Tissues were sterilely collected, homogenized, and serial dilutions of each homogenate (into PBS) were spread onto brain heart infusion media (Sigma) agar plates. For isolating fungi, brain heart infusion media used for agar plates were supplemented with ampicillin (2.5 µg/mL, Sigma), gentamicin (2.5 µg /mL, Sigma), metronidazole (2.5 µg /mL Sigma), neomycin (2.5 µg/mL, Sigma), vancomycin (1.25 µg /mL, MP Biomedicals). Colony forming units were enumerated after incubation for 24 hours at 37°C.
Brain heart infusion media
Manufactured by Merck Group
Brain-Heart Infusion media is a complex microbiological culture medium used for the cultivation and isolation of a wide range of fastidious microorganisms, including streptococci, pneumococci, and other pathogenic bacteria. It provides nutrients essential for the growth of these organisms.
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Lab products found in correlation
Yeast extract, by Merck Group (2 mentions)
Rifampicin, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Todd hewitt broth, by Merck Group (1 mentions)
β glycerophosphate disodium, by Merck Group (1 mentions)
Dfc3000 g microscope, by Leica (1 mentions)
Vancomycin, by MP Biomedicals (1 mentions)
Neomycin, by Merck Group (1 mentions)
Metronidazole, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Chocolate agar plates, by Hardy Diagnostics (1 mentions)
Hemin, by Merck Group (1 mentions)
5 protocols using brain heart infusion media
1
Fungal Isolation from Tissue Samples
2
Anaerobic Cultivation of Bacteroides spp.
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Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were routinely cultured in Brain Heart Infusion media (Sigma-Aldrich), supplemented with hemin (5 μg ml−1), cysteine (0.1% (w/v)), and sodium bicarbonate (0.2% (w/v)), and grown under anaerobic conditions at 37 °C. E. coli EPI300 was grown, aerobically at 37 °C, in Lysogeny broth (LB), supplemented, where appropriate, with ampicillin (100 μg ml−1) and chloramphenicol (12.5 μg ml−1).
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Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were routinely cultured in Brain Heart Infusion media (Sigma-Aldrich), supplemented with hemin (5 μg ml−1), cysteine (0.1% (w/v)), and sodium bicarbonate (0.2% (w/v)), and grown under anaerobic conditions at 37 °C. E. coli EPI300 was grown, aerobically at 37 °C, in Lysogeny broth (LB), supplemented, where appropriate, with ampicillin (100 μg ml−1) and chloramphenicol (12.5 μg ml−1).
3
Genotyping and culture conditions for S. pneumoniae
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Genotypes used in this study are described in Supplementary Table S1 . Unless otherwise stated, encapsulated S. pneumoniae were cultured statically at 35°C in 5% CO2. Culturing on solid media used Todd-Hewitt broth supplemented with 0.5% yeast extract and 1.5% agar (Sigma-Aldrich). Media were supplemented antibiotics for selection of mutated genotypes: rifampicin (Fisher Scientific) at 4 μg ml−1; kanamycin (Sigma-Aldrich) at 400 μg ml−1, or chloramphenicol (Sigma-Aldrich) at 4 μg ml−1. Phase contrast microscopy of colonies used a Leica DFC3000 G microscope.
Unless otherwise specified, culturing in liquid media used 10 ml of a 2:3 ratio mixture of Todd-Hewitt broth (Sigma-Aldrich) with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich); this is referred to as ‘mixed’ media. Transformation experiments with S. pneumoniae R6 derivatives used a chemically-defined medium, consisting of disodium β-glycerophosphate (20 g l−1; Sigma-Aldrich), sodium pyruvate (0.1 g l−1; Fluorochem), choline (0.001 g l−1; Alfa Aesar), cysteine (0.4 g l−1; Tokyo Chemical Industry UK), glucose (3.8 mM; Sigma-Aldrich) and galactose (12 mM; Sigma-Aldrich). Carbon source supplements were added to liquid media at a final concentration of 30 mM, unless otherwise specified.
Unless otherwise specified, culturing in liquid media used 10 ml of a 2:3 ratio mixture of Todd-Hewitt broth (Sigma-Aldrich) with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich); this is referred to as ‘mixed’ media. Transformation experiments with S. pneumoniae R6 derivatives used a chemically-defined medium, consisting of disodium β-glycerophosphate (20 g l−1; Sigma-Aldrich), sodium pyruvate (0.1 g l−1; Fluorochem), choline (0.001 g l−1; Alfa Aesar), cysteine (0.4 g l−1; Tokyo Chemical Industry UK), glucose (3.8 mM; Sigma-Aldrich) and galactose (12 mM; Sigma-Aldrich). Carbon source supplements were added to liquid media at a final concentration of 30 mM, unless otherwise specified.
4
Isolation of S. pneumoniae ΔϕOXC141 Mutant
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S. pneumoniae were grown statically at 35 °C in 5% CO2, unless otherwise stated. Genotypes used in this study are listed in Additional file 5 : Table S4. Liquid cultures were grown in a 2:3 ratio mixture of Todd-Hewitt media with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich), dissolved in milliQ (18MΩ) water, henceforth referred to as mixed media [63 ]. Culturing on solid media used Todd-Hewitt media supplemented with 0.5% yeast extract.
S. pneumoniae 99-4038 was passaged with the aim of isolating a ΔϕOXC141 mutant. For each round of the passage, an overnight culture was diluted 1:9 in fresh mixed media supplemented with 40 μg mL−1 catalase to a total volume of 10 mL. At an OD600 of between 0.3 and 0.4, mitomycin C was added to a final concentration of 0.1 μg mL−1. After three such passages, 24 colonies were picked and screened for the loss of ϕOXC141 by PCR amplification of the attLOXC and attBOXC sequences.
S. pneumoniae 99-4038 was passaged with the aim of isolating a ΔϕOXC141 mutant. For each round of the passage, an overnight culture was diluted 1:9 in fresh mixed media supplemented with 40 μg mL−1 catalase to a total volume of 10 mL. At an OD600 of between 0.3 and 0.4, mitomycin C was added to a final concentration of 0.1 μg mL−1. After three such passages, 24 colonies were picked and screened for the loss of ϕOXC141 by PCR amplification of the attLOXC and attBOXC sequences.
5
NTHi Strain 1479 Protein Extraction
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NTHi strain 1479 (a gift from Dr Brahm Segal, University of Buffalo) was grown overnight on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA) and then used to inoculate 20 ml of brain–heart infusion media (Sigma-Aldrich) containing 10 μg ml−1 NAD and 10 μg ml−1 Hemin (both from Sigma-Aldrich). The culture was incubated for 4 h at 36 °C with constant shaking and then used to inoculate an additional 200 ml of liquid media. After another 4 h of growth, bacteria were pelleted, boiled for 1 h, sonicated twice for 1 min each and filtered through a 0.22-μM polyethylsulfone filter (EMD Millipore, Darmstadt, Germany) using 1 ml of added PBS for each pellet generated from 50 ml of liquid culture. Protein concentration was adjusted to 1.5 mg ml−1 in PBS using Bradford assay (Pierce, Rockford, IL).
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