Blood agar base no 2

Manufactured by Merck Group

Sourced in Germany

Blood agar base no. 2 is a microbiological culture medium used for the isolation and cultivation of a variety of microorganisms, particularly those that require the presence of blood or blood components for growth. It provides a solid substrate for the growth of bacteria and fungi.

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7 protocols using blood agar base no 2

Bacterial Profiling: Abstinence Effects

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To assess changes in the bacterial profiles following 2 days and 2 h of abstinence, 100 µL of each specimen was inoculated on a selection of sterile agars (blood agar base no. 2, malt extract agar, trypticase soy agar; Merck, Darmstadt, Germany) and incubated under aerobic conditions at 36 ± 2 °C for 24 h. Grown bacterial colonies were counted and re-inoculated and incubated again under aerobic conditions at 37 ± 1 °C for 24 h [22 (link)].
Pure bacterial colonies were identified with the matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) Biotyper mass spectrometry (Brucker Daltonics, Bremen, Germany), previously described by Tvrdá et al. [22 (link)]. Briefly, purified cultures re-suspended in distilled water were treated with 99.8% ethanol (Sigma-Aldrich, St. Louis, MO, USA) and air-dried. The pellet was mixed thoroughly with a solution comprising acetonitrile (Sigma-Aldrich, St. Louis, MO, USA), 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA), centrifuged and transferred to the MALDI identification plate. Dried specimens were covered with a working solution of MALDI matrix and assessed with the Microflex LT instrument and the flexControl software (version 3.4). Obtained data were processed with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [22 (link)].

Tvrdá E., Ďuračka M., Benko F., Kováčik A., Lovíšek D., Gálová E., Žiarovská J., Schwarzová M, & Kačániová M. (2023). Ejaculatory Abstinence Affects the Sperm Quality in Normozoospermic Men—How Does the Seminal Bacteriome Respond?. International Journal of Molecular Sciences, 24(4), 3503.

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Standardized Bacterial Strain Preparation

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The bacterial strains used in this study are summarized in Table 1. The deletion mutants and knock‐back (KB) strains had been generated by markerless gene editing in a previous study (Rørvik et al., 2020 (link)). Bacteria were, unless otherwise stated, grown in tryptone soya broth (TSB) (Oxoid) at 37°C in a humidified atmosphere containing 5% CO₂. Pre‐cultures were prepared by growing bacteria from glycerol stocks (stored at −80°C) on blood agar plates [Blood agar base No.2 (Merck) + 5% defibrinated sheep blood (Oxoid)] overnight, followed by inoculation and growth in TSB to an OD₆₀₀ ≈ 0.5. Glycerol was added to a final concentration of 15%, before aliquotation into Eppendorf tubes and storage at −80°C until further use.
For experiments, bacteria were inoculated in TSB, either directly from blood agar plates or by diluting pre‐cultures 1:10, and grown under standard conditions to mid‐exponential phase (OD₆₀₀ ≈ 0.5). The OD₆₀₀ was then adjusted to 0.1, and these bacterial suspensions (referred to as “synchronized cultures”) were used to start the respective experiments.
Viability assays were performed by making 10‐fold dilutions of samples, spotting five µl of each dilution in duplicates on blood agar plates, and incubating the plates at 37°C, in an atmosphere supplemented with 5% CO₂ for 24 h. The number of colony‐forming units (CFU) was determined.

Rørvik G.H., Naemi A., Edvardsen P.K, & Simm R. (2021). The c‐di‐AMP signaling system influences stress tolerance and biofilm formation of Streptococcus mitis. MicrobiologyOpen, 10(4), e1203.

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Identifying Bacterial Species in Turkey Semen

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For the identification of the bacterial colonies and species in turkey semen, 100 µL of each sample were inoculated onto a sterile blood agar (BA, Blood Agar Base No. 2; Merck, Darmstadt, Germany) and tryptone soya agar (TSA, Soyabean Casein Digest Agar; Merck, Darmstadt, Germany). Subsequently, the plates were incubated under aerobic conditions at 36 ± 2 °C for 24 h. Following cultivation, the colonies were counted and transferred to a fresh TSA to obtain pure cultures, which were incubated again for 24 h at 37 ± 1 °C under aerobic conditions.

Lenický M., Slanina T., Kačániová M., Galovičová L., Petrovičová M., Ďuračka M., Benko F., Kováč J, & Tvrdá E. (2021). Identification of Bacterial Profiles and Their Interactions with Selected Quality, Oxidative, and Immunological Parameters of Turkey Semen. Animals : an Open Access Journal from MDPI, 11(6), 1771.

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Cultivation of Anaerobic and Aerobic Microbes

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P. gingivalis ATCC 33277, Prevotella intermedia ATCC 49046, Fusobacterium nucleatum ATCC 49256, and Aggregatibacter actinomycetemcomitans ATCC 43718 were routinely grown on agar (Blood agar base no. 2, Sigma‐Aldrich NV) supplemented with 5% horse blood (Horse Blood Defibrinated, Oxoid NV), hemin (5 μg/ml; Sigma) and menadione (1 μg/ml; Sigma) at 37 °C under anaerobic conditions (90% N₂, 5% H₂, and 5% CO₂) using an Anoxomat AN2OP system (Mart Microbiology, Drachten, the Netherlands). Streptococcus mutans ATCC 25175,

Staphylococcus aureus

SH1000 (O'Neill, 2010),

Staphylococcus epidermidis

CH (Costerston, Marrie, & Cheng, 1985),

Escherichia coli

TG1 (Carter, Bedouelle, & Winter, 1985), Pseudomonas aeruginosa (Lee et al., 2006), and Salmonella enterica serovar Typhimurium SL1344 (Hoiseth & Stocker, 1981) were grown routinely on solid trypticase soy broth (TSB, Becton Dickinson Benelux) containing 1.5% agar at 37 °C. AM404, arvanil, 4′‐hydroxystearanilde, and R(+)‐methanandamide were purchased from Sigma‐Aldrich and stock solutions of 20 mM were prepared in dimethyl sulfoxide (DMSO).

Gerits E., Spincemaille P., De Cremer K., De Brucker K., Beullens S., Thevissen K., Cammue B.P., Vandamme K., Fauvart M., Verstraeten N, & Michiels J. (2017). Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis. Clinical and Experimental Dental Research, 3(2), 69-76.

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Quantifying H. pylori Colonization in Mice

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H. pylori infection levels within mouse gastric tissues were quantified by colony-forming assay. Briefly, stomachs were opened along the inner curvature and bisected longitudinally. One half was placed in BHI and homogenized (GmbH Polytron homogeniser, Kinematica, Switzerland) and the other half rapidly frozen for RNA extraction. Ten-fold serial dilutions were prepared in BHI broth and aliquots spread over GSSA selective agar plates [Blood Agar Base No. 2 with 5% horse blood, vancomycin (12 mg/mL), polymyxin B (0.40 mg/mL), bacitracin (24 mg/mL), nalidixic acid (1.3 mg/mL), and amphotericin B (3.75 mg/mL), all from Sigma]. After 5 days culture as above, colonies were counted, and the number of colony-forming units was calculated per stomach (McGuckin et al., 2007 (link)). Colonies were confirmed to be H. pylori by the rapid urease test as previously described (Sutton et al., 2000 (link)).

Sheng Y.H., Ng G.Z., Summers K.M., Every A.L., Price G., Hasnain S.Z., Sutton P, & McGuckin M.A. (2020). Influence of the MUC1 Cell Surface Mucin on Gastric Mucosal Gene Expression Profiles in Response to Helicobacter pylori Infection in Mice. Frontiers in Cellular and Infection Microbiology, 10, 343.

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Pneumococcal Strain Growth Assay

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S. pneumoniae strains used in this study are listed in Supplementary Table 1. Thirty of them (PG01 to PG30) belong to a surveillance study done in a Nijmegen hospital, the Netherlands^{23 (link)}, while the other six are lab strains available at our lab.
Different strains were grown on tryptic soy agar or blood agar base no. 2 (Sigma-Aldrich) plates supplemented with 5% defibrinated sheep’s blood at 37 °C in a 5% CO₂ atmosphere. Liquid cultures were grown statically in THY, C+Y or semi-defined minimal media (SDMM) at pH 7.3, with 5 µl ml⁻¹ oxyrase (Oxyrase)^{2 (link)} at the same incubation conditions as plates. For growth curve assays, strains were grown in THY until an optical density (OD)₆₀₀ of ~0.5, pelleted and resuspended in the same volume of phosphate saline buffer (PBS). OD₆₀₀ was adjusted to 0.05 in PBS and 20 μl of this suspension were diluted in 180 μl of different media conditions in wells of flat-bottom 96-well plates. OD₆₀₀ measurements were taken on a BioSpa 8 plate reader (BioTek) and experiments were repeated at least three times. Different modifications to SDMM were assayed as described in figure legends and text.

Rosconi F., Rudmann E., Li J., Surujon D., Anthony J., Frank M., Jones D.S., Rock C., Rosch J.W., Johnston C.D, & van Opijnen T. (2022). A bacterial pan-genome makes gene essentiality strain-dependent and evolvable. Nature Microbiology, 7(10), 1580-1592.

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Growth Kinetics of S. pneumoniae Strains

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S. pneumoniae strains used in this study are listed in Supplementary Table 1. Thirty of them (PG01 to PG30) belong to a surveillance study done in a Nijmegen hospital, NL 25 , while the other six are lab strains available at our lab.
Different strains were grown on Tryptic Soy Agar or Blood Agar Base no. 2 (Sigma-Aldrich) plates supplemented with 5% defibrinated sheep's blood at 37 o C in a 5% CO2 atmosphere. Liquid cultures were grown statically in THY, C+Y or semi-defined minimal media (SDMM) at pH 7.3, with 5 µl/ml Oxyrase (Oxyrase, Inc) 2 at the same incubation conditions as plates. For growth curve assays, strains were grown in THY until an OD600 of ~0.5, pelleted and resuspended in same volume of phosphate saline buffer (PBS). OD600 was adjusted to 0.05 in PBS and 20 µL of this suspension were diluted in 180 µL different media conditions in wells of flat bottom 96-well plates. OD600 measurements were taken on a BioSpa 8 plate reader (BioTek), and experiments were repeated at least three times. Different modifications to SDMM were assayed as described in figure legends and text.

Rosconi F., Rudmann E., Li J., Surujon D., Anthony J., Frank M.W., Jones D.S., Rock C.O., Rosch J.W., Johnston C.D., & Opijnen T.v. (2022). A bacterial pan-genome makes gene essentiality strain-dependent and evolvable.

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