Inhibition of Escherichia coli O157:H7 on stainless steel using Pseudomonas veronii biofilms.

We produced a Pseudomonas veronii biofilm on the surface of a stainless steel that is inhibitory to Escherichia coli O157:H7. Pseudomonas veronii strain KACC 81051BP, isolated from lettuce, readily formed biofilm on the surface of stainless steel coupons (SSCs) immersed in tryptic soy broth at 25°C. Cells showed significantly (P ≤ 0·05) enhanced tolerance to desiccation stress (43% relative humidity (RH)) and retained antimicrobial activity against E. coli O157:H7. The number of E. coli O157:H7 (control; 4·1 ± 0·1 log CFU per coupon) on sterile SSCs decreased to 2·7 ± 0·2 log CFU per coupon after exposure to 43% RH at 25°C for 48 h, while the population of E. coli O157:H7 (4·1 ± 0·0 log CFU per coupon) on SSCs containing P. veronii biofilm decreased to below the theoretical detection limit (1·5 log CFU per coupon) within 24 h. The antimicrobial biofilm produced on stainless steel may have application in preventing cross-contamination by E. coli O157:H7 on other abiotic surfaces in food-contact environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Escherichia coli O157:H7 on environmental surfaces of food manufacturing, transportation and storage facilities is a significant food safety concern because it can result in cross-contamination of food products. In this study, we developed a Pseudomonas veronii biofilm on the surface of a stainless steel that inhibits the growth of E. coli O157:H7. Since P. veronii in biofilm resists desiccation, it provides persistent antimicrobial activity. Information presented here provides novel and practical insights to developing biological strategies to inactivate E. coli O157:H7 on diverse surfaces in food processing and handling environments.

Reduction of Bacillus cereus spores in sikhye, a traditional Korean rice beverage, by modified tyndallization processes with and without carbon dioxide injection.

AIMS: The objective of this study was to inactivate Bacillus cereus spores in sikhye using a modified tyndallization process involving injection with carbon dioxide (CO2). METHODS AND RESULTS: Heat tolerance of B. cereus spores in tryptic soy broth and sikhye was evaluated. The D(95°C) values of the B. cereus spores were 2·8-4·9 min, dependent of type of heating medium or inoculum level. The lethality of conventional heat treatment and modified tyndallization with or without CO2 injection against B. cereus spores in sikhye was determined. The order of effectiveness was modified tyndallization with CO2 > modified tyndallization without CO2 > conventional heat treatment. Modified tyndallization with CO2 reduced the number of B. cereus spores in sikhye by 5·8 log CFU ml⁻¹. The increased CO2 concentration and decreased pH of sikhye resulting from CO2 injection rapidly reverted to near-normal values after heat treatment. CONCLUSIONS: Modified tyndallization with CO2 was more effective than conventional heat treatment or modified tyndallization without CO2 in reducing B. cereus spores in sikhye. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study will be useful when developing strategies to control B. cereus spores in sikhye and may have application to other beverages.

Survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity.

AIMS: To determine survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity (r.h.). METHODS AND RESULTS: Spinach leaves were inoculated with suspensions of E. coli O157:H7 in distilled water (DW) and 0.1% peptone water (PW) and incubated at 4, 12 and 25°C and 43, 85 and 100% r.h. The number of E. coli O157:H7 on leaves (5.6 or 1.9 log CFU per leaf) inoculated using DW as a carrier medium increased significantly at 25°C and 100% r.h. within 120 h but remained constant or decreased significantly under other test conditions. E. coli O157:H7 on leaves (5.4 log CFU per leaf) inoculated using PW as a carrier increased significantly within 72 and 24 h, respectively, at 12 or 25°C and 100% r.h.; counts using a low inoculum (2.2 log CFU per leaf) increased significantly within 24 h at 25°C. CONCLUSIONS: Escherichia coli O157:H7 can colonize on spinach leaves at 12 or 25°C in a 100% r.h. environment. Organic matter in the inoculum carrier may provide protection and nutrients which enhance survival and colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of E. coli O157:H7 on spinach leaves as affected by organic matter in the inoculum, temperature and r.h. was determined. These observations will be useful when developing strategies to prevent growth of E. coli O157:H7 on pre- and postharvest spinach.

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry-heat treatment.

AIMS: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO(2) ) and dry heat in reducing the number of Escherichia coli O157:H7. METHODS AND RESULTS: Radish seeds containing E. coli O157:H7 at 5.5 log CFU g(-1) were treated with 500 µg ml(-1) ClO(2) for 5 min and subsequently heated at 60 °C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4.8 log CFU g(-1) after 12 h dry-heat treatment. The pathogen was inactivated after 48 h dry-heat treatment, but the germination rate of treated seeds was substantially reduced from 91.2 ± 5.0% to 68.7 ± 12.3%. CONCLUSIONS: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO(2) and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry-heat treatment should be investigated, and conditions for drying and dry-heat treatment should be optimized. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sequential treatment with ClO(2) and dry-heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.

Identification of Yersinia enterocolitica using a random genomic DNA microarray chip.

AIMS: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. METHODS AND RESULTS: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. CONCLUSIONS: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. SIGNIFICANCE AND IMPACT OF THE STUDY: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.

Comparison of dry sheet media and conventional agar media methods for enumerating yeasts and molds in food.

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DG18) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81), PYM versus DG18 (0.80), CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (alpha = 0.05) when samples were plated on DRBC, DG18, PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 = CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (alpha = 0.05) or significantly lower (alpha = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested; respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall, DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis.

Inhibitory effect of oxalic acid on bacterial spoilage of raw chilled chicken.

Oxalic acid was evaluated as a treatment for reducing populations of naturally occurring microorganisms on raw chicken. Raw chicken breasts were dipped in solutions of oxalic acid (0, 0.5, 1.0, 1.5, and 2.0%, wt/vol) for 10, 20, and 30 min, individually packed in oxygen-permeable polyethylene bags, and stored at 4 degrees C. Total plate counts of aerobic bacteria and populations of Pseudomonas spp. and Enterobacteriaceae on breasts were determined before treatment and after storage for 1, 3, 7, 10, and 14 days. The pH and Hunter L, a, and b values of the breast surface were measured. Total plate counts were ca. 1.5 and 4.0 log CFU/g higher on untreated chicken breasts after storage for 7 and 14 days, respectively, than on breasts treated with 0.5% oxalic acid, regardless of dip time. Differences in counts on chicken breasts treated with water and 1.0 to 2.0% of oxalic acid were greater. Populations of Pseudomonas spp. on chicken breasts treated with 0.5 to 2.0% oxalic acid and stored at 4 degrees C for 1 day were less than 2 log CFU/g (detection limit), compared with 5.14 log CFU/g on untreated breasts. Pseudomonas grew on chicken breasts treated with 0.5% oxalic acid to reach counts not exceeding 3.88 log CFU/g after storage for 14 days. Counts on untreated chicken exceeded 8.83 log CFU/g at 14 days. Treatment with oxalic acid caused similar reductions in Enterobacteriaceae counts. Kocuria rhizophila was the predominant bacterium isolated from treated chicken. Other common bacteria included Escherichia coli and Empedobacter brevis. Treatment with oxalic acid caused a slight darkening in color (decreased Hunter L value), retention of redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Oxalic acid has potential for use as a sanitizer to reduce populations of spoilage microorganisms naturally occurring on raw chicken, thereby extending chicken shelf life.

Protein quality of several multi-component, commercially prepared foods.

Eleven commercial food products that may be considered by consumers to be major sources of protein were evaluated. Most of these items contained significant amounts of carbohydrate in the form of cereal products or purified sugars. Seven of the products were at least equal to casein in protein efficiency ratio (PER). Canned spaghetti and meat balls, the TV dinner, canned chicken-and-vegetable baby food, and food bars had PER values between 91 and 73 per cent of that of casein, while PER values for ground beef and high-protein cereal were significantly higher than casein.

Behavior of Aeromonas species at refrigeration temperatures.

The ability of many strains of Aeromonas hydrophila and A. sobria to produce several types of virulence factors has been documented. The presence of Aeromonas in drinking water, as well as in river and saline waters and on various finfish and shellfish taken from them, has caused some concern relative to the role this bacterium plays as a causative agent of human gastroenteritis. The fairly common occurrence of Aeromonas on red meats, poultry and fresh produce and its ability to grow at 4 degrees C gives rise to further concern over public health risks which may be associated with consumption of these foods. A brief overview of the behavior of Aeromonas species at refrigeration temperatures is presented.

Influence of organic acids on heat resistance characteristics of Talaromyces flavus ascospores.

Three strains of Talaromyces flavus were investigated for their tolerance to organic acids during and after exposure to elevated temperatures. Fumaric, sorbic and benzoic acids were clearly more lethal than acetic, malic, citric and tartaric acids, and lethality was enhanced as the pH of the heating medium was reduced from 5.0 to 2.5. The effects of sorbic and benzoic acids on viability of ascospores varied depending upon the strain and were influenced by other constituents in the heating medium. Ascospores of a T. flavus strain with greater heat tolerance were larger and less ellipsoid in shape than ascospores of a less heat-tolerant strain, as observed by scanning electron microscopy. While both strains are known to develop heat resistance with age, no external differences in shape, degree of ornamentation and size could be discerned between ascospores as influenced by age (11 and 51 day old) of cultures.

Selective media for detecting and enumerating foodborne yeasts.

No one medium is satisfactory for detecting, isolating and enumerating all yeasts in all foods. Antibiotic-supplemented media such as dichloran rose Bengal chloramphenicol agar, tryptone glucose yeast extract chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose Bengal chloramphenicol agar are superior to acidified potato dextrose agar and other acidified media for enumeration of the vast majority of spoilage yeasts. Dichloran glycerol (18%) agar performs well for enumerating moderately xerotolerant yeasts. Malt extract yeast extract glucose (up to 60%) can be used for detecting and enumerating moderate and extreme xerophiles. These media also support the growth of moulds. Lysine agar, Schwarz differential agar and Lin's wild yeast differential agar are used by the brewing industry to differentiate wild yeasts from brewer's strains. Lysine agar is selective for apiculate yeasts and ethanol sulfite yeast extract agar is selective for Saccharomyces. Both have application in wineries. Modified molybdate agar can be used to selectively isolate yeasts from tropical fruits. Preservative-resistant yeasts can be detected on malt acetic agar. The recommended incubation temperature is 25 degrees C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of yeasts to 10 days for more for xerotolerant yeasts. There is need for new and improved media for selectively isolating various groups, genera, species and strains of yeasts capable of growing only under specific environmental conditions in specific types of foods and beverages.

Media for detecting and enumerating yeasts and moulds.

Dilution plating techniques are designed to determine populations of viable fungal propagules per unit weight or volume of food. Direct plating techniques, on the other hand, are designed to assess the internal mycoflora of individual pieces of foods, e.g., seeds or dried fruits, and results are expressed as a percentage of infected pieces. Both techniques are used by industry and regulatory agencies to monitor levels of fungal contamination at various stages of food handling, storing, processing and marketing. Peptone (0.1%) water is commonly used as a diluent for samples to be homogenized or blended. Buffered diluents containing up to 30% glucose or 60% sucrose are recommended for enumerating xerophiles. No one medium is satisfactory for detection or enumeration of yeasts and moulds in all foods. Dichloran rose bengal chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose bengal chloramphenicol agar are superior to acidified potato dextrose agar for enumeration of yeasts and moulds. Dichloran 18% glycerol agar performs well for enumerating moderately xerophilic yeasts and moulds. Fastidious xerophiles require media containing high concentrations of sugars and/or sodium chloride. Media have been formulated to detect potentially aflatoxigenic aspergilli and mycotoxigenic strains of penicillia and fusaria, but increased selectivity and specificity of media for detecting mycotoxigenic moulds are needed. Heat-resistant mould ascospores often require heat treatment prior to plating in order to activate the germination process. The spread-plate technique is strongly preferred over the pour-plate technique for enumerating yeasts and moulds. The recommended incubation temperature is 25 degrees C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of mycoflora to 4 weeks or more for fastidious xerophiles. There is a need for new and improved media for selectively isolating various groups, genera, species and/or strains of fungi capable of growing only under specific environmental conditions, e.g., low aw or, in the case of sublethally injured cells, under conditions which facilitate resuscitation. Improved media are needed which accurately detect moulds producing specific mycotoxins in a wide range of food types.

Comparison of chemical treatments to kill Salmonella on alfalfa seeds destined for sprout production.

Outbreaks of salmonellosis in the US, Canada and Finland linked to alfalfa sprouts have been attributed to Salmonella stanley in 1995 and Salmonella newport in 1996. This study was undertaken to compare the efficacy of chemical treatments in killing a mixture of five Salmonella serovars inoculated onto alfalfa seeds. Solutions containing calcium hypochlorite or sodium hypochlorite at concentrations of 1800 and 2000 micrograms/ml active (available) chlorine respectively, as well as 6% hydrogen peroxide or 80% ethanol were effective in reducing Salmonella populations by more than 1000 fold. However, viable Salmonella cells were detected in seeds treated for 10 min in these solutions. The inaccessibility of Salmonella cells in crevices and between the cotyledon and testa of seeds to lethal concentrations of these chemicals are thought to be the reason for the lack of effectiveness.

Efficacy of a lactic acid/sodium benzoate wash solution in reducing bacterial contamination of raw chicken.

Raw chicken wings inoculated with Salmonella, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, or Escherichia coli O157:H7 were washed in water (control) or a solution of a 0.5% lactic acid/0.05% sodium benzoate (LB) (pH 2.64) for 30 min. Viable cells of pathogenic bacteria and naturally occurring psychrotrophic bacteria on wings were enumerated after 0, 2, 4, 6, and 8 days of storage at 4 degrees C. Lower populations of pathogenic and psychrotrophic bacteria were detected on wings immediately after washing with LB compared to populations detected on control wings. LB solution was more effective in killing Salmonella, C. jejuni, and E. coli O157:H7 than L. monocytogenes, and S. aureus. During refrigerated storage, populations of Salmonella, C. jejuni, L. monocytogenes, and E. coli O157:H7 decreased significantly on LB-washed wings, as compared to populations of respective pathogens on control wings. The growth of psychrotrophic bacteria on LB-washed wings was significantly retarded as compared to growth on control wings during refrigerated storage. Washing chicken wings with a solution containing 0.5% lactic acid and 0.05% sodium benzoate can greatly reduce the populations of pathogenic and psychotrophic bacteria, thus enhancing safety and extending shelf life.

Changes in chemical composition and sensory qualities of peanut milk fermented with lactic acid bacteria.

The effects of fermentation of aqueous extracts of peanuts (peanut milk) with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus, separately and in combination, on selected chemical and sensory qualities were investigated. Changes in pH, titratable acidity and viable cell populations indicated that there was a synergistic interaction between L. delbrueckii ssp. bulgaricus and S. salivarius ssp. thermophilus during fermentation. Analysis of headspace volatiles revealed that hexanal, which is one of the compounds responsible for undesirable green/beany flavor in peanut milk, completely disappeared as a result of fermentation. S. salivarius ssp. thermophilus was more effective than L. delbrueckii ssp. bulgaricus in reducing the hexanal content. The acetaldehyde content of peanut milk increased during fermentation. Changes in concentrations of these volatile compounds were correlated with sensory evaluation scores which showed that a significant (P less than or equal to 0.05) decrease in green/beany flavor and a significant increase in creamy flavor occurred as a result of fermentation.

Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods.

Many methods and media currently exist for the detection and enumeration of Listeria monocytogenes. However, the suitability of any specific method or media is influenced by the purpose of the analysis and the type of food being analyzed. Food which are likely to contain high populations of contaminating microorganisms require highly selective media for enumeration. Moreover, media which contain indicator systems are also helpful in distinguishing L. monocytogenes colonies. In contrast, less selective media may be adequate for less contaminated foods.

Evaluation of diluents and media for enumerating Zygosaccharomyces rouxii in blueberry syrup.

Combinations of diluents and enumeration media were evaluated for their efficacy in enumerating Zygosaccharomyces rouxii in blueberry syrup (aw 0.818, 0.870 and 0.921). Diluents consisted of deionized water containing 0.1% peptone, 20%, 30%, 40% or 50% glucose, 50% glucose plus 0.05% Tween 80, and 12%, 18%, 26% or 35% glycerol, all calculated on a w/w basis. Enumeration media were dichloran rose bengal chloramphenicol (DRBC) agar, tryptone glucose yeast extract (TGY) agar, dichloran 18% glycerol (DG18) agar, plate count agar containing 52% sucrose (PCA52S) and malt extract yeast extract 50% glucose (MY50G) agar. Two test strains of Z. rouxii grown in blueberry syrup for 7 or 14 days responded similarly to diluent/enumeration medium combinations. The use of 0.1% peptone diluent or DRBC agar in combination with other enumeration media or diluents was inferior for recovering Z. rouxii. As the aw of the blueberry syrup decreased, the sensitivity of Z. rouxii to diluent containing decreased concentrations of glucose increased. The use of glycerol as a solute in diluent did not result in a similar trend, indicating that the protective effect of glycerol against osmotic stress to Z. rouxii cells is greater than that of glucose at high aw and that cells adapted to low aw in blueberry syrup are more sensitive to high aw in diluents. The addition of 0.05% Tween 80 to 50% glucose diluent did not influence performance. Overall, diluents containing 50% glucose (aw 0.898), 18% glycerol (aw 0.956) or 26% glycerol (0.934), in combination with TGY agar (aw 0.982), resulted in the highest recovery of Z. rouxii from blueberry syrup at aw 0.818 to 0.921.

Enrichment in Fraser broth supplemented with catalase or Oxyrase, combined with the microcolony immunoblot technique, for detecting heat-injured Listeria monocytogenes in foods.

The microcolony immunoblot technique using monoclonal antibodies to Listeria monocytogenes was evaluated for its suitability to detect heat-injured cells. Pasteurized milk and filtrates of homogenized raw ground beef slurry and cabbage were inoculated with L. monocytogenes Scott A, heated, diluted, inoculated into Fraser broth (FB) supplemented with 400 micrograms of catalase ml-1 or 0.01 unit of Oxyrase ml-1, and incubated at 30 degrees C for 6 h. Three inoculum populations (high, medium, and low) were used. The extent of injury was dependent on the heating menstruum. Forty percent of the cells were injured in beef slurry filtrate, whereas 79 and 94% were injured in milk and cabbage filtrate, respectively, when foods were heated at 52 degrees C for 20 min. Populations of viable cells were determined using the immunoblot technique and by surface plating on modified Oxford (mMOX) agar. Recovery of cells from heated foods was enhanced in FB supplemented with catalase or Oxyrase compared to recovery in control broth. Essentially all unheated (control) cells could be detected within about 30 h using enrichment and the immunoblot technique; 54 h were required to easily detect colonies on mMOX. In most cases, the number of cells detected in heated milk or filtrates of homogenized beef after enrichment in FB supplemented with catalase or Oxyrase was significantly higher than populations detected using unsupplemented FB; however, enrichment in FB supplemented with catalase or Oxyrase did not significantly increase cell populations in heated cabbage filtrate. Within each heat treatment and level of inoculum, cell populations detected on mMOX agar after incubating plates for 48 h or on immunoblots after 24 h were not significantly different. Results indicate that the immunoblot technique in conjunction with enrichment in FB containing either catalase or Oxyrase can be successfully used to detect healthy and heat-injured cells of L. monocytogenes in diverse types of foods within 34 h.

Evaluation of simplified and commercial systems for identification of foodborne yeasts.

Commercial identification kits (API 20C, API Yeast-Ident and API-Zym) were compared with a conventional but simplified identification method (SIM) for identifying seventy-two yeast isolates from fresh sweet corn. SIM failed to provide identification of two isolates. Of the twenty species identified, only eleven were included in the API 20C profile index. Three isolates were identified at the species level and three were identified at the genus level with 100% accuracy. The enzyme kit (Yeast-Ident) gave rather unreliable results, in that identification of only four isolates with 75 to 85% of appropriate reactions was made. The API 20C kit could be used to identify non-clinical yeasts, provided they were included in its database and a few additional tests (urease reaction, nitrate assimilation and glucose fermentation) were also performed.

Comparison of dilution schemes for enumerating fungi in ground pepper: a collaborative study.

Ten lots of ground pepper were analyzed for fungal populations using 1:5 and 1:10 dilutions in sterile 0.1% peptone. Duplicate samples (0.1 ml) diluted 1:5 were surface plated on dichloran rose bengal chloramphenicol (DRBC) agar whereas quadruplicate samples (0.1 ml) diluted 1:10 were surface plated on DRBC agar. Nine collaborators from six countries participated in the study. Significantly (p < 0.05) higher fungal populations were detected in 7 of the 10 lots of pepper diluted using the 1:5 dilution scheme; the other three showed no significant difference. Using 1:5 and 1:10 dilution schemes, among-laboratory variability in mean fungal populations detected was not significant (p < 0.05) for 7 of 10 and 2 of 10 lots of peppers, respectively. Coefficients of variation for reproducibility (among-laboratory variation) were 2.8% and 6.2%, respectively, using 1:5 and 1:10 dilution schemes. Analysis of collapsed data from all ten lots of pepper revealed that significantly higher populations of fungi were detected using a 1:5 dilution scheme.