For qualitative bacteremia culture analysis, whole blood was streaked over blood agar plates (Hardy Diagnostics, Santa Maria, CA, USA) and observed to detect growth and morphology consistent with B. anthracis after a minimum incubation of 48 h at 37 °C ± 2 °C. Quantitative counts were achieved by 10-fold serial dilutions of the blood samples in Dulbecco’s phosphate buffered saline (Hardy Diagnostics) from 1 × 10−1 to 1 × 10−9 and spread plating 100 microliters (µL) of each dilution onto tryptic soy agar (Hardy Diagnostics) in triplicate. The limit of detection (LOD) and lower limit of quantification (LLOQ) for detecting bacteremia were 100 CFU/mL and 2500 CFU/mL, respectively.
Tryptic soy agar
Manufactured by Hardy Diagnostics
Sourced in United States, Canada
Tryptic soy agar is a general-purpose microbial growth medium. It provides nutrients and growth factors essential for the cultivation of a wide range of microorganisms, including bacteria and fungi.
Automatically generated - may contain errors
Lab products found in correlation
Tryptic soy broth (tsb), by Hardy Diagnostics (2 mentions)
Blood agar plate, by Hardy Diagnostics (1 mentions)
Clindamycin hydrochloride monohydrate, by Tokyo Chemical Industry (1 mentions)
Mannitol salt agar, by Hardy Diagnostics (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Auranofin, by Chem-Impex (1 mentions)
Tryptic soy broth tsb, by BD (1 mentions)
Clavulanic acid, by Merck Group (1 mentions)
Mueller hinton agar (mha), by Merck Group (1 mentions)
Maxsignal β lactam elisa kit, by PSC Biotech (1 mentions)
β lactamase, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Sheep s blood, by Hardy Diagnostics (1 mentions)
Sheep blood agar, by Hardy Diagnostics (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
10 protocols using tryptic soy agar
1
Quantitative Bacteremia Culture Analysis
2
Bacterial Strain and Culture Conditions
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Strains and plasmids used in this study are listed in Additional file 10 : Table S5, except Bbsl strains which are listed in Additional file 6 : Table S3. S. pyogenes were grown at 37 °C in Todd–Hewitt broth or agar plates, or on plates of 5 % sheep blood in tryptic soy agar (Hardy Diagnostics) when determining hemolytic activity by functional expression. For antibiotic selection, 5 μg ml−1 erythromycin was used. E. coli strains were grown at 37 °C with aeration in Luria–Bertani broth or on Luria–Bertani agar plates; antibiotic selection utilized 500 μg ml−1 erythromycin and 50 μg ml−1 kanamycin where relevant.
3
Staphylococcal Pathogen Characterization
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Staphylococcal clinical isolates were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) or the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA, USA). Auranofin (Chem-Impex International, Wood Dale, IL, USA), clindamycin hydrochloride monohydrate (Tokyo Chemical Industry Co., Tokyo, Japan), and mupirocin (PanReac AppliChem ITW Reagents, Darmstadt, Germany) were purchased from commercial vendors and dissolved either in sterile water or in dimethyl sulfoxide (DMSO) to prepare stock 10 mg/mL solutions. Cation-adjusted Mueller Hinton broth (CA-MHB, Becton, Dickinson and Company, Sparks, MD, USA), Tryptic soy agar (TSA, Hardy Diagnostics, Santa Maria, CA, USA), Tryptic soy broth (TSB, Becton, Dickinson and Company, Sparks, MD, USA), mannitol salt agar (Hardy Diagnostics, Santa Maria, CA, USA), phosphate-buffered saline (PBS, Corning, Manassas, VA, USA), hydrochloric acid (HCl, Fisher Scientific, Fair Lawn, NJ, USA), petroleum jelly, rare earth magnets (Magcraft, Vienna, VA, USA), betadine solution (Fisher Scientific, Fair Lawn, NJ, USA), Tegaderm (3 M, St. Paul, MN, USA), Uro-bond V (Urocare, Pomona, CA, USA), and 96-well plates were all purchased from commercial vendors. Buprenorphine was provided by the Purdue Translational Pharmacology Core at Purdue University.
4
Quantifying Heat-Injured Bacteria in Chicken
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After cooking, chicken samples were immediately placed individually into sterile WhirlPak food sample filter bags (19 × 30 cm, Nasco, Modesto, CA) containing 100 mL of refrigerated TSB plus 0.1% sodium pyruvate (Fisher Scientific, Fair Lawn, NY) for enumeration of bacteria survival populations including heat injured cells. The sample bags with chicken meat were homogenized in a blender (Microbiology International, Frederick, MD) for 2 min. The liquid solution from the filtered side of sample bags was then 10- or 100-fold serial diluted in 9.0 or 9.9 mL of 0.1% BPW. One tenth mL of this solution was spread-plated onto XLT-4 and BEA-NaL agars for Salmonella and E. faecium, respectively. After spread plating, 12 mL of tempered, melted tryptic soy agar (Hardy Diagnostics) was overlaid onto the surface of each plate before incubating at 35°C for 48 h. After incubation, colonies were manually counted to determine the recovery of heat injured cells. All bacterial counts were transformed to log10 CFU/g with the detection limit of 0.3 log10 CFU/g.
5
Antibiotic Susceptibility Testing
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Tryptic soy broth and tryptic soy agar were obtained from Hardy Diagnostics (Santa Monica, CA). Antibiotics, Mueller Hinton agar, clavulanic acid, β-lactamase (from Enterobacter cloacae), and ethanol were obtained from Sigma-Aldrich Chemicals. The β-lactam ELISA kit (MaxSignal® β-Lactam ELISA Kit) was obtained from Bioo Scientific.
6
Evaluating 3DPIP's Particle Counting of Citrobacter freundii
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Gram negative bacteria Citrobacter freundii (ATCC 8090) were used to evaluate the 3DPIP's particle counting capabilities in a clinical setting. Bacteria stored at -80 °C were revived twice in Tryptic Soy Broth (TSB, Hardy Diagnostics, Santa Maria, CA) and incubated at 35 °C for 24 h. Next, the bacteria were streaked on non-selective culture media Tryptic Soy Agar (TSA, Hardy Diagnostics, Santa Maria, CA) and incubated at 35 °C for another 24 h. Isolated colonies were selected from the TSA media and suspended in sterile phosphate buffered saline (1× PBS, pH 7.4, Alfa Aesar, Tewksbury, MA). The bacterial suspension was adjusted according to the 0.5 McFarland standard solution turbidity, resulting in an initial concentration of approximately 1.5 × 10 8 CFU per mL (colony forming units per milliliter). Then, 2 µL of BactoView™ fluorescent staining solution (Biotium, Fremont, CA) was added to 1 mL of the bacteria suspension and incubated at 37 °C for 30 min. Next, the stained bacteria suspension was centrifuged at 3073g-force for 5 min and the stained bacteria cells were resuspended in 1× PBS. Serial dilutions were performed and evaluated using the 3DPIP.
7
Quantification of Translocated Bacteria
8
Isolation and Cryopreservation of S. enterica
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When agglutination reaction of a colony was positive, it was streaked onto 60 × 15 mm plate of Tryptic Soy Agar (TSA; Hardy Diagnostics), and incubated at 35 ± 2.0°C for 24 ± 2.0 h. The isolated colony was transferred into conical tube containing 10‐ml Tryptic Soya Broth (TSB; Hardy Diagnostic) and incubated at 35 ± 2°C for 18–24 h. The TSB inoculated broth was then used to create a lawn of S. enterica culture onto TSA using a sterilized cotton swab. The TSA plates then were incubated at 35 ± 2.0°C for 18–24 h. After incubation, the lawn was harvested using a sterile 10 µl loop and transferred the growth into cryobeads (Key Scientific Products Inc.) following manufacturer's instruction. The cryobeads were stored at −80 ± 2.0°C at the National Animal Health Diagnostic and Investigation Center (NAHDIC), Ethiopia for further analysis.
9
Preparing Bacterial Cultures for Experiments
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Fresh cultures of Staphylococcus aureus, Enterococcus hirae and Pseudomonas aeruginosa were prepared by inoculating 35 mL Nutrient Broth (General Laboratory Products Inc.) with a single colony isolated on a Tryptic Soy Agar (TSA, Hardy Diagnostics) plate stored at 4ºC. For S. aureus, the same streak plate and Nutrient broth batch were used for all experiments. We observed a standard error in pHAgg (± 0.6 pH unit) with varying batches of streak plate and Nutrient broth. The S. aureus liquid cultures were then incubated overnight at 35 °C while shaking at 150 rpm (incubating Minishaker 12620-942, VWR International). The E. hirae and P. aeruginosa liquid cultures were incubated overnight at 37 °C while shaking at 150 rpm. For S. aureus, the concentration of the overnight bacterial cultures was determined by serial dilution and plating on TSA plates. Colony forming units of bacteria per mL (CFU mL−1) were counted after 24 h of incubation at 35 °C. The final bacterial concentration in the overnight culture was about 8 log10CFU mL−1 for S. aureus (OD600 nm ~ 0.3). The concentrations of E. hirae and P. aeruginosa, were ~ 7.3 (Nutrient Broth)/8.1(water with NaCl) and ~ 9.5 log10CFU mL−1, respectively.
10
Characterizing E. coli Mastitis Isolates
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E. coli isolates used in this study were a part of the mastitis pathogen culture collection (MPCC) across Alberta, Ontario, Quebec, and Atlantic provinces (Prince Edward Island, Nova Scotia, and New Brunswick) [12 (link)]. Each isolate was obtained as previously described [4 (link), 32 (link)]. The metadata including number and location of the herd, cow ID, quarter position, sampling date, mastitis severity score, days in milk (DIM) at sampling, and cow’s parity is summarized in Supplementary table S1 [33 ].
Single colonies of 113 bacterial isolates grown in Tryptic Soy Agar (TSA) plates containing 5 % sheep blood agar (Hardy Diagnostics, Canada) was inoculated in Mueller-Hinton broth (MHB) (Millipore Sigma, Canada) and kept for incubation at 37 °C under shaking (4 x g) for 18 h for obtaining freshly grown bacterial cells for conducting assays.
Single colonies of 113 bacterial isolates grown in Tryptic Soy Agar (TSA) plates containing 5 % sheep blood agar (Hardy Diagnostics, Canada) was inoculated in Mueller-Hinton broth (MHB) (Millipore Sigma, Canada) and kept for incubation at 37 °C under shaking (4 x g) for 18 h for obtaining freshly grown bacterial cells for conducting assays.
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