Three different bacterial strains were selected: Aeromonas hydrophila (ATCC 7966), enterotoxigenic Escherichia coli (ETEC, H10407 LT/ST O78:H11), and Vibrio parahaemolyticus (IMA635, derived from a 1994 outbreak Selleckchem Anti-diabetic Compound Library in Lima, Peru). An inoculum of each strain was prepared by culturing each separately in duplicate on tryptic soy agar (TSA) plates (lot 6228427, BDDIFCO) and incubating overnight at 37°C. Strains were harvested and suspended separately in phosphate-buffered saline (PBS; pH 7.2). Serial dilutions were obtained to give a final concentration of 1 × 108 CFU/mL. Bacterial growth was determined by measuring the absorbance at 600 nm (with optical densities of each 1/100 dilution between 0.106 and 0.111)

and by plate count on TSA. The infectious dose for each pathogenic strain was considered before obtaining the final inocula to confirm that each 450 g portion would contain sufficient bacteria to be potentially infectious if consumed. Prior to the addition of cebiche ingredients, we inoculated each fish filet sample (450 g) with a 50 mL bacterial suspension13 containing approximately 1 × 108 CFU/mL of each organism. The bacterial suspension remained in contact with the surface of the fish for 10 minutes at room temperature.14 Ten grams of portions Selleckchem LDK378 were then collected and blended for 2 minutes in an electric blender with 90 mL PBS. To determine the initial bacterial count in the fish,

100 µL aliquots of diluted homogenate were streaked in duplicate onto TSA, MacConkey agar (ETEC, A hydrophila), and TCBS agar (V parahaemolyticus) plates and incubated overnight at 37°C.

Before and after the addition of lime juice but prior to the addition of the remaining cebiche ingredients, baseline pH levels of the samples were determined by obtaining 10 g from the sample and blending it with 40 mL of distilled water. A typical Peruvian cebiche recipe was used combining 450 g of toyo (Mustelus whitney, Mustelus lunulatusi), a common fish found in all warm and temperate coastal seas with cilantro, garlic, hot peppers, sweet potatoes, and corn (all ingredients were obtained from a retail market) marinated together with lime juice for 10 and 30 minutes (which are typical marination times for Peruvian cebiche).15 After the 10- and 30-minute marination ASK1 periods, all ingredients were homogenized in a blender. A 10 g aliquot was transferred to a blender jar containing 90 mL of PBS and blended for 2 minutes, resulting in a 1 : 10 dilution. Serial dilutions of the original homogenate were prepared to 1 : 1,000, 1 : 10,000, and 1 : 100,000 concentrations in PBS. Hundred microliters of aliquots of each dilution were transferred using a pipette into separate and duplicate TSA, MacConkey agar (ETEC, A hydrophila), and TCBS agar (V parahaemolyticus) plates. Plates were incubated overnight at 37°C. The pH was then measured using an electronic pH meter as described previously.