Escherichia coli O157:H7 ATCC 35150, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 7644, Klebsiella pneumoniae ATCC 13833, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 43387, Salmonella enteritidis ATCC 13076 and Candida albicans ATCC 14053 were the reference human pathogens used in this study (American Type Culture Collection, USA). All strains were maintained in Tryptic Soy Agar (TSA, Oxoid, Milan, Italy) at 37 °C, while C. albicans ATCC 14053 was grown in Sabouraud Dextrose Agar (SDA, Oxoid). All stock cultures were kept at −80 °C in nutrient broth with glycerol 15%.
Tryptic soy agar tsa
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Italy, United States
Tryptic Soy Agar (TSA) is a general-purpose microbiological growth medium. It is composed of tryptone, soy peptone, sodium chloride, and agar. TSA supports the growth of a wide range of microorganisms, including bacteria and fungi.
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Lab products found in correlation
Tryptic soy broth (tsb), by Thermo Fisher Scientific (23 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (3 mentions)
Tryptic soya broth tsb, by Thermo Fisher Scientific (2 mentions)
Microbank beads, by Pro-Lab Diagnostics (2 mentions)
Brucella selective supplement, by Thermo Fisher Scientific (2 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Klebsiella pneumoniae, by American Type Culture Collection (1 mentions)
Escherichia coli o157 h7, by American Type Culture Collection (1 mentions)
Listeria monocytogenes, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa, by American Type Culture Collection (1 mentions)
Enterococcus faecalis, by American Type Culture Collection (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Salmonella enteritidis, by American Type Culture Collection (1 mentions)
Candida albicans, by American Type Culture Collection (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Ciprofloxacin, by AppliChem (1 mentions)
Tryptone soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
62 protocols using tryptic soy agar tsa
1
Characterization of Pathogenic Microbes
2
Evaluating Bean Accessions in Soils
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Seeds of the eight bean accessions were surface-sterilized twice with sodium hypochlorite (0.5%) during 3 min and rinsed in sterile water four times. One hundred microliters of the last rinsing step was cultured in Tryptic Soy Agar (TSA, Oxoid) and in Potato Dextrose Agar (PDA, Difco) media by triplicate in order to check the growth of bacteria and fungi, respectively. Disinfected seeds were germinated on filter paper with sterile tap water; after 2 to 5 days, all the seeds had germinated. The native and agricultural soils were air-dried, passed through a 2-mm-mesh sieve, and distributed into 1 L PVC pots, with 700 g of dried soil per pot. Seedlings were transferred to the pots, with one plant per pot and four replicates per bean accession and per soil. The plants were cultivated in a growth chamber for 1 week and then arranged randomly in a screenhouse with an average temperature of 25 °C, 12 h of daylight, and daily watered with sterile tap water up to 70% of the maximum water holding capacity. Four pots with native soil and four pots with agricultural soil, both without plants were used as bulk soils.
3
Bacterial Strain Assay Protocol
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The bacterial strains assayed in this study were obtained from the Spanish Collection of Type Cultures (CECT) included and maintained frozen at −80 °C in cryovials until the sensitivity tests. Three gram-positive bacteria, Listeria monocytogenes (CECT 911), Enterococcus faecium (CECT 410) and Staphylococcus aureus (CECT 435), and two gram-negative bacteria, Salmonella Typhimurium (CECT 443) and Escherichia coli (CECT 516), were selected for the assays.
Broth subcultures were prepared by inoculating, with one single colony from a Tryptic Soy Agar (TSA, Oxoid, Madrid, Spain) plate, a test tube containing 10 mL of sterile Tryptic Soy Broth (TSB, Oxoid, Madrid, Spain). The inoculated tubes were incubated overnight (16 hours) at 37 °C. Then, the bacterial concentration was adjusted to an absorbance between 0.08 to 0.1 using a spectrophotometer (Jenway 3600, Tirana, Albania) with a wavelength of 620 nm which corresponds to 1 × 108 UFC/mL according to McFarland Turbidity scale (Standart N1 0.5, Becton Dickinson and Company, Madrid, Spain). Additionally, inocula concentration was confirmed by colony counting in agar plates after performing 1:10 dilutions in peptone water (Buffered peptone water, Oxoid, Madrid, Spain).
Broth subcultures were prepared by inoculating, with one single colony from a Tryptic Soy Agar (TSA, Oxoid, Madrid, Spain) plate, a test tube containing 10 mL of sterile Tryptic Soy Broth (TSB, Oxoid, Madrid, Spain). The inoculated tubes were incubated overnight (16 hours) at 37 °C. Then, the bacterial concentration was adjusted to an absorbance between 0.08 to 0.1 using a spectrophotometer (Jenway 3600, Tirana, Albania) with a wavelength of 620 nm which corresponds to 1 × 108 UFC/mL according to McFarland Turbidity scale (Standart N1 0.5, Becton Dickinson and Company, Madrid, Spain). Additionally, inocula concentration was confirmed by colony counting in agar plates after performing 1:10 dilutions in peptone water (Buffered peptone water, Oxoid, Madrid, Spain).
4
Antimicrobial Activity of D. binata Extracts
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We investigated the antimicrobial activity of D. binata extracts and AgNPs towards two multidrug-resistant clinical strains of S. aureus (MRSA 43300 and 703k) obtained from the Laboratory of Microbiology at the Provincial Hospital in Gdansk, Poland. The S. aureus ATCC 13420 strain was used as a reference strain in all experiments. Tested S. aureus strains were characterized by their susceptibility to antibiotics: oxacillin (Fluka Analytics), ciprofloxacin (AppliChem) and vancomycin (Sigma-Aldrich) accordingly to the CLSI standard broth microdilutions method [30] . All strains mentioned are deposited at the IFB, UG & MUG, Poland. Except for experiments concerning antimicrobial susceptibility testing bacteria were grown in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar (TSA) (Oxoid, UK) at 37°C. Overnight bacterial cultures were diluted to obtain the initial inocula, equivalent to 0.5 McFarland turbidity standard (1.5−5×107 Colony Forming Units [CFU]/ml) measured by densimeter (DensiMeter II, EMO, Brno).
5
Phenotypic and Genomic Analysis of Burkholderia Strains
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The bacterial strains phenotypically studied and genome sequenced in this study were drawn from the Burkholderia Table 1 ). A complete list of the 283 isolates and their genomes analysed within the study is provided in Table S1, available in the online version of this article. The isolates studied were recovered from a range of sources, including CF, chronic granulomatous disease (CGD), non-CF clinical infections (NON-CF), the natural environment (ENV) and the healthcare environment (ENVH). Stock cultures were stored at −80 °C in cryogenic vials by resuspension of fresh growth in tryptic soya broth (TSB; Oxoid) containing 8 % (v/v) dimethyl sulfoxide (Sigma-Aldrich). Culture purity was determined by plating frozen stocks onto tryptic soy agar (TSA) (Oxoid) and incubating plates for 24–48 h at 37 °C. Overnight cultures were made by taking a swab from a fresh TSA plate and transferring into 3 ml of TSB. Cultures were grown for 18–20 h at 37 °C using continuous shaking on a rotating platform set to 150 r.p.m.
6
Isolation and Characterization of Bacterial Pathogens
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K. pneumoniae KP/01, used as a host for bacteriophage infection, was isolated from a human clinical diabetic-foot sample from a male patient in May 2016 and identified by National Institute of Diabetes using the VITEK method for identification (Cairo, Egypt). Other clinical isolates of K. pneumoniae (n = 21), Proteus mirabilis (n = 18) and E. coli (n = 15) were also isolated by National Institute of Diabetes, for bacteriophage host-range analysis, from wound infection samples and provided to the microbiology research lab at Zewail City. Isolates were kept in tryptone soy broth (TSB; Oxoid, England) containing (w/v) 20% of glycerol, at −80°C. In the following experiments, bacterial strains were grown on tryptic soy agar (TSA; Oxoid, England) overnight, and isolated colonies of bacteria were grown at 37°C, in TSB, to reach OD600 approximately 0.3.
7
Cultivation of Livestock-Associated MRSA ST398
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The whole genome sequenced wild type (WT) livestock-associated methicillin-resistant S. aureus ST398 (Genbank accession AM990992) [21] (link) and S. aureus RN4220 were grown in Brain Heart Infusion (BHI) (Oxoid, Difco) broth at 37°C with aeration. S. aureus SH1000 pMARGH2b, S. aureus SH1000 pFA545 and S. aureus RN4220 pFA545gen were grown in BHI or Tryptic Soy Broth (TSB) (Oxoid) with 5 mg/l erythromycin (Sigma), 5 mg/l tetracycline (Sigma) and 16 mg/l gentamicin (Sigma) respectively, at 30°C with aeration. For solid growth BHI agar, sheep blood agar plates (Oxoid) or Tryptic Soy Agar (TSA) (Oxoid) were applied and supplemented with the appropriate antibiotic if needed. Escherichia coli DH10 was cultured in Luria Broth (LB) at 37°C with aeration or on LB agar plates (Sigma).
8
Brucella pinnipedialis Strain Isolation
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The strains used were a B. pinnipedialis HS field isolate (strain 17a-1; [5 (link)]) and the B. pinnipedialis reference strain (NCTC 12890T, BCCN 94-73T) from harbour seal [2 (link)]. Bacteria were grown on Tryptic Soy Agar (TSA, Oxoid, Basingstoke, UK) at 37°C in an atmosphere of air plus 5% CO2, with the exception of fecal and water samples which were grown on modified Farrell medium (one vial of Brucella selective supplement (Oxoid) per TSA litre + 5% foetal calf serum (FCS)). The strains were kept at -80°C on Microbank™ beads (Pro-Lab Diagnostics, Round Rock, TX, USA). Before the infection a bead was plated and the bacteria were grown for 2–4 days and subsequently sub-cultured for 96 h.
9
Listeria monocytogenes Inoculation on Smoked Salmon
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The bacterial strains used were four; Listeria monocytogenes ATCC 19114, Listeria monocytogenes ATCC 15313, Listeria monocytogenes ATCC 19111 and Listeria monocytogenes ATCC 7644.
The individual microorganisms were rehydrated in 9 mL of Buffered Peptone Water (BPW, Oxoid), and after 20 minutes the cultures were streaked on Tryptic Soy Agar (TSA, Oxoid) and incubated at 35°C for 22 h. A colony was taken from each TSA plate, diluted in 10 mL of Tryptic Soy Broth (TSB, Oxoid) and incubated at 35°C for 22 h. Cells were collected by centrifugation at 4000 rpm for 20 minutes at 4°C and washed three times with BPW. The individual bacterial strains were resuspended in BPW to obtain a final concentration of about 108 cfu/mL. The final concentration of the inoculum was determined by a spectrophotometer at a wavelength of 550 nm. The bacterial cocktail was prepared by mixing equal volumes of each ATCC strain and was tested before use by the pour plate technique according to ISO 11290-2 standard method. The smoked salmon was weighed, maintaining sterile conditions, to give samples of 25 g. A spot inoculation method was then used to inoculate L. monocytogenes on smoked salmon samples (Mahmoud, 2010 (link)). Subsequently, the samples have been packaged under vacuum and subjected to sonication and thermosonication treatments within 10 min from inoculation.
The individual microorganisms were rehydrated in 9 mL of Buffered Peptone Water (BPW, Oxoid), and after 20 minutes the cultures were streaked on Tryptic Soy Agar (TSA, Oxoid) and incubated at 35°C for 22 h. A colony was taken from each TSA plate, diluted in 10 mL of Tryptic Soy Broth (TSB, Oxoid) and incubated at 35°C for 22 h. Cells were collected by centrifugation at 4000 rpm for 20 minutes at 4°C and washed three times with BPW. The individual bacterial strains were resuspended in BPW to obtain a final concentration of about 108 cfu/mL. The final concentration of the inoculum was determined by a spectrophotometer at a wavelength of 550 nm. The bacterial cocktail was prepared by mixing equal volumes of each ATCC strain and was tested before use by the pour plate technique according to ISO 11290-2 standard method. The smoked salmon was weighed, maintaining sterile conditions, to give samples of 25 g. A spot inoculation method was then used to inoculate L. monocytogenes on smoked salmon samples (Mahmoud, 2010 (link)). Subsequently, the samples have been packaged under vacuum and subjected to sonication and thermosonication treatments within 10 min from inoculation.
10
Screening Exopolymer-Producing INA Bacteria
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The exopolymer-producing INA bacteria were screened by plating the selected INA isolates onto Tryptic Soy Agar (TSA; Oxoid, Basingstoke, United Kingdom). TSA plates were then incubated at 30 °C for 24 h. The bacteria were screened for their ability to synthesis EPS based on colony morphology indicated by mucoid phenotypes from colony growth.
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