bayesian method provided unbiased estimates of the concentration distribution parameters for all data sets. We provide computer code for the Bayesian fitting method.

Ferrate treatment for inactivation of bacterial community in municipal secondary effluent

This paper demonstrates the effect of ferrate [Fe(VI)-compound], an environmental friendly multi-purpose reagent, in municipal secondary effluent treatment. The purpose was to study the inactivation capability of ferrate and for the first time to compare the effect and efficiency of Fe(VI) with the widely used disinfectant, chlorine gas on the indigenous bacterial community in the case of secondary effluents. The most probable number technique (MPN) was applied for the determination of cultivable heterotrophic bacterial abundance and terminal restriction fragment length polymorphism (T-RFLP) analysis for comparing bacterial communities. The study demonstrated that (i) ferrate and chlorine had different effect on the total bacterial community of secondary effluents, (ii) low ferrate dose [5mgL−1 Fe(VI)] was sufficient for >99.9% reduction of indigenous bacteria, and (iii) a similar dosage was also effective in the inactivation of chlorine-resistant bacteria.

Effect of repeated irrigation with water containing varying levels of total organic carbon on the persistence of Escherichia coli O157:H7 on baby spinach.

The California lettuce and leafy greens industry has adopted the Leafy Greens Marketing Agreement (LGMA), which allows for 126 most-probable-number (MPN) Escherichia coli per 100 ml in irrigation water. Repeat irrigation of baby spinach plants with water containing E. coli O157:H7 and different levels of total organic carbon (TOC) was used to determine the epiphytic survival of E. coli O157:H7. Three irrigation treatments (0 ppm of TOC, 12 or 15 ppm of TOC, and 120 or 150 ppm of TOC) were prepared with bovine manure containing E. coli O157:H7 at either low (0 to 1 log CFU/100 ml) or high (5 to 6 log CFU/100 ml) populations, and sprayed onto baby spinach plants in growth chambers by using a fine-mist airbrush. MPN and direct plating techniques were used to determine the E. coli O157:H7 populations on the aerial plant tissue. Plants irrigated with high E. coli O157:H7 populations, regardless of TOC levels, showed a 3-log reduction within the first 24 h. Low levels of E. coli O157:H7 were observed for up to 16 days on all TOC treatments, ranging from 76.4 MPN per plant (day 1) to 0.40 MPN per plant (day 16). No viable cells were detected on spinach tissue 24 h after irrigation with water containing fewer than 126 CFU/100 ml E. coli O157:H7. Under growth chamber conditions in this study, E. coli O157:H7 populations in irrigation water that complies with the LGMA standards will not persist for more than 24 h when applied onto foliar surfaces of spinach plants.