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.
Eosin methylene blue agar
Manufactured by Hopebio
Sourced in China
Eosin methylene blue agar is a selective and differential culture medium used for the isolation and identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It contains eosin Y and methylene blue as dyes, which inhibit the growth of Gram-positive bacteria and differentiate between lactose-fermenting and non-lactose-fermenting Gram-negative bacteria.
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4 protocols using eosin methylene blue agar
1
Microbiological Analysis of Lettuce Samples
2
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.
3
Isolation and Identification of E. coli from Swine
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During May 2015 to June 2017, 456 E. coli isolates (270 from healthy pigs, 186 from diarrheal pigs) were isolated from fecal samples of different swine in ten pig farms, which are widely dispersed across Shaanxi and Gansu provinces. Fecal samples were collected from individual pigs using a sterile cotton swab and transported to laboratory within 12 h. All samples were immediately seeded on MacConkey agar (Beijing Land Bridge Technology Co., Ltd, Beijing, China). After incubation at 37°C for 18 to 24 h, three colonies with typical E. coli morphology (bright pink with a dimple) were randomly selected and transferred to Eosin Methylene Blue agar (Qingdao Hope Bio Technology Co., Ltd, Qingdao, Shandong, China) for further purification. Finally, the suspect E. coli isolates on Eosin Methylene Blue agar (green colonies with a metallic sheen) were subjected to biochemical tests (indole, methyl red, oxidase, citrate, and triple sugar iron) as described previously (Liu et al., 2017 (link)). All confirmed E. coli isolates were stored at −80°C in Tryptic Soy broth medium containing 30% glycerol for later study.
4
Isolating Salmonella and E. coli from Chicken
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Two swabs were taken from each chicken. One swab was emulsified in 2 mL of selenite cystine broth (SC) (Hopebio, Qingdao, China) and then further inoculated on chromogenic Salmonella agar (second generation) plates supplemented with 0.25 mg/L of ENR. Salmonella represented a typical purple single colony on the chromogenic Salmonella agar (second generation). The colonies were randomly screened for further amplification of qnrS and repA genes to identify SE211-qnrS strains. In the meantime, all the Salmonella and putative SE211-qnrS isolates were stored at –20°C. Another swab was weighed, which was emulsified in 1 mL of sterile 0.9% NaCl and then further diluted 105 times. An aliquot (50 μL) of the appropriate dilution was spread onto eosin-methylene blue agar (Hopebio, Qingdao, China) plates supplemented with ENR (32 mg/L). These plates were incubated at 37°C overnight to detect the colonization level of E. coli E2.
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