The following viruses were obtained from the Southeast Poultry Research Laboratory (SEPRL) repository: low virulent (lentogenic) NDV LaSota/1946 (vaccine strain, ICPI = 0.4), virulent (mesogenic) NDV Pigeon/1984 (ICPI = 1.45), virulent (velogenic) NDV CA/2002 (ICPI = 1.85); and HPAIVs viruses A/chicken/Queretaro/14588-1988 (H5N2) and A/chicken/Jalisco/CPA-12283-12/2012 (H7N3). The viruses were propagated in specific pathogen free (SPF) embryonating chicken eggs (ECE), as previously described [23 ]. Virus-infected allantoic fluid was diluted in brain heart infusion (BHI) medium (BD Bioscience, Sparks, MD) in order to obtain an inoculum with titers of 104 to 107 50% egg infectious dose (EID50) per 0.1 mL/bird. Sham inoculum was made using non-infected allantoic fluid from SPF ECE diluted 1:300 in brain heart infusion (BHI) medium (BD Bioscience, Sparks, MD, USA). The experiments were performed in biosecurity level-3 enhanced (BSL-3E) facilities in accordance with procedures approved by the SEPRL's Institutional Biosecurity Committee.
Brain heart infusion medium
Manufactured by BD
Sourced in United States, Ireland, Japan
Brain Heart Infusion medium is a microbiological culture medium used for the cultivation of a variety of microorganisms, including bacteria and fungi. It provides essential nutrients and growth factors required for the optimal growth of these microorganisms.
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Lab products found in correlation
Hemin, by Merck Group (3 mentions)
L cysteine, by Merck Group (2 mentions)
Yeast extract, by BD (2 mentions)
Mrs medium, by BD (2 mentions)
Sodium lactate, by Thermo Fisher Scientific (2 mentions)
Sucrose, by Merck Group (1 mentions)
Uv 1800 spectrophotometer, by Shimadzu (1 mentions)
Mucin, by Merck Group (1 mentions)
Baa 835, by American Type Culture Collection (1 mentions)
Gaspak 100 system, by BD (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
0.45 m filter, by Merck Group (1 mentions)
Tryptic soy agar tsa, by BD (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
C acnes atcc 6919, by American Type Culture Collection (1 mentions)
Vitamin k1, by Merck Group (1 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Avantor (1 mentions)
Penicillin, by Avantor (1 mentions)
38 protocols using brain heart infusion medium
1
Characterization of Virulent Avian Influenza Viruses
2
Virulence of Avian Influenza Viruses
3
Bacterial Transformation and Cultivation
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C. glutamicum (ATCC 13032) transformed to encode kanamycin resistance was cultured in brain heart infusion medium (BD, Sparks, MD, USA) with 0.5 M sucrose (Sigma-Aldrich, St Louis, MO, USA; BHIS medium) containing 50 μg ml−1 of kanamycin at 37 °C. B. subtilis JMA222 was cultured in Lysogeny broth (LB) medium at 37 °C. P. syringae ESC 10 (ATCC 55389) was cultured at nutrient broth at 30 °C. All cells were cultured stationary phase. Optical density at 600 nm (OD600) of the cells was measured with ultraviolet–vis spectrophotometer (UV-1800 spectrophotometer, Shimadzu Scientific Inc., Kyoto, Japan) and then diluted in BHIS medium (for C. glutamicum), LB medium (for B. subtilis) or nutrient broth medium (for P. syringae) to OD600 equal to one. C. glutamicum was transformed with the plasmid pEC-K18mob2 using the method described by M.E. van der Rest et al.50 (link) to express kanamycin resistance as the selection marker. The transformed cells were recovered in LBHIS agar plates (per liter of LBHIS agar consists of 12.5 g LB broth, 18.5 brain heart infusion powder, 15 g agar and 91 g sorbitol) containing 50 μg ml−1 of kanamycin. B. subtilis and P. syringae were not transformed. However, when using B. subtilis and P. syringae, negative controls were conducted to confirm that experiments were not contaminated from the environment.
4
Oral Administration of A. muciniphila
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A. muciniphila (BAA-835, ATCC) were cultured on brain heart infusion medium (BD Biosciences) supplemented with 0.4% mucin (Sigma-Aldrich) and anaerobically incubated using the GasPak 100 system (BD Biosciences) at 37°C. Cultures were concentrated, suspended in anaerobic PBS, and administered to mice (8 × 108 CFU per dose) every day for 4 weeks by oral Zonde needle (Fuchigami).
5
Antioxidant Catalase Activity in Oral Bacteria
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Facultative anaerobes such as Streptococcus mutants (S. mutants), Streptococcus gordonii DL1 (S. Gordonni), Moraxella catarrhalis (M. catarrhalis) were grown in brain heart infusion medium (BD Bioscience, USA) at 37 ℃ and Obligate anaerobe Porphyromonas gingivalis (P. gingivalis) was grown anaerobically in trypticase soy broth medium (BD Bioscience, USA) supplemented with 10% sheep blood, hemin (5 μg mL−1) and menadione (1 μg mL−1) and incubated at 37 °C in an atmosphere of N2/H2/CO2 (90:5:5). A single colony of each bacterium was added to 5% or 15% of H2O2 and qualitatively analyzed for the presence of antioxidant catalase. For the survival assay, a 10% 2,3,5-Triphenyl-tetrazolium chloride solution (TTC, Sigma Aldrich, USA) was added to cultures wells and incubated for 6–12 h or until red color change was apparent. Color intensity was quantified by using a microplate reader (SpectraMax i3x, Molecular device, USA) at 480 nm.
6
Metabolite Extraction of Nontypeable H. influenzae
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Nontypeable H. influenzae strain 86-028NP was grown in 7 mL brain–heart infusion medium (BD biosciences) supplemented with 1 μg/mL hemin and 2 μg/mL ß-nicotinamide adenine dinucleotide (Merck) to an optical density at 620 nm (OD620) of 0.5 in a 50 mL tube. Bacteria were pelleted by centrifugation with 3220 g for 10 min at 4°C. Bacterial pellet was suspended into 2 mL wash buffer (75 mM ammonium carbonate, pH 7.4) and pelleted by centrifugation with 3220 g for 10 min at 4°C. For metabolite extraction, the pellet was suspended into 1 mL 40:40:20 acetonitrile:methanol:water, transferred to a 1.5 mL tube and incubated 5 min at −20°C. Bacteria were pelleted by centrifugation at 16,100 g for 3 min at 4°C. The supernatant was transferred to a new 1.5 mL tube and was dried in a vacuum centrifuge and the tubes stored at −80°C until analysis.
7
Microbial Cultivation and Extracellular Vesicle Purification
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Methicillin-susceptible S. aureus ATCC1718, methicillin-resistant S. aureus ATCC1717, P. aeruginosa ATCC15692, Acinetobacter baumannii ATCC19606, Enterococcus faecalis ATCC700802, Salmonella enterica subsp. enterica serovar χ3306 (Sashinami et al., 2006 (link)), E. coli ATCC25922, Listeria monocytogenes 1b1684, and Candida albicans NBRC1385 were cultivated at 37°C for 16–24 h in tryptic soy broth (TSB; BD Bioscience, Sparks, MD) and maintained in tryptic soy agar (TSA, BD Bioscience). For experiments, bacterial and yeast cells were inoculated into TSB and cultured for 4–6 h to reach an exponential phase before use. The numbers of bacterial and yeast cells were adjusted in corresponding to optical density (OD) measurement at a wavelength of 600 nm. For PaEV purification, P. aeruginosa was cultivated in Brain Heart Infusion medium (BD Bioscience) for 8 h. After removal of bacterial cells by centrifugation at 5,000 × g, 4°C for 30 min twice, the supernatant was collected, filtrated through 0.45 µm filter (Merck Millipore Ltd, Tullagreen, Ireland), and stored at -80°C until use.
8
Isolation and Characterization of MRSA Exosomes
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A clinical MRSA isolate, S. aureus 834 [12 (link)] was cultivated and maintained at 37°C for 16–24 h in tryptic soy broth (BD Bioscience, Sparks, MD) and tryptic soy agar, respectively. The activated bacterial cells were inoculated into Brain Heart Infusion medium (BD Bioscience) and cultured under aerobic condition at 37°C for 8 h. The supernatant was then collected by centrifugation twice at 5000×g, 4°C for 20 min, filtrated through 0.45 μm filter (Merck Millipore Ltd., Tullagreen, Ireland) to remove all remaining bacterial cells and kept at −80°C until SaEV purification. For mouse infection model, MRSA 834 were cultured in tryptic soy broth as mentioned above. The bacterial cells were harvested by centrifugation at 5000×g for 20 min and washed with phosphate-buffered saline (PBS). After suspension in PBS, the bacterial cells were adjusted spectrophotometrically at 550 nm to be 5 × 108 CFU/mL (OD550nm of 1.0 correlates to 3.3 × 108 CFU/mL).
9
C. acnes Cultivation Protocol
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C. acnes (ATCC 6919; American Type Culture Collection, Rockville, MD, USA) were obtained from the Korean Culture Center of Microorganisms (Seoul, Korea) and cultured at 37 °C on Brain Heart Infusion Medium (BD Diagnostics, Sparks, MD, USA) under anaerobic conditions at 37 °C until reaching OD600 = 1.0 (logarithmic growth phase). The log phase bacterial culture was centrifuged at 5000× g at 4 °C for 15 min. Subsequently, the culture supernatant was removed.
10
Broth Microdilution Assay for C. difficile
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The broth microdilution assay was utilized following the Clinical and Laboratory Standards Institute (CLSI) guidelines with slight modifications35 . Briefly, C. difficile strains were grown on anaerobic blood agar plates (Becton Dickinson, BD) and suspended in brain heart infusion supplemented broth (Brain heart infusion medium, BD, supplemented with yeast extract, L-cysteine, Vitamin K1 and Hemin, Sigma) to achieve a bacterial concentration of ~105 CFU/ml. The bacterial suspension was seeded in 96-well plates containing drugs/compounds, in duplicate, at the required concentration. Plates were then incubated at 37 °C anaerobically for 48 hours. MICs reported are the lowest concentration of each agent that inhibited the visual growth of bacteria.
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