Beta-lactamase production of representative SHV and CTX-M positive isolates and all CTX-M and SHV negative isolates was analyzed by IEF as described previously.11 (link) The isolates with known beta-lactamases (TEM-1, OXA-1, SHV-3, SHV-12, CTX-M-3, and CTX-M-15) were used as controls. The hydrolytic activity of individual beta-lactamase bands was assessed by bioassay.11 (link) Two consecutive agar overlays were laid on the gel: first 0.5% tryptic soy agar (Becton Dickinson) containing the respective beta-lactam (cefotaxime 2 mg/L or ceftazidime 1 mg/L), followed (after 2hours of incubation at 35°C) by a second tryptic soy agar overlay containing the susceptible indicator strain E. coli K12:W3110 (RifR lac-, 1.2×107 CFU/mL). After an overnight incubation at 35°C, the growth of the indicator strain on the gel was determined the bands with hydrolytic activity.
Tryptic soy agar
Manufactured by BD
Sourced in United States, Canada, Cameroon, France, China
Tryptic soy agar is a type of culture medium used in microbiology laboratories. It provides nutrients necessary for the growth of a wide range of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Tryptic soy broth (tsb), by BD (46 mentions)
Tryptic soy broth tsb, by BD (13 mentions)
Mueller hinton broth (mhb), by BD (6 mentions)
Sheep blood, by BD (5 mentions)
Yeast extract, by BD (5 mentions)
Gentamicin, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Chloramphenicol, by Merck Group (4 mentions)
Schaedler agar, by BD (3 mentions)
Brain heart infusion agar, by BD (3 mentions)
Mueller hinton broth (mhb), by Merck Group (3 mentions)
Ciprofloxacin, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Tryptic soy broth (tsb), by Merck Group (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Pseudomonas aeruginosa reference strain atcc 27853, by American Type Culture Collection (2 mentions)
Brucella broth, by BD (2 mentions)
Nutrient agar, by BD (2 mentions)
Chef mapper, by Bio-Rad (2 mentions)
186 protocols using tryptic soy agar
1
Isoelectric Focusing for Beta-Lactamase Detection
2
Quantifying Gut Microbial Populations
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Whole duodenum, ileum, and both ceca were aseptically removed, separated into sterile bags, and homogenized. Samples were weighed and 1:4 wt/vol dilutions were made with sterile 0.9% saline. Ten-fold dilutions of each sample, from each group were made in a sterile 96 well Bacti flat bottom plate and the diluted samples were plated on three different culture media; to evaluate total number of lactic acid bacteria (LAB) in Man Rogosa Sharpe (Difco Lactobacilli MRS Agar VWR Cat. No. 90004-084 Suwanee, GA 30024, USA); total Enterobacteriaceae in MacConkey; and total anaerobes in tryptic soy agar with sodium thioglycolate plates (tryptic soy agar, catalog no. 211822, Becton Dickinson, Sparks, MD, USA).
3
Isolation and Characterization of Duck Virus
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Twenty-day-old Pekin ducks that died at a farm in Yucheng, Shandong Province were necropsied. Pathological examination revealed mild to moderate swollen liver and intestinal mucosa haemorrhagic plaques. Samples of liver, spleen and brain tissue were inoculated onto tryptic soy agar (BD, Sparks, MD, USA) plates containing 2% fetal bovine serum and incubated at 37 °C for 48 h for bacterial examination. Virus isolation was performed later. The intestinal mucus, liver and spleen samples were pooled and homogenised in sterile phosphate-buffered saline solution (pH 7.2) to give a 20% suspension (w/v). After centrifugation at 8,000 × g for 10 min, the supernatant was filtered through a 0.22 μm pore-size sterile filter and inoculated into five 9-day-old duck embryos (0.2 mL/embryo) by the chorioallantoic membrane route. Embryos were candled daily and were chilled at 4 °C to death on 5 days post-inoculation (dpi) to harvest allantoic fluid for subsequent passage. Since no consistent embryo death was observed during the serial embryo-passage, we inoculated the allantoic fluid of the fourth passage onto confluent monolayers of duck embryo fibroblasts. The syncytium formation of the infected cells was visualised by Giemsa staining. To test the growth of the isolate in Vero cells, the DEF-adapted virus suspension was inoculated onto the monolayer of Vero cells using the conventional method.
4
Quantification of Bacterial Burden and Histopathology
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On day 21 post infection, the livers, spleens, lungs, hearts and kidneys were harvested for the determination of the bacterial burden. The bacterial numbers in the organs were enumerated by preparing organ homogenates in PBS and plating 10-fold serial dilutions on tryptic soy agar (BD Diagnosis System). The colonies were counted after 24 h of incubation at 37 °C. Meantime, the colonization by MRSA was quantified by real-time PCR using the TaqMan method, amplifying mecA of MRSA as previously described52 (link). For histopathology, the organs were fixed with 10% phosphate buffered formalin and embedded in paraffin. Four-micrometer thick sections were prepared and stained with hematoxylin and eosin for microscopic examination.
5
Bacterial Culture Verification Protocol
6
Growth Curve Determination of Bacterial Strains
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Overnight cultures grown in BHIB at 37°C were standardized to 109 CFU/ml using spectrophotometric methods, and diluted in pre-chilled BHIB to yield a final density of 103 CFU/ml and stored at 4°C (previously described in Arguedas-Villa et al., 2010 (link)). The bacterial density was enumerated daily for the first 4 days and then bi-weekly for up to 5 weeks by plating on tryptic soy agar (BD, Fisher Scientific) +6% yeast extract (BD, Fisher Scientific). The resulting growth curves were fitted using a four parameter logistic model described by Dalgaard and Koutsoumanis (2001 (link)).
7
Isolation and Characterization of Escherichia callanderi
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E. callanderi KGMB02377 has been isolated from the feces of a healthy Korean individual as described in our previous study (14 (link)). The identification of the strain was verified by 16S rRNA gene sequencing and phylogenetic and phenotypic analyses. Bacterial isolation and cultivation were performed in an anaerobic chamber (Coy Laboratory Products, MI, USA) that was filled with 86% N2, 7% CO2, and 7% H2. E. callanderi KGMB02377 was subcultured every 2 days on tryptic soy agar (BD, NJ, USA) supplemented with 5% sheep blood (TSAB). Bacteria were preserved at −80°C in 10% (vol/vol) skim milk solution. EcCFS was prepared as previously described (14 (link)). Briefly, E. callanderi KGMB02377 was inoculated into 20 mL of reinforced clostridial medium (RCM; MB cell, Seoul, Republic of Korea) broth and incubated under anaerobic conditions for 48 h. The cells were removed by centrifugation at 6,000 × g for 30 min, and the supernatant was harvested. To remove the effect of acidic pH, pH was adjusted using 1 M NaOH to 6.8, and filtered through a 0.22 μm pore size hydrophilic polyethersulfone membrane filter. The prepared EcCFS was stored at −20°C until use.
8
Oral B. suis S2 Vaccine Efficacy
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The lyophilized B. suis S2 vaccine was purchased from Jinyu Baoling Bio-pharmaceutical Co., Ltd (Inner Mongolia, China). To examine its accuracy in counting live bacteria, the number of colony-forming units (CFU) were confirmed retrospectively by counts of distinct colonies of the tryptic soy agar (BD Biosciences, NJ, USA) at 37 °C for 3–5 days. In the control group, three sheep were inoculated with 1 mL sterilized phosphate-buffered saline (PBS, pH 7.2). The remaining sheep in the T group and the vaccinated group were immunized with the lyophilized B. suis S2 vaccine (with an amount of 2 × 1010 CFU in 1 mL sterilized PBS) via oral administration. The control group and vaccinated group were monitored at 0, 7, 14, 21, and 30 days post-immunization (dpi) individually.
9
Bactericidal Effect of UVC Irradiation on S. aureus Skin Infections
10
Identification of ESBL and VRE Bacteria
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The presence of ESBL and VRE was established by plating aliquots of the frozen samples onto either ESBL CHROMagarTM or VRE CHROMagarTM plates, according to the manufacturer’s instructions (Biotrading Benelux, Mijdrecht, The Netherlands). Colonies resembling the proper morphology (according to instructions) were picked and streaked onto Blood Agar plates (40 gr/L Tryptic Soy Agar, 2 gr/L glucose, 5% defibrinated sheep blood) to check their purity. Each isolate was Gram’s stained and was subsequently subcultured in TSB. Each ESBL positive culture was spread onto Tryptic Soy Agar (BD) and incubated with an E-test® ESBL (BioMérieux Benelux B.V., Zaltbommel, The Netherlands) to confirm resistance towards cefepime (MIC ≥ 1.0 μg/ml) in the presence and absence of clavulanic acid [17 (link)]. VRE positives isolates were confirmed using an MIC-test towards vancomycin (0–16 mg/L vancomycin).
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