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.
Xylose lysine deoxycholate agar
Manufactured by Hopebio
Sourced in China
Xylose lysine deoxycholate agar is a selective and differential culture medium used for the isolation and identification of Salmonella and Shigella species in clinical and food samples. It contains xylose, lysine, and deoxycholate as the key components that inhibit the growth of Gram-positive bacteria and most Gram-negative bacteria, while allowing the growth of Salmonella and Shigella.
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6 protocols using xylose lysine deoxycholate agar
1
Microbiological Analysis of Lettuce Samples
2
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.
3
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.
4
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.
5
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.
6
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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