A strain of E. coli, designated as E. coli-R168, possesses resistance to rifampicin (rif, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and was used in this study. The original strain was isolated from lettuce and was artificially induced in our laboratory to develop resistance against 100 mg/L rifampicin. Bacterial cultures with 15% glycerin were stored at −80 °C. The frozen cultures were activated and propagated in tryptone soya broth (TSB, Termo Fisher Scientific, Waltham, MA, USA) plates supplemented with 100 mg/L rifampicin (TSB/R) by aerobic incubation at 37 °C for about 12 h. A single representative colony was inoculated into 10 mL TSB containing 100 mg/L rifampicin. The strain was incubated at 37 °C (190 rpm) for approximately 12–13 h to achieve the stationary phase. Then, it was centrifuged (8000 rpm, 10 min at 4 °C), the supernatant was discarded, and sterile buffer peptone water (BPW, Termo Fisher Scientific, Waltham, MA, USA) was added.
Tryptic soy broth (tsb)
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Italy, Australia, Germany, Canada, France, Denmark
TSB is a general-purpose growth medium used for the cultivation of a wide range of aerobic bacteria. It provides essential nutrients and growth factors required for the optimal growth of microorganisms in a laboratory setting.
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Lab products found in correlation
Tryptic soy agar, by Thermo Fisher Scientific (23 mentions)
Tryptic soy agar tsa, by Thermo Fisher Scientific (23 mentions)
Yeast extract, by Thermo Fisher Scientific (9 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (7 mentions)
Macconkey agar, by Thermo Fisher Scientific (6 mentions)
Glycerol, by Merck Group (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Glucose, by Merck Group (5 mentions)
Nutrient agar, by Thermo Fisher Scientific (4 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (4 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Gentamicin, by Merck Group (4 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (4 mentions)
Ampicillin, by Merck Group (4 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (4 mentions)
Camhb, by Thermo Fisher Scientific (3 mentions)
Nutrient broth, by Thermo Fisher Scientific (3 mentions)
390 protocols using tryptic soy broth (tsb)
1
Rifampicin-Resistant E. coli Strain Propagation
2
Construction of S. aureus Gene Knockout Strains
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S. aureus USA500 (Diep et al., 2006 (link)) was used for construction of gene knockout and complementation strains. E. coli DC10B (Monk et al., 2012 (link)) was used for shuttle plasmid construction. Luria Broth medium was composed of 1% tryptone (Oxoid), 0.5% yeast extract (Oxoid) and 0.5% NaCl; BM (B-Medium) was composed of 1% tryptone, 0.5% yeast extract, 0.5% glucose, 0.1% K2HPO4 and 0.5% NaCl; BM and TSB (Tryptic soy broth, Oxoid) were used for S. aureus cultivation. Bacterial strains were inoculated in BM, and their growth rate at 37°C was monitored by measuring the OD values at 600 nm. Anhydrotetracycline (ATc) was used for induction of secY antisense RNA during gene knockout. Antibiotics were added to medium at the following concentrations: chloramphenicol, 10 μg/ml; ampicillin, 100 μg/ml, levofloxacin, 50 μg/ml.
3
Bacterial Efflux Dynamics in E. coli Strains
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Two strains of E. coli were used to study their bacterial efflux: ŽM 370 (ATCC 11229) is a pathogenic reference strain while E. coli ŽM 513 (VF 3584) was isolated from steak tartare; both isolates were from the Veterinary Faculty of the University of Ljubljana. Stock cultures of these strains were stored at −80°C, then sub-cultured twice and maintained on Tryptic Soy Agar (Oxoid, Basingstoke, Hampshire, United Kingdom). Overnight cultures were prepared in the third passage inoculated in Tryptic Soy Broth (Oxoid, Basingstoke, Hampshire, UK), and incubated for 24 h, without carvacrol at 37°C.
The overnight cultures had been diluted in fresh Tryptic Soy Broth at OD600 = 0.1 before the fluorescent assays. Three physiological states were tested: fast-, slow-, and non-growing phases. To achieve them, freshly diluted cell cultures were incubated at 37°C for 0.5 h, 4 h, and 12–16 h, respectively. The final test cultures used in fluorescence assays had the same concentrations (OD600 = 0.2). The incubation times were decided according to the growth curves measured at 600 nm and 37°C using a Tecan Safire 2 microplate reader (Tecan, Zürich, Switzerland).
The overnight cultures had been diluted in fresh Tryptic Soy Broth at OD600 = 0.1 before the fluorescent assays. Three physiological states were tested: fast-, slow-, and non-growing phases. To achieve them, freshly diluted cell cultures were incubated at 37°C for 0.5 h, 4 h, and 12–16 h, respectively. The final test cultures used in fluorescence assays had the same concentrations (OD600 = 0.2). The incubation times were decided according to the growth curves measured at 600 nm and 37°C using a Tecan Safire 2 microplate reader (Tecan, Zürich, Switzerland).
4
Campylobacter Detection in Chicken Samples
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All oligonucleotides (Denis et al., 1999 (link))
were synthesized and purified by Bionics (Seoul, Korea;Table 1 ). For the inclusivity test, some C.
jejuni (A total of four C. jejuni strains: ATCC
33560 and 3 wild type strains from chicken carcasses) and C.
coli strains (A total of four C. coli strains:
ATCC 33559 and 3 wild type strains from chicken carcasses) were used.
Campylobacter strains were isolated from chicken carcasses
from a local slaughterhouse and a poultry farm.
Non-Campylobacter strains for the exclusivity test
including Salmonella Enteritidis and Escherichia
coli, were also isolated from the collected chicken carcasses.
All Campylobacter strains were incubated in Bolton broth (Oxoid,
Hampshire, UK) at 42°C for 42 h under microaerobic condition (5%
O2, 10% CO2, and 85% N2),
while non-Campylobacter strains were incubated in tryptic soy
broth (Oxoid) at 37°C for 24 h. Additionally, phosphate-buffered saline
was applied to dilute samples in the sensitivity test of AuNP-PCR and 4 Log CFU
of Campylobacter and non-Campylobacter strains
were incubated in 30 g of chicken meat samples with 30 mL of Bolton broth
(42°C for 42 h) and tryptic soy broth (37°C for 24 h). Thereafter,
10 μL of cultured bacteria was used for AuNP-PCR direct detection
test.
were synthesized and purified by Bionics (Seoul, Korea;
jejuni (A total of four C. jejuni strains: ATCC
33560 and 3 wild type strains from chicken carcasses) and C.
coli strains (A total of four C. coli strains:
ATCC 33559 and 3 wild type strains from chicken carcasses) were used.
Campylobacter strains were isolated from chicken carcasses
from a local slaughterhouse and a poultry farm.
Non-Campylobacter strains for the exclusivity test
including Salmonella Enteritidis and Escherichia
coli, were also isolated from the collected chicken carcasses.
All Campylobacter strains were incubated in Bolton broth (Oxoid,
Hampshire, UK) at 42°C for 42 h under microaerobic condition (5%
O2, 10% CO2, and 85% N2),
while non-Campylobacter strains were incubated in tryptic soy
broth (Oxoid) at 37°C for 24 h. Additionally, phosphate-buffered saline
was applied to dilute samples in the sensitivity test of AuNP-PCR and 4 Log CFU
of Campylobacter and non-Campylobacter strains
were incubated in 30 g of chicken meat samples with 30 mL of Bolton broth
(42°C for 42 h) and tryptic soy broth (37°C for 24 h). Thereafter,
10 μL of cultured bacteria was used for AuNP-PCR direct detection
test.
5
Cultivation and Maintenance of Bacterial Strains
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The plasmids and bacterial strains used in this study were listed in Supplementary Table 1
The plasmid pBT2 and S. aureus RN4220 were kindly provided by Prof. Baolin Sun (University of Science and Technology of China). B. pseudomallei strain BPC006 was isolated from the clinical specimen of a patient hospitalized in Hainan People’s Hospital (28 (link)), and kindly provided by Prof. Xu-hu Mao (Army Medical University).
The plasmid pBT2 and S. aureus RN4220 were kindly provided by Prof. Baolin Sun (University of Science and Technology of China). B. pseudomallei strain BPC006 was isolated from the clinical specimen of a patient hospitalized in Hainan People’s Hospital (28 (link)), and kindly provided by Prof. Xu-hu Mao (Army Medical University).
6
Experimental Colonization of BALB/c Mice with S. aureus
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BALB/c female mice (Taconic Farms, Denmark) were experimentally colonised with S. aureus strain termed ‘SaF_1’; an isolate obtained from a stool sample of one of the 4 BALB/c mice (Harlan, UK),which were sequenced as described above. Three to four individual colonies of SaF were picked and cultured overnight in TSB (Tryptic Soy Broth, Oxoid Ltd) at 37 °C with shaking at 130 rpm followed by 1:100 dilution into fresh TSB to culture for 2.5 hours at 37 °C, without shaking. Bacteria were washed and resuspended in PBS. Mice received either SaF (108 cfu in 100 µl PBS) or PBS via an oral feeding tube (Instech, Plymouth, PA, USA) and stool samples were subsequently taken as described above to monitor carriage levels.
7
Isolation and Identification of Staphylococcus aureus
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Samples were cultivated in nutrient broth (Oxoid, UK) and on nutrient agar (Oxoid, UK) plates. Isolated colonies were observed under microscope by Gram staining and by colonial morphology. After preliminary identification of S. aureus isolates, stocks were made using 25% v/v glycerol in TSB (Tryptic Soy Broth, (Oxoid, UK) and stored at -80°C.
The specimens were revived from the glycerol stock by using TSB according to manufacturer’s instructions. Sub culturing was done on nutrient agar plates. Isolated colonies on nutrient agar were sub cultured on mannitol salt agar to assess mannitol fermentation.
The specimens were revived from the glycerol stock by using TSB according to manufacturer’s instructions. Sub culturing was done on nutrient agar plates. Isolated colonies on nutrient agar were sub cultured on mannitol salt agar to assess mannitol fermentation.
8
Evaluating Antibiotic Activity of AuNPs
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The procedure for determining antibiotic activity followed a previous publication [27 (link)]. Escherichia coli ATCC 25922 (ATCC, Manassas, VA, USA) was sub-cultured in Tryptic soy broth (Thermo Scientific, Waltham, MA, USA) at 37 °C for 12 h. E. coli approximately at 2.4 × 106 CFU, as determined by a spectrophotometer (ELx808 absorbance reader; BioTek, Shoreline, WA) at optical density 600 nm at 0.003 (OD 600 nm at 0.003), were diluted in Tryptic soy broth (Thermo Scientific) and added with AuNP in different concentrations: 12.5, 25 and 50 ppm, respectively. Then the solutions were incubated at 37 °C for 4 h. After that, the supernatant in serial dilution was plated in Tryptic soy agar (Thermo Scientific), kept at 37 °C overnight before bacterial colony enumeration. E. coli in Tryptic soy broth alone or in 100 μg/ml of gentamicin were used as the positive and negative control group, respectively.
9
Quantifying S. aureus Biofilm Formation
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Biofilm formation assays were performed with S. aureus isolates (n = 30) following the protocol that was described in our previous work [19 (link)]. Briefly, the bacteria were grown overnight at 37 °C in 5 mL of tryptic soy broth (TSB; Oxoid) for 18 h. Following 18 h of incubation, the bacteria were diluted in TSB that was supplemented with 20% glucose to reach the final concentration of 107 CFU/mL. Aliquots of bacteria (1 mL, 107 CFU/mL) were placed in a 12-well cell culture plate and incubated at 37 °C for 48 h. After incubation, the non-adherent cells were removed and the produced biofilms on the surface of the wells were fixed at 60 °C for 1 h. The biofilm was stained with 0.1% crystal violet solution and rinsed with PBS. The OD was measured at 550 mm using a spectrophotometer. Each assay was performed in triplicate. S. aureus 15 AL was used as positive control.
10
Isolating Salmonella from Egg Samples
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All broken or cracked egg samples were discarded. Eggshell surface was swabbed using a sterile cotton swab soaked in sterile normal saline solution. The swabbed stick was immersed into 10 mL Tryptic soy broth (TSB ) (Oxoid) and incubated at 37°C for 24 h. For the egg yolk, isolation was done as recommended by the US Food and Drug Administration Bacteriological Analytical Manual, and by a previous study (Zhang et al., 2013 (link)). Briefly, the egg sample surface was disinfected by immersing whole eggs in a 3:1 disinfectant solution consisting of 3 parts 70% ethyl alcohol and 1 part iodine/potassium iodide solution for 10 s (Zhang et al., 2013 (link)). Surface-disinfected eggs were air-dried at room temperature before eggshell was cracked. Egg yolks were stirred in a stomacher for thorough mixing. 10 mL of the mixed egg yolk content was then inoculated into 90 mL of TSB (Oxoid) and incubated at 37°C for 24 h.
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