Columbia blood agar plates

Manufactured by BD

Sourced in Germany

Columbia blood agar plates are a type of microbiological culture medium used for the isolation and cultivation of a variety of microorganisms, including both aerobic and anaerobic bacteria. The plates contain Columbia agar base supplemented with 5-10% sheep or horse blood.

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Lab products found in correlation

Yeast extract, by BD (2 mentions) Todd hewitt broth, by BD (2 mentions) Anaerobic conditions, by Coy Laboratory Products (1 mentions) Bhi broth, by Carl Roth (1 mentions) Tissue homogenizer, by Kinematica (1 mentions) Dynamo sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions) Steponeplus rt pcr system, by Thermo Fisher Scientific (1 mentions) Microbank system, by Pro-Lab Diagnostics (1 mentions)

15 protocols using columbia blood agar plates

Reconstitution of Gut Microbiome in Mice

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CBBP bacterial strains (Clostridium bolteae, Blautia producta, Bacteroides sartorii, Parabacteroides distasonis) were initially isolated from a mouse colony as described elsewhere (Caballero et al., 2017 (link)). Strains were separately maintained in glycerol stock at −80°C. To generate the inoculum to be administered to mice, bacteria were thawed and 50 μl of stock were spread onto pre-reduced Columbia blood agar plates (BD) at 37°C in anaerobic conditions (Coy Laboratory Products) for 48h. Bacterial lawns were collected by scraping and resuspended in pre-reduced PBS to an OD of ~2, equal amounts of suspensions were mixed for the 4 bacteria, and 200 μl of mix were administered to ampicillin-treated mice by oral gavage.
Mice were administered ampicillin in drinking water (0.5 g/l) starting for 4 days prior to reconstitution with CBBP (with the exception of experiments carried on GF mice, for which amipicillin was not used).

Becattini S., Sorbara M.T., Kim S.G., Littman E.L., Dong Q., Walsh G., Wright R., Amoretti L., Fontana E., Hohl T.M, & Pamer E.G. (2021). Rapid transcriptional and metabolic adaptation of intestinal microbes to host immune activation. Cell host & microbe, 29(3), 378-393.e5.

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Pneumococcal Protein Precipitation Protocol

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S. pneumoniae strain DSM20566 (serotype 1) was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). Bacteria were grown on Columbia blood agar plates (BD Biosciences, Heidelberg, Germany) at 37°C in 5% CO₂ overnight or cultured in brain heart infusion (BHI) broth (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37°C as described elsewhere (Walther et al., 2015 (link)).
Precipitation of pneumococcal total proteins was described previously (Richter et al., 2015 (link)). Briefly, 200 μL of a pneumococci culture (grown overnight in BHI broth) were precipitated by 1.8 mL of ice-cold absolute ethanol (−20°C for 16 h). Aliquots were centrifuged at 3000 × g for 20 min at 4°C. The precipitate was washed with 1 mL of ice-cold 70% ethanol. After centrifugation at 3000 × g for 5 min at 4°C, the supernatant was removed and the pellet dried. The ethanol-precipitated proteins were dissolved in 50 μL of NA assay buffer and stored at −20°C until use.

Hoffmann A., Richter M., von Grafenstein S., Walther E., Xu Z., Schumann L., Grienke U., Mair C.E., Kramer C., Rollinger J.M., Liedl K.R., Schmidtke M, & Kirchmair J. (2017). Discovery and Characterization of Diazenylaryl Sulfonic Acids as Inhibitors of Viral and Bacterial Neuraminidases. Frontiers in Microbiology, 8, 205.

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Pneumococcal Infection Model in Mice

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Streptococcus pneumoniae serotype 19F (strain BHN100) [18 (link)] was provided by Birgitta Henriques-Normark (Karolinska Institutet, Stockholm). Bacteria were grown in Todd-Hewitt yeast (THY) medium as previously described [19 (link)]. LPS- and IFN-γ-treated C57BL/6JRj mice and control groups were infected i.n. with 10⁸S. pneumoniae 19F in 10 µl PBS 48 h after the first treatment. Mice were sacrificed 18 h post-infection and lung, trachea, NALT, and nasopharynx were homogenized using a tissue homogenizer (KINEMATICA AG, Switzerland). Samples were plated onto Columbia blood agar plates (BD Diagnostic Systems, Germany) and incubated over night at 37 °C, 5% CO₂. CFU were counted to determine the bacterial burden.

Pausder A., Fricke J., Schughart K., Schreiber J., Strowig T., Bruder D, & Boehme J.D. (2021). Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract. Lung, 200(1), 119-128.

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Anaerobic Culturing of Mesenteric Lymph Nodes

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Mesenteric lymph nodes (MLNs) were harvested from mice and
homogenized in PBS and cultured anaerobically on BHI plates containing yeast
extract and 5% sterilized rumen fluid (Fisher Scientific) and Columbia blood
agar plates (BD) for 4 days at 37°C. Colony-forming units (CFUs) were
counted and adjusted per organ. Bacteria were identified by MALDI
Biotyper.

Hayase E., Hayase T., Jamal M.A., Miyama T., Chang C.C., Ortega M.R., Ahmed S.S., Karmouch J.L., Sanchez C.A., Brown A.N., El-Himri R.K., Flores I.I., McDaniel L.K., Pham D., Halsey T., Frenk A.C., Chapa V.A., Heckel B.E., Jin Y., Tsai W.B., Prasad R., Tan L., Veillon L., Ajami N.J., Wargo J.A., Galloway-Peña J., Shelburne S., Chemaly R.F., Davey L., Glowacki R.W., Liu C., Rondon G., Alousi A.M., Molldrem J.J., Champlin R.E., Shpall E.J., Valdivia R.H., Martens E.C., Lorenzi P.L, & Jenq R.R. (2022). Mucus-degrading Bacteroides link carbapenems to aggravated graft-versushost disease. Cell, 185(20), 3705-3719.e14.

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Anaerobic co-culture and oxidative stress

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BP, CB, PD as well as MNC+ E. coli and WT E. coli were cultured on Columbia blood agar plates (BD) for 24h and then inoculated in BHI+ medium (brain heart infusion broth supplemented with yeast extract (5 g/l) and l-cysteine (1 g/l)) in anaerobic conditions. Overnight cultures of BP, CB and PD, respectively, were mixed with an equal volume of overnight culture of either MNC+ E. coli or WT E. coli, doubled in volume with fresh medium and co-cultured anaerobically for 4h in the presence of IPTG 1 mM. Cultures were then challenged with 500 μM H₂O₂ for 1h, and viability of CB, BP, PD was assessed through plating on BHI+ plates supplemented with 100 μg/ml nalidixic acid, to eliminate E. coli.

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Pneumococcal Virulence Factor Cultures

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For in vitro studies, D39 wildtype (Spn WT) and PLY-deficient D39Δply (Spn Δply) strains were used. Bacteria were cultivated in Todd-Hewitt broth (BD, Heidelberg, Germany) supplemented with 0.5% yeast extract (BD, Heidelberg, Germany) to mid-log phase (A₆₀₀ = 0.3–0.4) at 37 °C and 5% CO₂ or grown on Columbia blood agar plates (BD, Heidelberg, Germany). PLY [wild type and non-lytic (NL) PLY] was produced and purified as described^{61 (link)}.

Letsiou E., Teixeira Alves L.G., Fatykhova D., Felten M., Mitchell T.J., Müller-Redetzky H.C., Hocke A.C, & Witzenrath M. (2021). Microvesicles released from pneumolysin-stimulated lung epithelial cells carry mitochondrial cargo and suppress neutrophil oxidative burst. Scientific Reports, 11, 9529.

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Quantifying Gut Microbiome Members

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DNA extracted from fecal pellets or cecal content was subjected to quantitative PCR using the following custom CBBP member-specific primers.
BS1 (B. sartorii): fw: 5’-ATCCAACCTGCCTTATACTCCC-3’; rev: 5’-TACCGAAGTTCTTTAATCACGAGA-3’; LanB1 (B. producta): fw: 5’-ACTGCCGTTTTTACCTGTGG-3’; rev: 5’-CCATGGGAACTCAGACCACT-3’; CB4 (C. bolteae): fw: 5’-CATCCTTTTGACCGGCGT-3’; rev: 5’-GATTTGCTCCACATCACTGTC-3’; PD3 (P. distasonis): fw: 5’-GGGATGAAGGTTCTATGGATCG-3’; rev: 5’- CGCTGACTTAAAAGGCCGC-3’.
Standard curves were generated by amplification of DNA extracted from pure cultures (48h on Columbia Blood Agar plates, BD) of the respective bacteria (six serial 1:10 dilutions, from 3.3 ng/μl, 3 μl, ~ 10 ng total DNA). A control consisting of equal amounts DNA from the 4 members was always added to confirm accuracy of the quantification. DyNAmo SYBR Green qPCR Master Mix (Fisher# F-410L) was used to set up the reactions. Reactions were run on a StepOne Plus RT PCR system (Applied Biosystems). The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 52°C for 30s, and 72°C for 1min.

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Cultivation of Streptococcus pneumoniae Strains

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The S. pneumoniae strains used in this study are the D39 wildtype strain, a D39 derived capsule locus (cps) deletion mutant D39Δcps, the cps/pneumolysin double mutant D39ΔcpsΔply, and a GFP-expressing D39 strain (D39::GFP). S.pn strains were cultivated in Todd-Hewitt broth (BD, Heidelberg, Germany) supplemented with 0.5% yeast extract (BD, Heidelberg, Germany) to mid-log phase (A₆₀₀ = 0.35–0.40) at 37 °C and 5% CO₂ or grown on Columbia blood agar plates (BD, Heidelberg, Germany).

Nerlich A., Mieth M., Letsiou E., Fatykhova D., Zscheppang K., Imai-Matsushima A., Meyer T.F., Paasch L., Mitchell T.J., Tönnies M., Bauer T.T., Schneider P., Neudecker J., Rückert J.C., Eggeling S., Schimek M., Witzenrath M., Suttorp N., Hippenstiel S, & Hocke A.C. (2018). Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA. Scientific Reports, 8, 182.

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Duodenal Microbiome and Lymph Node Analysis

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The contents from 1 cm of duodenum were harvested in 500 μL of PBS. Homogenized duodenum contents were diluted and cultured on Lysogeny Broth (LB) plates containing yeast extract for overnight at 37°C. Gastric draining lymph nodes were harvested from mice and homogenized in PBS and cultured on Columbia blood agar plates (BD) for 48 hours at 37°C. Colony-forming units were counted and adjusted per total contents in 1 cm of duodenum or per organ.

Hayase E., Ara T., Saito Y., Takahashi S., Yoshioka K., Ohigashi H., Ogasawara R., Yokoyama E., Yamakawa T., Ebata K., Hasegawa Y., Tomizuka K, & Teshima T. (2023). R-Spondin1 protects gastric stem cells and mitigates gastric GVHD in allogeneic hematopoietic stem cell transplantation. Blood Advances, 8(3), 725-731.

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Bacterial Biofilm Formation on Titanium Implants

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Thin, disc-shaped implant samples (diameter, 11 mm Â 2 mm) that were unilaterally polished (DOT GmbH, Rostock, Germany) and comprised commercial pure titanium (cp-Ti, grade 2) and titanium-aluminum-vanadium (Ti6Al4V, grade 5) were used. The samples were sterilized by autoclaving prior to biofilm formation. Streptococcus sanguinis ATCC 10556 as a facultative anaerobic species and Porphyromonas gingivalis W83 as an anaerobic species were used for all experiments. The bacterial strains were commercially purchased (Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and available for long-term storage in the MicrobankTM system of Pro-Lab Diagnostics (Neston, England), according to the manufacturer's instructions. Streptococcus sanguinis (S. sanguinis) was grown under aerobic conditions (5% CO 2 , 20% O 2 ), and Porphyromonas gingivalis (P. gingivalis) was grown under anaerobic conditions (80% N 2 , 10% CO 2 , 10% H 2 ) at 37 C for at least 48 h on Columbia blood agar plates (BD, Franklin Lakes, USA).

Weller J., Vasudevan P., Kreikemeyer B., Ekat K., Jackszis M., Springer A., Chatzivasileiou K, & Lang H. (2022). The role of bacterial corrosion on recolonization of titanium implant surfaces: An in vitro study. Clinical implant dentistry and related research, 24(5).

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