Initially, a stock of Streptococcus sanguis (HiMedia Laboratories, Mumbai) was streaked onto a plate of tryptic soy agar (HiMedia Laboratories Mumbai) with 5% defibrinated sheep blood. The plate was cultured under anaerobic condition for 48 h at 37°C. Single colony was obtained from this plate and inoculated into tryptic soy broth in a test tube and cultured overnight under the same condition. Bacteria obtained from this broth were used to coat the patency files.
Tryptic soy agar
Manufactured by HiMedia
Sourced in India
Tryptic Soy Agar is a general-purpose culture medium used for the growth and isolation of a wide variety of microorganisms. It provides the necessary nutrients and supports the growth of both aerobic and anaerobic bacteria, as well as some fungi.
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Lab products found in correlation
Tryptic soy broth (tsb), by HiMedia (2 mentions)
R2a agar, by HiMedia (2 mentions)
Dimethyl sulfoxide (dmso), by HiMedia (2 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Brain heart infusion agar, by Thermo Fisher Scientific (1 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (1 mentions)
Amies transport medium, by Thermo Fisher Scientific (1 mentions)
Chocolate agar, by HiMedia (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
Bacteriological agar, by HiMedia (1 mentions)
Tryptone, by HiMedia (1 mentions)
Peptone, by HiMedia (1 mentions)
Yeast extract, by HiMedia (1 mentions)
Colistin sulfate, by HiMedia (1 mentions)
6 gingerol, by Merck Group (1 mentions)
Ethidium bromide, by HiMedia (1 mentions)
Sodium chloride (nacl), by HiMedia (1 mentions)
Actinomycetes isolation agar, by HiMedia (1 mentions)
Streptomyces agar, by HiMedia (1 mentions)
16 protocols using tryptic soy agar
1
Culturing Streptococcus sanguis for Patency Files
2
Standardized Bacterial Strain Testing
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The following seven standard strains were tested: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes ATCC 12344, and Salmonella enterica NCTC 6017. The strains were grown in a test tube containing 10 mL sterile Mueller–Hinton broth (Oxoid Ltd., Basingstoke, Hampshire, England), Tryptone Soya broth (CM0129, Thermo Fisher Scientific, USA) and Brain Heart Infusion broth (M210, HiMedia Laboratories, Pvt. Ltd.), respectively, at 37 °C for 24 h. A loopful of grown inoculum was streaked on growth agar medium: Tryptic Soy agar (M1968, HiMedia Laboratories, Pvt. Ltd.) for E. coli and P. aeruginosa, Mueller–Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, England) for S. epidermidis, S. aureus, S. enterica, and Brain Heart Infusion agar for E. faecalis and S. pyogenes. Plates were incubated for 24 h at 37 °C. Bacterial morphology was confirmed by optical microscopy.
3
Throat Swab Collection and Bacterial Culture
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Throat swabs were taken by the attending pediatricians from each patient using a sterile cotton swab. Visible exudates or hyperemic areas on the tonsillar walls were swabbed with a sterile cotton swab, while the tongue was depressed by a wooden spatula when necessary. All swab samples were immediately transported to the Microbiology Department of HGH using Amie's transport medium (Oxoid, England). Swabs were simultaneously plated onto Tryptic Soy Agar (Himedia, India) containing 5% sheep blood, chocolate agar (CA), and MacConkey (MAC) Agar (Himedia, India) and incubated for 48 h at 37°C. chocolate agar was incubated in a candle jar to get 5% CO2, while BA and MAC were incubated under a normal atmosphere.
4
Antimicrobial Activity Evaluation
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Culture media (Tryptic Soy Broth, Tryptic Soy Agar, Peptone, Yeast Extract, Tryptone, Glucose, and Bacteriological Agar) were purchased from HiMedia (Mumbai, India); ABTS from Sigma Aldrich Co. (St. Louis, MO, USA); and ciprofloxacin disks from Bioanalyse® (Ankara, Turkey).
5
Isolation of Bacterial Endophytes from P. harmala
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P. harmala samples were collected in March 2014, from Taif region, Saudi Arabia. After collection, plant specimens were placed in a sterile bag and transferred to laboratory within 24 h for bacterial isolation. These plant samples were washed with sterile distilled water to remove soil and root, leaf and fruit of plant were separated. Bacteria were isolated from each part after cutting 1.0 g of tissue from each part mentioned above. These plant tissue samples were ground using sterile mortar and pestle. This homogenate was used to make serial dilutions using autoclaved distilled water and 0.1 ml aliquots were plated out on two different isolation media, 1/2 Tryptic soy agar and 1/2 R2A agar (HIMEDIA) supplemented with amphotericin B 25 μg/ml to inhibit fungal growth. These plates were then incubated at 28 °C for 1–2 weeks. Bacterial colonies were selected based on morphological features such as size, color and appearance. For pure culture, colonies were re-streaked and stocks were maintained in 15% (v/v) glycerol solution and stored at −80 °C for further use.
6
Antimicrobial Susceptibility Assay
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Cation adjusted Mueller Hinton broth (CaMHB), Luria Bertani agar (LBA), Luria Bertani broth (LB), Tryptic soy agar (TSA), dimethyl sulfoxide (DMSO), carbonyl cyanide m-chloro phenylhydrazine (CCCP), ethidium bromide (EtBr), colistin sulfate, and triphenyl tetrazolium chloride (TTC) were procured from Himedia, India, and 6-gingerol was purchased from Sigma-Aldrich, India.
7
Identification and Characterization of Vibrio and Staphylococcus Strains
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Bacterial strains growing on the selective mediums plates (Green and yellow colonies growing on TCBS plates, mauve, green blue/turquoise blue and white colonies from CHROMagar™ Vibrio plates) were purified on Tryptic soy agar plates supplemented with 1% NaCl (HiMedia Laboratories Pvt, Ltd., Mumbai, India), while the pink to mauve colonies on CHROMagar™ S. aureus medium were purified on Tryptic soy agar. All pure colonies were subjected to standard morphological, physiological, biochemical plate and tube tests [73 (link)]. A commercial miniaturized Api 20E and Api 20NE systems (Bio Mérieux SA, Marcy l’Etoile, France) was used to identify all the strains isolated from TCBS and CHROMagar™ Vibrio media. The Api Staph strips (Bio Mérieux) were used to identify the bacterial strains isolated from the CHROMagar™ S. aureus plates.
For Exoenzymes production, all strains were tested for haemolysin (Human blood agar, Pronadisa Laboratories Coda, S.A) and DNA hydrolysis (DNase Agar, Biolife, S.r.l., Milano, Italy). The enzymes amylase, caseinase and lecithinase were detected on media prepared with Phosphate Buffer Saline (PBS) supplemented with 0.5% casein peptone, 5% skim milk powder and 5% egg yolk emulsion, respectively [32 (link)].
For Exoenzymes production, all strains were tested for haemolysin (Human blood agar, Pronadisa Laboratories Coda, S.A) and DNA hydrolysis (DNase Agar, Biolife, S.r.l., Milano, Italy). The enzymes amylase, caseinase and lecithinase were detected on media prepared with Phosphate Buffer Saline (PBS) supplemented with 0.5% casein peptone, 5% skim milk powder and 5% egg yolk emulsion, respectively [32 (link)].
8
Bacterial Isolation and Enumeration Protocols
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The samples were immediately processed by serial dilution and plating technique using normal saline (0.45%) by plating on to general purpose media like TSA (Tryptic Soy Agar; Hi-Media), NA (Nutrient Agar; Hi-Media), minimal media involving TSA 1:10 and 1:100 dilutions (TSA; Hi-Media) and selective media involving AIA (Actinomycetes Isolation Agar; Hi-Media), SMA (Streptomyces Agar; Hi-Media) followed by incubation at 30°C for a week subsequently monitoring bacterial growth at 24 h, 48 h, 72 h, and so on. Quantitative analysis for bacterial growth in all the samples was examined using colony counting and determination of colony forming units (CFU) per milliliter. Qualitative analysis of colonies involved identification of bacterial species and their enumeration from all the samples. The colony morphotypes were selected using four parameters: colony size, form, color, and texture. A phenotypic variant was considered when it differed in at least one of the referred morphological parameters and further selected for identification.35 (link)
9
Antimicrobial Resistance Characterization of K. pneumoniae
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Mueller–Hinton agar (MHA), cation-adjusted Mueller–Hinton broth (CAMHB) and agar were procured from Difco (Becton Dickinson, Franklin Lakes, NJ, USA. Tryptone soy broth was from Hi-media, India and was used for Tryptic soy agar (TSA) preparation. Ceftazidime, cefepime, imipenem and meropenem were acquired from commercial manufacturers. Various β-lactamase inhibitors and β-lactam enhancer (zidebactam) used in this study were kind gifts from Wockhardt Research Center, India. Ten K. pneumoniae included in the study were collected from Indian tertiary care hospitals during 2018, and were genomically characterized for blaOXA-181/blaOXA-232, ESBLs and class C β-lactamases, based on WGS. The species-level identification for all the parent strains and their respective mutants was undertaken by using VITEK 2.
10
Irradiated Brucella ovis Vaccine Preparation
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B. ovis strain was grown in 2YT to mid-log phase, and aliquots of 5×109–1×1010 colony-forming unit (CFU)/mL were then stored at -80℃ until use. Three weeks before immunization, an aliquot of the B. ovis was subjected to γ-irradiation using a 60Co source gamma irradiator (Gammacell 220 irradiator; Issledovatel Gamma Irradiator, Techsnabexport Co. Ltd., Moscow, Russia). The inability of irradiated bacteria to replicate was confirmed by culturing on Tryptic Soy Agar (HiMedia, Mumbai, India) and incubating for at least 7 days. The irradiated bacteria were stored at 4℃ until used for immunization.
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