The inoculation procedure was carried out according to our previous report [9] , [25] . Single colonies of E. coli O157:H7 (NCTC12900), L. monocytogenes (ATCC19115), and Salmonella Typhimurium (ATCC14028) were inoculated into nutrient broth (Hopebio, Qingdao, China) and shaken overnight at 37 °C. The bacterial suspension was adjusted to 109 CFU/mL, and 5 mL of this culture was then added to a stomacher bag containing 200 mL sterilized 0.85% NaCl solution. Then, 10 g of the lettuce sample was placed into the bag and massaged for 20 min. The sample was then placed on a sterilized plastic tray in a biosafety cabinet and air dried.
Nutrient broth
Manufactured by Hopebio
Sourced in China
Nutrient broth is a type of liquid growth medium used in microbiology and other laboratory settings to culture various microorganisms. It provides a balanced source of nutrients essential for the growth and proliferation of a wide range of bacterial, fungal, and other microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Modified sorbitol macconkey agar, by Hopebio (2 mentions)
Nutrient agar, by Hopebio (2 mentions)
Potato dextrose broth (pdb), by Beijing Land Bridge Technology (1 mentions)
Propidium iodide solution, by Merck Group (1 mentions)
Lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (1 mentions)
Fungal rna kit, by Omega Bio-Tek (1 mentions)
Superreal premix plus sybr green, by Tiangen Biotech (1 mentions)
Potato dextrose agar (pda), by Beijing Land Bridge Technology (1 mentions)
Fluorescein diacetate fda, by Solarbio (1 mentions)
Propidium iodide, by Solarbio (1 mentions)
Eosin methylene blue agar, by Hopebio (1 mentions)
Xylose lysine deoxycholate agar, by Hopebio (1 mentions)
Neutral resin, by Solarbio (1 mentions)
Paraffin, by Sinopharm (1 mentions)
Potato dextrose agar pda, by Hopebio (1 mentions)
Sabouraud dextrose broth (sdb), by Hopebio (1 mentions)
Mueller hinton agar (mha), by Hopebio (1 mentions)
Glacial acetic acid, by Sinopharm (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
14 protocols using nutrient broth
1
Bacterial Inoculation and Lettuce Sampling
2
Inoculation of Blueberries with E. coli O157:H7 and Salmonella Typhimurium
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Blueberry (Joyvio, Beijing, China) was purchased from a local market on the day of the experiment, and samples with no apparent rotting, wounds, and bruises with a weight of 3.5 ± 0.2 g were selected for the experiment. The sample was rinsed for 30 s under tap water to remove dirt.
E. coli O157:H7 (NCTC12900), a non-toxic strain previously used in fresh produce inoculation experiments [27] (link), [28] (link), was selected for this experiment. Salmonella Typhimurium (ATCC14028), a quality control strain recommended by the FDA for food safety testing [29] , was also selected. One colony strain was inoculated into the nutrient broth (Hopebio, Qingdao, China) and incubated overnight at 37 °C shaking at 150 rpm. After centrifuging at 4000×g for 10 min, the obtained cell pellet was washed with sterilized 0.85% NaCl three times and resuspended in sterilized distilled water to adjust the counts to ∼ 109 CFU/mL. Ten blueberries and 150 mL of inoculation solution were placed in a sterilized stomacher bag and manually massaged for 15 min. The inoculated samples were placed in a biosafety cabinet and air-dried for 3 h. The sample was placed under 4 °C for 12 h for bacterial adhesion, and an inoculated sample with 5.8 and 5.7 log CFU/g of E. coli O157:H7 and S. Typhimurium were obtained, respectively.
E. coli O157:H7 (NCTC12900), a non-toxic strain previously used in fresh produce inoculation experiments [27] (link), [28] (link), was selected for this experiment. Salmonella Typhimurium (ATCC14028), a quality control strain recommended by the FDA for food safety testing [29] , was also selected. One colony strain was inoculated into the nutrient broth (Hopebio, Qingdao, China) and incubated overnight at 37 °C shaking at 150 rpm. After centrifuging at 4000×g for 10 min, the obtained cell pellet was washed with sterilized 0.85% NaCl three times and resuspended in sterilized distilled water to adjust the counts to ∼ 109 CFU/mL. Ten blueberries and 150 mL of inoculation solution were placed in a sterilized stomacher bag and manually massaged for 15 min. The inoculated samples were placed in a biosafety cabinet and air-dried for 3 h. The sample was placed under 4 °C for 12 h for bacterial adhesion, and an inoculated sample with 5.8 and 5.7 log CFU/g of E. coli O157:H7 and S. Typhimurium were obtained, respectively.
3
Mycotoxin Detection in Food Samples
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Nutrient agar (NA) and nutrient broth (NB) were purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China). Potato dextrose agar (PDA) and potato dextrose broth (PDB) were provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). LC-MS grade acetonitrile and acetic acid were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Standard solutions of TeA, AOH, AME, and TEN were purchased from Romar Labs Division Holding GmbH (Getzersdorf, Austria). Propidium iodide (PI) solution was provided by Sigma-Aldrich, Co., Ltd. (Burlington, MA, USA). The Fungal RNA Kit was purchased from Omega Bio-tek, Inc. (Norcross, GA, USA). The FastKing Rt Kit (With gDNase) and SuperReal PreMix Plus (SYBR Green) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China).
4
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.
5
Staining and Fluorescence Analysis of Bacteria
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0.4 mL inoculum containing ~1.5 × 106 CFU/mL of either E. coli or S. aureus were incubated in nutrient broth (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China) and 7.5% sodium chloride broth (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China), respectively. E. coli and S. aureus were stained using 25 μg/mL propidium iodide (PI) (Solarbio Life Science, Beijing, China) for 20 min and 25 μg/mL fluorescein diacetate (FDA) (Solarbio Life Science, Beijing, China) for 20 min, respectively, at the ambient temperature (25 °C) prior to fluorescence analysis. Fluorescence analysis was carried out using Fluorescent microplate reader (FlexStation II, NanoDrop, USA). Control groups comprised bacteria incubated in broth only for 24 h at 37 °C.
6
Microbial Growth Media Optimization
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For the sediment, seawater, and marine plant roots, we used the following three media: (1) 1:2 diluted Marine Broth 2216 (50% of manufacturer's suggested concentration, Difco, Franklin Lakes, NJ, USA); (2) 1:10 diluted R2A Broth (10% of the manufacturer's suggested concentration, Hopebio, Qingdao, China); and (3) 1:100 diluted Nutrient Broth (1% of the manufacturer's suggested concentration, Hopebio, Qingdao, China). All media were supplemented with additional artificial sea salt (Instant Ocean, USA) to a final concentration of 2%. For the soil sample, we used one medium, 1:10 diluted R2A Broth (10% of the manufacturer's suggested concentration, Nihon Seiyaku, Japan).
7
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.
8
Antibacterial Activity of A. ordosica Essential Oil
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The antibacterial activity of the A. ordosica essential oil was evaluated using microorganism strains, including Staphylococcus aureus ATCC 6538 (Gram-positive bacteria), Escherichia coli ATCC 8739 (Gram-negative bacteria), and Salmonella abony NTCC 6017 (Gram-negative bacteria). The antibacterial assay was conducted by the broth microdilution method [37 (link)]. Colonies of the microbial strains were maintained on nutrient agar (Hopebio, Qingdao, China) at 37 °C in the incubator. Using a twofold dilution approach, emulsions were created with dimethyl sulfoxide (DMSO) for each essential oil sample to establish a concentration serial from 40 to 2.5 μL/mL in the final test. The MIC of each pathogen was determined using a test tube containing 3460 μL of nutrient broth (Hopebio, Qingdao, China). After adding the essential oil emulsion (500 μL) and broth, 40 μL of inoculum at a concentration of 3 × 109 CFU/mL was added to each test tube. In the bacterial growth control group, neither essential oil nor detergent was applied to the test tubes. Each tube was visually checked for the existence of turbidity after 18 h of incubation at 37 °C. The MIC value is the concentration at which observable microorganism growth is inhibited relative to the control group. All tests were conducted in triplicate under aerobic conditions.
9
Isolation and Characterization of L. rhamnosus Strains
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L. rhamnosus X253, which was isolated from fermented milk in Xinjiang, China, was provided by Junlebao Dairy Group (Shijiazhuang, China). L. rhamnosus GG was purchased from Chr. Hansen A/S (Hørsholm, Denmark). L. rhamnosus X253 and L. rhamnosus GG were grown in de Man, Rogosa, and Sharpe Broth (MRS; HopeBio, Qingdao, China) at 37 °C under static conditions. The pathogenic bacterium Staphylococcus aureus ATCC 6538 was cultured in Nutrient Broth (HopeBio, Qingdao, China) at 37 °C without shaking.
10
Pathogenic Bacterial Inoculation on Cherry Tomatoes
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Cherry tomatoes were purchased from a local market on the day of the experiment, and samples without rotting and apparent bruises were selected for the experiment. S. Typhimurium (ATCC14028) recommended by the FDA in food safety testing and non-toxic E. coli O157:H7 (NCTC12900) used for fresh produce disinfection experiments were selected in this study [15] , [16] , [17] , [18] . The inoculation process was performed as described by Huang et al. [19] (link), with some modifications. Briefly, a single colony of the two pathogens was inoculated in nutrient broth (Hopebio, Qingdao, China) and incubated for 12 h at 37 °C with shaking at 120 rpm. The bacterial suspension was washed three times using sterile 0.85% NaCl solution and resuspended in sterile distilled water to achieve ∼109 CFU/mL cell concentration. Then, 10 cherry tomatoes and adjusted bacterial suspension were added into sterile stomacher bags at a ratio of 1:8 (w/v) and manually massaged for 15 min. The samples were transferred into a biosafety cabinet for air drying for 3 h. Finally, the inoculated samples were stored at 4 °C for 24 h to allow sufficient bacterial attachment.
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