P. aeruginosa isolates were seeded on LB agar (Neogen, Lansing, MI, USA) and incubated at 37 °C for 24 h. One colony of each isolate was inoculated in the presence (1/2, 1/4, and 1/8 MIC) or absence (control) of subinhibitory concentrations of bio-AgNPs at the bottom of twitching agar plates containing 1.0% w/v tryptone (Acumedia, Lansing, MI, USA), 0.5% w/v yeast extract (BD, Sparks, MD, USA), 1.0% w/v sodium chloride (Merck, Darmstadt, Germany), and 1.0% w/v agar (BD, Sparks, MD, USA). Plates were inverted and incubated at 37 °C for 24 h. Posteriorly, the agar was removed and stained with 2% w/v crystal violet (Laborclin, Pinhais, PR, Brazil) for 2 h [24 (link)]. The motility halo was measured to the nearest millimeter. As a negative control, each isolate was inoculated in tryptone soy agar (BD, Sparks, MD, USA) under the same conditions.
Tryptone soy agar
Manufactured by BD
Sourced in United States
Tryptone soy agar is a general-purpose microbiology media used for the cultivation and isolation of a wide range of aerobic bacteria. It provides the necessary nutrients for the growth of a variety of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Tryptic soy broth (tsb), by BD (2 mentions)
Tryptone, by Acumedia (1 mentions)
Crystal violet, by Laborclin (1 mentions)
Lb agar, by Neogen (1 mentions)
Yeast extract, by BD (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ceftazidime, by Merck Group (1 mentions)
Macconkey broth, by Thermo Fisher Scientific (1 mentions)
Tryptone soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Tsa sb, by BD (1 mentions)
Microflex lt, by Bruker (1 mentions)
5 protocols using tryptone soy agar
1
Twitching Motility of P. aeruginosa under Bio-AgNPs
2
Culturing and Standardizing S. aureus
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S. aureus strain ATCC 25923 provided by Infectious Diseases Department, Southern Medical University was verified by PCR amplification. S. aureus was cultured in tryptic soy broth (BD Biosciences, San Jose, CA, USA) at 37°C in a shaking incubator at 200 rpm overnight for 16 h. Bacteria in the log phase were harvested by centrifugation at 3000 rpm for 10 min, resuspended in sterile phosphate-buffered saline (PBS), and washed 3 times. The S. aureus concentration was determined by serial dilution on tryptone soy agar (BD Biosciences) containing 5% sheep blood.
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S. aureus strain ATCC 25923 provided by Infectious Diseases Department, Southern Medical University was verified by PCR amplification. S. aureus was cultured in tryptic soy broth (BD Biosciences, San Jose, CA, USA) at 37°C in a shaking incubator at 200 rpm overnight for 16 h. Bacteria in the log phase were harvested by centrifugation at 3000 rpm for 10 min, resuspended in sterile phosphate-buffered saline (PBS), and washed 3 times. The S. aureus concentration was determined by serial dilution on tryptone soy agar (BD Biosciences) containing 5% sheep blood.
3
Quantification of Oyster Bacterial Diversity
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Ten microliters each from each fresh tissue homogenate was spread on a marine salt agar-blood (MSA-B) plate and a thiosulphate-citrate-bile salts-sucrose (TCBS) agar (Oxoid, UK) plate separately, using sterile disposable spreaders (Copan, Brescia, Italy). MSA-B was made using 20 g of BD® tryptone soy agar, 7.5 g NaCl, 500 ml of deionized, ultrapure water and 15 ml of sterile, anticoagulated, sheep blood for each 500 ml of MSA-B (Buller, 2014 ). The inoculated culture plates were incubated at 23 °C for 24h (MSA-B) or 48hr (TCBS agar) in a refrigerated incubator (LMS Ltd, UK).
Bacterial colonies on MSA-B plates and TCBS plates were counted and the number of colony forming units (CFU)/g of oyster tissue were calculated (total cultivable bacterial count, TCBC; total cultivable Vibrio count, TCVC). Colony morphology on TCBS plates was recorded based on visual assessment of size and colour. Single colonies of dominant morphotypes on TCBS agar were directly inoculated into nutrient broth with 2% NaCl and incubated at 23 °C overnight. Broth cultures (0.85 ml) were mixed with glycerol (0.15 ml) and stored at -80 °C. Species identification of select cryopreserved Vibrio cultures was later performed at the Animal Health Laboratories, Department of Agriculture, Western Australia, using biochemical methods.
Bacterial colonies on MSA-B plates and TCBS plates were counted and the number of colony forming units (CFU)/g of oyster tissue were calculated (total cultivable bacterial count, TCBC; total cultivable Vibrio count, TCVC). Colony morphology on TCBS plates was recorded based on visual assessment of size and colour. Single colonies of dominant morphotypes on TCBS agar were directly inoculated into nutrient broth with 2% NaCl and incubated at 23 °C overnight. Broth cultures (0.85 ml) were mixed with glycerol (0.15 ml) and stored at -80 °C. Species identification of select cryopreserved Vibrio cultures was later performed at the Animal Health Laboratories, Department of Agriculture, Western Australia, using biochemical methods.
4
Cultivation of Pseudomonas aeruginosa
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Pseudomonas aeruginosa (P. aeruginosa; ATCC 9027) and Pseudomonas aeruginosa clinical strain isolated from ocular swabs were maintained in glycerol stock cultures at −80 °C prior to use and cultured onto Tryptone soy agar (TSA) (Becton Dickinson and Company). Single colonies of bacteria from the overnight cultures were inoculated into tryptone soy broth (TSB) (Becton Dickinson and Company) and incubated in a shaking incubator at 37 °C.
5
Screening for 3GCs-R Enterobacteriaceae
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The inoculated Tryptone Soy Broth (Oxoid) medium was transferred into 5 mL of MacConkey Broth (Oxoid) containing 8 mg/L of ceftazidime (Sigma-Aldrich, Merck, St. Louis, MO, USA) and incubated at 37 °C for 24 h under agitation. Then, a loopful (10 μL) of the mixture was plated onto MacConkey Agar (BioCen, BioCubaFarma, Bejucal, Cuba) supplemented with 8 mg/L of ceftazidime (Sigma-Aldrich) for the screening of 3GCs-R Enterobacteriaceae and reincubated overnight. Lactose-positive (pink colonies) were selected and streaked three times on selective MacConkey Agar plates to obtain pure culture. Single pink colonies from each selective plate were streaked onto Tryptone Soy Agar plates containing 5% sheep blood (TSA-SB; Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37 °C. The colonies were identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS, Microflex LT; Bruker Daltonics, Billerica, MA, USA) and frozen at −80 °C in glycerol stocks.
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