Each 10 g lettuce sample was transferred to a stomacher bag containing 150 mL sterilized 0.85% NaCl solution and homogenized for 90 s. A bacterial suspension series (0.1 mL) was prepared and surface-plated on modified sorbitol MacConkey agar (Hopebio), Listeria chromogenic agar (Land Bridge, Beijing, China), eosin methylene blue agar (Hopebio), and xylose lysine deoxycholate agar (Hopebio) to analyze E. coli O157:H7, L. monocytogenes, non-O157 E. coli, and Salmonella typhimurium, respectively. All plates were incubated for 24 h at 37 °C. To detect naturally present microbes, a 1 mL bacterial suspension was pour-plated onto plate count agar and incubated at 37 °C for 2 days to obtain the AMC and at 7 °C for 10 days to obtain the APC. In addition, 0.1 mL of the diluted bacterial suspension was surface-plated on Rose Bengal agar (Hopebio) and incubated at 30 °C for 3 days to quantify M&Y.
Modified sorbitol macconkey agar
Manufactured by Hopebio
Sourced in China
Modified sorbitol MacConkey agar is a microbiological culture medium used for the selective isolation and differentiation of Escherichia coli O157:H7 from other Gram-negative bacteria. It contains sorbitol as the primary carbohydrate source instead of lactose, which helps identify E. coli O157:H7 strains that are unable to ferment sorbitol.
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7 protocols using modified sorbitol macconkey agar
1
Microbiological Analysis of Lettuce Samples
2
Sanitizing E. coli O157:H7 with Organic Acids
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A single colony of E. coli O157:H7 (NCTC12900) was inoculated into nutrient broth (Hopebio, Qingdao, China) and cultured overnight at 37 °C. After adjusting the culture to 107–108 CFU/mL, 5 mL was centrifuged at 12,000× g for 10 min to obtain the cell pellet, followed by three washing steps using 0.85% NaCl solution. Then, the cells were resuspended in 1 mL of sterilized distilled water and supplemented with 4 mL of sanitizer to obtain the desired sanitizer concentration. The treatment groups were treated with 0.8%LA+0.2%AA, 0.6%LA+0.4%AA, 1%LA, and 1% AA, and the control group was treated with sterilized distilled water. After reaction for 0, 20, 40, 60, and 90 s, 1 mL of the above mixture was mixed with 5 mL of 0.04 M K2HPO4·3H2O to neutralize the sanitizer [21 ].
After serial dilution, the suspension (0.1 mL) was surface-plated on modified sorbitol MacConkey agar (Hopebio, Qingdao, China) for the quantification of E. coli O157:H7.
After serial dilution, the suspension (0.1 mL) was surface-plated on modified sorbitol MacConkey agar (Hopebio, Qingdao, China) for the quantification of E. coli O157:H7.
3
Microbiological Analysis of Food Samples
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Microbiological analysis was performed on days 0, 3, and 7. A sample (15 g) was placed in a stomacher bag containing 135 mL of 0.85 % NaCl and homogenized for 2 min, followed by serial dilution. Then, 0.1 mL of the diluted bacterial suspension was surface-plated on modified sorbitol MacConkey agar (Hopebio, Qingdao, China) or xylose lysine deoxycholate agar (Hopebio) and incubated at 37 °C for 24 h to count E. coli O157:H7 and Salmonella Typhimurium, respectively. For naturally present microbes, 0.1 mL of suspension was surface-plated on Rose Bengal agar (Hopebio) and incubated at 30 °C for 3 days to quantify molds and yeasts (M&Y). In addition, 1 mL of the suspension was pour-plated onto plate count agar (Hopebio) and incubated at 37 °C for 2 days to obtain the aerobic mesophilic count (AMC). All results are expressed as log CFU/g.
4
Bacterial Inoculation of Jujubes
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E. coli O157:H7 (NCTC12900), a non-toxic strain that was previously used in fresh produce inoculation experiments [19] (link), [20] (link), [21] (link), was selected in this experiment. Non-O157 E. coli (ATCC25922) and Salmonella Typhimurium (ATCC14028), two quality control strains recommended by the FDA for food safety testing [22] , [23] , were selected as well. The inoculation experiment was performed according to our previous study [6] , with minor modifications. Pure cultures of E. coli O157:H7, non-O157 E. coli, and Salmonella Typhimurium stored in 50% glycerol were cultured on modified sorbitol MacConkey agar (Hopebio, Qingdao, China), eosin methylene blue agar (Hopebio), and xylose lysine deoxycholate agar (Hopebio), respectively. After incubation for 24 h at 37 °C, one bacterial colony was cultured in nutrient broth (Hopebio) overnight at 37 °C, and the cell density of the suspension was adjusted to 109 colony forming units (CFU)/mL. The adjusted suspension (6.5 mL) was added to a stomacher bag containing sterilized 0.85% NaCl (200 mL) and 10 jujubes and massaged for 20 min. After air drying in a biological safety cabinet, infected samples were placed at 4 °C for 24 h. The cell counts of the pathogen on the sample were 105–106 CFU/g.
5
Microbial Analysis of Food Samples
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Samples were analyzed at 0, 3, and 7 days. A 25-g sample was homogenized with 225 mL sterile NaCl solution for 1.5 min in a stomacher bag. Then, the suspension was serially diluted. The suspension (0.1 mL) was surface-plated on modified sorbitol MacConkey agar (Hopebio), Listeria chromogenic agar (Land Bridge, Beijing, China), and xylose lysine deoxycholate agar (Hopebio) to analyze E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium, respectively, and incubated for 24 h at 37 °C. For naturally present microbes, 0.1 mL of the diluted bacterial suspension was surface-plated on Rose Bengal agar (Hopebio) and incubated at 30 °C for 3 days to quantify molds and yeasts (M&Y). In addition, 1 mL of the suspension was pour-plated onto plate count agar (Hopebio) and incubated at 7 °C for 10 days to obtain the aerobic psychrotrophic count (APC) and or at 37 °C for 2 days to obtain the aerobic mesophilic count (AMC). All results are expressed as log CFU/g.
6
Rapid Enumeration of E. coli and Salmonella
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Eight samples were randomly selected from the package, transferred to a sterile stomacher bag, diluted 1:9 (w/v) in sterile 0.85% NaCl solution, and homogenized in a stomacher for 2 min. Then, 1 mL of the diluted bacterial suspension was spread-plated on modified sorbitol MacConkey agar (Hopebio) and xylose lysine deoxycholate agar (Hopebio) and incubated for 24 h at 37 °C to analyze E. coli O157:H7 and S. Typhimurium, respectively. For naturally-present microbes, 1 mL of the bacterial suspension was pour-plated in plate count agar (Hopebio) and incubated at 37 °C for 2 d to obtain the AMC, and 1 mL was pour-plated in rose bengal agar (Hopebio) and incubated at 28 °C for 5 days to quantify the M&Y.
7
Enumeration of Foodborne Pathogens and Spoilage Microbes
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Ten blueberries and 0.85% sterile NaCl solution at a ratio of 1:9 (w/v) were added to a stomacher bag and homogenized for 2 mins under 250 rpm. The bacterial suspensions were then serially diluted. The diluted suspension was surface-plated on modified sorbitol MacConkey agar (Hopebio) and xylose lysine deoxycholate agar (Hopebio) and incubated for 24 h at 37 °C to analyze E. coli O157:H7 and S. Typhimurium, respectively. For naturally present microbes, 1 mL of suspension was pour-plated on plate count agar (Hopebio) and incubated 48 h at 37 °C to analyze aerobic mesophilic counts (AMC). In addition, 1 mL suspension was pour-plated on Rose Bengal agar (Hopebio) and incubated for 5 days at 28 °C to quantify the amount of molds and yeast (M&Y).
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