Cryptosporidium parvum studies with dairy products

Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice-cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation-based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air-drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at -20 degrees C. Although approximately 20% (25 and 18%) of oocysts were viable before hardening, none were viable after 24 h at -20 degrees C. Control samples of oocysts suspended in distilled water and stored at -20 degrees C were taken at the same time intervals and 8% of the oocysts were still viable after 24 h.     

Optimization of DNA extraction and molecular detection of Cryptosporidium oocysts in natural mineral water sources

The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65 degrees C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65 degrees C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.     

Behavior of enteroaggregative Escherichia coli in bottled spring and mineral water

The ability of enteroaggregative Escherichia coli (EAEC) to survive in bottled mineral and spring water at common storage temperatures was investigated. Filtered mineral and spring waters were inoculated with EAEC (ca. 10(4) CFU/ml) and stored at 4, 10, and 23 degrees C. Water samples were analyzed every 3 days for viable EAEC by plating on tryptic soy agar plates over 60 days of storage. EAEC survived for the duration of the study in both mineral and spring waters. EAEC survival levels were significantly higher (P < 0.01) at 23 and 10 degrees C than at 4 degrees C. Furthermore, EAEC survival levels were significantly higher (P < 0.01) in mineral water than in spring water at 4 and 10 degrees C. The results of this study indicate that EAEC can survive in bottled mineral and spring waters for long periods of storage at 4, 10, and 23 degrees C. The ability of EAEC to survive in bottled water indicates that the source water for bottling industries must be kept free of contamination. Furthermore, the refrigeration of bottled water is recommended to minimize the growth of EAEC in water.     

Inactivation of Cryptosporidium parvum oocysts in cider by flash pasteurization

Cryptosporidium parvum is a well-recognized pathogen of significant medical importance, and cider (apple juice) has been associated with foodborne cryptosporidiosis. This study investigated the effect of flash pasteurization on the viability of contaminant C. parvum oocysts. Cider inoculated with oocysts was heated at 70 or 71.7 degrees C for 5, 10, or 20 s, and oocyst viability was measured by a semiquantitative in vitro infectivity assay. By infecting multiple wells of confluent Madin-Darby bovine kidney cells with serial dilutions of heat-treated oocysts and examining infected cells by indirect fluorescent antibody staining, the most probable number technique was applied to quantify log reduction of oocyst viability. Heating for 10 or 20 s at either temperature caused oocyst killing of at least 4.9 log (or 99.999%), whereas oocyst inactivation after pasteurization for 5 s at 70 and 71.7 degrees C was 3.0 log (99.9%) and 4.8 log (99.998%), respectively. Our results suggested that current practices of flash pasteurization in the juice industry are sufficient in inactivating contaminant oocysts.     

Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.     

Microwave inactivation of Cyclospora cayetanensis sporulation and viability of Cryptosporidium parvum oocysts

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23 degrees C for 2 weeks, and sporulation rates were then determined. The 4',6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80 degrees C or higher were reached in the microwave ovens.     

THE POTENTIAL FOR CRYPTOSPORIDIUM PARVUM OOCYST SURVIVAL IN BEVERAGES ASSOCIATED WITH CONTAMINATED TAP WATER

Cryptosporidium parvum is an enteric coccidian protozoan which produces an environmentally stable oocyst that is excreted in the feces of infected individuals. There have been ten documented water borne outbreaks in North America. If food or beverages were prepared from contaminated water, that food or beverage would also be a hazard. The objective of this study was to evaluate the survival of Cryptosporidium parvum in beverages. Viability of oocysts, as determined by morphology decreased over 24 h exposure in carbonated beverages. Uptake of vital dyes indicated a loss of >85% of oocyst viability in beer or cola stored at 4C. Loss of viability in tap water, orange juice or infant formula was ± 35%. It is likely that the low pH of the carbonated beverages was involved in the loss of oocyst viability and premature excystation of the sporozoites .     

Phylogenetic analysis implicates birds as a source of Cryptosporidium spp. oocysts in agricultural watersheds

Cryptosporidium parvum and C. hominis are protozoan parasites responsible for cryptosporidiosis, an acute gastrointestinal illness that can be life-threatening for immunocompromised persons. Sources and genotypes of Cryptosporidium oocysts were investigated in two agricultural areas within the Wachusett Reservoir watershed, a drinking water source for Boston, Massachusetts. Two brooks (denoted Brook SF and Brook JF, respectively), each downgradient from a dairy farm, were chosen as sample sites. For one year, Brooks SF and JF were sampled monthly; oocysts were detected in 6 (50%) out of 12 samples from Brook JF, and no oocysts were detected in Brook SF. Oocyst genotypes from agricultural surface waters were compared to oocyst genotypes from Genbank, as well as fecal samples of cattle and birds, using phylogenetic analysis of a hypervariable region of the 18S rRNA gene by both neighbor-joining and parsimony methods. Results show extensive heterogeneity among Cryptosporidium spp. 18S rRNA sequences, and also suggest that birds are an oocyst source in this watershed. Principal components analysis showed oocyst presence correlating strongly with seasonal factors, and oocysts in surface waters were only detected in the summer through late fall, co-incident with the presence of migratory birds in this watershed. If birds are confirmed to be an important source of oocysts infectious to humans, the data suggest that protection of raw drinking water supplies in some agricultural areas may depend upon management and control of resident and migratory bird populations.     

Effect of wine phenolic compounds on Lactobacillus hilgardii 5w viability.

After 6 days of Lactobacillus hilgardii 5w incubation at 4 degrees C, the viable cell counts diminish 31.9, 45.6, and 89.0% when suspended in control wine (2,600 mg/liter gallic acid equivalents [GAE]), three-fold concentrated wine (6,150 mg/liter GAE), and six-fold concentrated wine (13,000 mg/liter GAE), respectively. At 20 degrees C in the same conditions, the cell viabilities decrease 74.2, 80.5, and 100.0%, respectively. In decolorized wines, which result in tannin losses, the viable cell counts increase. There is a relationship between L. hilgardii 5w tannin binding and its viability loss.     

Effect of a commercial freeze/tempering process on the viability of Cryptosporidium parvum oocysts on lean and fat beef trimmings

Lean and fat beef trimmings (25 cm⁻²) were inoculated with approximately 250,000 Cryptosporidium parvum oocysts, placed in commercial packages (28 kg boxes) and subjected to normal commercial processes i.e. blast frozen (to −20 °C within 60 h), stored (−20 °C, 21 days), tempered (48 h at −3 °C), and held at 0 °C for 10 h. Inoculated areas were then excised, pulsified (30 s in 50 ml PBST), and centrifuged (2500×g, 15 min). The resultant pellet was resuspended in 10 ml water and subjected to immunomagnetic separation and viability dye assay. Following the commercial freeze/tempering process the viability of the oocysts had decreased from 90.6% viable in the working stock suspension to 7.17% and 9.46% viable on lean and fat trimmings, respectively. The results of this study indicate that if C. parvum oocysts were present on beef trimmings their viability would be substantially reduced as a result of the freeze/tempering process.     

Behavior of aeromonas hydrophila in bottled mineral waters.

The growth and survival of Aeromonas hydrophila in three types of natural mineral waters were investigated. Mineral waters with different levels of mineral content (low, medium, and high) were experimentally contaminated with A. hydrophila, stored at different temperatures (10 degrees C and 20 degrees C), and analyzed at intervals over a 60-day period. Water samples that were not experimentally contaminated were investigated for indigenous A. hydrophila. The results confirmed that A. hydrophila may occur naturally in mineral waters and showed that the level of mineral content, temperature, length of storage, and, in some cases, the type of container used may favor the growth of A. hydrophila. The greatest proliferation was observed in water with a low mineral content stored in PET bottles at 10 degrees C, in which A. hydrophila peaked at day 28 (4.47 +/- 0.01 log CFU/100 ml). At 20 degrees C, the same load was observed at day 60. The presence of high densities of A. hydrophila in bottled mineral water can constitute a risk for some groups of consumers, such as elderly and immunocompromised persons.     

Effect of vinegar on the viability of Giardia duodenalis cysts

The inactivation of Giardia duodenalis cysts by vinegar was investigated. Experiments were carried out in 100 ml volume of vinegar (acetic acid 4%), undiluted or diluted in distilled water in ratios of 1:1, 1:15.6, and 1:62.5 (vol/vol), which were inoculated with 5x10(5) cysts obtained from human feces. Experiments were performed at room temperature (21+/-1 degrees C) and at 4 degrees C. After contact times of 1.5 min, 10, 30, and 60 min, the cysts were recovered from the treatment fluid and subjected to an in vitro excystation assay to determine their viability. The relative viability, which was calculated in relation to controls (maximum excystation percentage), was significantly affected (p<0.1) by the vinegar concentration, contact time, and temperature. At 21+/-1 degrees C, no cysts remained viable after being treated with undiluted vinegar for 60 min, while the treatment with 1:1, 1:15.6, and 1:62.5 vinegar-water mixtures decreased the relative viability to 1.8%, 19.4%, and 56.4%, respectively. The relative viability after corresponding treatments at 4 degrees C also decreased, but 23.6% to 48.8% remained viable after 60 min, and thus complete inactivation was not obtained with any treatment at that temperature.     

Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.     

Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157: H7 for 24+/-1 h at 4 degrees C. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degrees C. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3+/-16.2%) that had penetrated 30 to 40 microm from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2+/-15.8% survival), while cells that penetrated 0 to 10 microm from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/-13.5 and 45.6+/-9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.     

High hydrostatic pressure and UV light treatment of produce contaminated with Eimeria acervulina as a Cyclospora cayetanensis surrogate

The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (10(6) oocysts) and low (10(4) oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40 degrees C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (10(4) oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (10(4) oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.     

Survival of bifidobacteria in yogurt and simulated gastric juice following immobilization in gellan-xanthan beads

A novel acid-stable bead made of gellan gum and xanthan gum was used to immobilize Bifidobacteria. The beads (0.75% gellan and 1% xanthan gum) had an average diameter of 3 mm and did not shrink in 25% lactic acid solution, pH 1.5, or 20% acetic acid solution, pH 1.5, after storage at 4 degrees C for 4 weeks. Bifidobacterium infantis ATCC 15697, the most acid-tolerant strain tested, was immobilized in gellan-xanthan beads and its survival in peptone water, pH 4, pasteurized yogurt, and simulated gastric juice was monitored. In peptone water, pH 4, the reduction in cell count of immobilized cells of B. infantis ATCC 15697 was not significantly different from that obtained with free cells during 6 weeks of storage at 4 degrees C. However, counts of immobilized cells of B. infantis ATCC 15697 remained significantly higher than free cells (P < 0.0001) when both were exposed to simulated gastric juices at pH 2.5, 2.0 and 1.5. At pH 2.5, the viable count of free cells dropped from 1.23 x 10(9) CFU/ml to an undetectable level (< 10 CFU/ml) in 30 min, while the viable count of immobilized cells decreased by only 0.67 log cycle in the same time period. Immobilized cells also survived significantly better than free cells (P <0.05) in pasteurized yogurt after refrigerated storage for 5 weeks.     

Effect of chlorine, blanching, freezing, and microwave heating on Cryptosporidium parvum viability inoculated on green peppers

Cryptosporidium parvum oocysts have been found on the surface of vegetables in both developed and developing countries. C. parvum can contaminate vegetables via various routes, including irrigation water. This study investigated the effect of individual treatments of chlorine, blanching, blast freezing, and microwave heating, as well as combined treatments of chlorine and freezing, and chlorine and microwave heating on the viability of C. parvum oocysts inoculated on green peppers. The viability of the oocysts after the treatments was assessed using propidium iodide and a flow cytometer. Based on the propidium iodide staining, the chlorine treatments did not affect the viability of the oocysts. Blast freezing significantly inactivated 20% of the oocysts. Microwave heating and blanching significantly inactivated 93% of oocysts. Treatment with chlorine followed by blast freezing did not affect the viability of the oocysts significantly. Treatment with chlorine and microwave heating was significantly more effective than microwave heating alone and inactivated 98% of the oocysts. The study indicates that C. parvum oocysts are sensitive to heat and, to some extent, to blast freezing, but are resistant to chlorine. Therefore, the use of chlorine during vegetable processing is not a critical control point for C. parvum oocysts, and the consumption of raw or minimally processed vegetables may constitute a health risk as C. parvum oocysts can still be found viable on ready-to-eat, minimally processed vegetables.     

Inactivation by ultrahigh-pressure homogenization of Escherichia coli strains inoculated into orange juice

The aim of this work was to evaluate the efficacy of ultrahigh-pressure homogenization (UHPH) for inactivation and/or sublethal injury of two strains of Escherichia coli (O58:H21 ATCC 10536 and O157:H7 CCUG 44857) inoculated into orange juice (pH 3.6). The effects of orange juice inlet temperature (6 and 20 degrees C) on the lethality values and the capacity of these strains for survival, repair, and growth during refrigerated storage after UHPH treatment also was evaluated. Samples of orange juice that had been treated with ultrahigh temperatures were inoculated with E. coli in the stationary phase of growth until a final concentration of approximately 7.0 log CFU/ml was reached. These samples were then treated for one cycle with a double-valve UHPH machine, with 300 MPa at the primary homogenizing valve and 30 MPa at the secondary valve. Counts of viable and injured bacterial cells were obtained for samples taken 2 h after UHPH treatment and after 3, 6, 9, 12, 15, 18, 21, 27, and 33 days of storage at 4 degrees C. The inlet temperature and the strain type both influenced significantly (P < 0.05) the lethality effect on E. coli, which was higher when the inlet temperature was 20 degrees C. No sublethal injuries were detected after any treatment. The changes in viable counts over time for both strains in pressurized and control samples were similar. The viable counts remained high from day 0 to day 18 and then tended to decrease. After 27 days of storage at 4 degrees C, E. coli O157: H7 was more resistant in orange juice samples pressurized at inlet temperatures of 6 and 20 degrees C, with viable counts of 3.41 and 3.20 log CFU/ml, respectively.     

Comparative evaluation of yogurt and low-fat cheddar cheese as delivery media for probiotic Lactobacillus casei

This study used Lactobacillus casei 334e, an erythromycin-resistant derivative of ATCC 334, as a model to evaluate viability and acid resistance of probiotic L. casei in low-fat Cheddar cheese and yogurt. Cheese and yogurt were made by standard methods and the probiotic L. casei adjunct was added at approximately 10(7) CFU/g with the starter cultures. Low-fat cheese and yogurt samples were stored at 8 and 2 degrees C, respectively, and numbers of the L. casei adjunct were periodically determined by plating on MRS agar that contained 5 microg/mL of erythromycin. L. casei 334e counts in cheese and yogurt remained at 10(7) CFU/g over 3 mo and 3 wk, respectively, indicating good survival in both products. Acid challenge studies in 8.7 mM phosphoric acid (pH 2) at 37 degrees C showed numbers of L. casei 334e in yogurt dropped from 10(7) CFU/g to less than 10(1) CFU/g after 30 min, while counts in cheese samples dropped from 10(7) CFU/g to about 10(5) after 30 min, and remained near 10(4) CFU/g after 120 min. As a whole, these data showed that low-fat Cheddar cheese is a viable delivery food for probiotic L. casei because it allowed for good survival during storage and helped protect cells against the very low pH that will be encountered during stomach transit.     

Survival and growth of Listeria monocytogenes and Escherichia coli O157:H7 in ready-to-eat iceberg lettuce washed in warm chlorinated water

Cut iceberg lettuce inoculated with Escherichia coli O157:H7 and Listeria monocytogenes before and after washing for 3 min in cold (4 degrees C) and warm (47 degrees C) water containing 100 mg/liter total chlorine was stored at I and 10 degrees C in oxygen-permeable film packages (6,000 to 8,000 cc/m2/24 h). Cold chlorinated water was detrimental to the survival of E. coli O157: H7 and L. monocytogenes at both storage temperatures. In contrast, washing in warm chlorinated water favored the growth of both pathogens in lettuce stored at 10 degrees C. There was no evidence of a relationship between the magnitude of spoilage microflora and the fate of either bacterium.