Frozen stocks of Clostridium scindens (ATCC 35704) or engineered Bacteroides strains were streaked on Columbia agar plates (BD) and grown at 37°C inside an anaerobic chamber. Bacteria were scraped from agar plates with bacteriological loops into sterile anaerobic PBS. Consortia were assembled and transported in airtight tubes to the animal facility and administered to experimental animals by oral gavage. Mice were housed in flexible PVC isolators (Park Bioservices) or Sentry Sealed positive pressure cages (SPP, Allentown Inc.) for the duration of the experiments. For experiments in SPP cages, animals were manipulated with sterile gloves using aseptic techniques inside biosafety cabinets.
Columbia agar plates
Manufactured by BD
Sourced in Germany, United States
Columbia agar plates are a type of culture media used for the isolation and growth of a wide range of microorganisms. They provide a standardized substrate for culturing bacteria, fungi, and other microbes in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Sheep blood, by BD (1 mentions)
Qiaamp dna purification kit, by Qiagen (1 mentions)
Gentamicin, by BD (1 mentions)
Densitomat 2, by bioMérieux (1 mentions)
Mueller hinton agar (mha), by Biomaxima (1 mentions)
Yeast extract, by BD (1 mentions)
Starch, by Merck Group (1 mentions)
11 protocols using columbia agar plates
1
Oral Administration of Anaerobic Bacterial Consortia
2
Gut Microbiome Reconstitution in GF Mice
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GF mice were maintained in isolators until the day of reconstitution. Upon transfer to the SPF facility, mice were reconstituted via oral gavage with comparable amounts of Clostridiales of interest (C. ramosum, C. saccharogumia, C. hathewayi, and B. producta [I]) of ∼1–3 × 106/bacterium/mouse, resuspended in 200 µl of reduced PBS. Alternatively, GF mice were reconstituted with a suspension in reduced PBS of a fecal pellet from MNVC-treated mice (pellet collected 1 d after clindamycin treatment, 1 pellet per ml, 200 µl/mouse). Engraftment was confirmed by plating of fecal pellets onto Columbia Agar Plates with 5% Sheep Blood (BD) in anaerobic chamber, 2 d after reconstitution.
3
Cultivation and Biofilm Formation of S. epidermidis
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S. epidermidis RP62A (ATCC 35984; American Type Culture Collection, Manassas, USA) was routinely grown in tryptic soya broth medium (TSB, CASO-bouillon, ROTH GmbH, Karlsruhe, Germany). Static cultures were incubated at 37 °C and under a 20% O2/5% CO2 atmosphere. Columbia agar plates (BD, Heidelberg, Germany) were used for CFU determination and incubated for 24 h prior to counting. Biofilms were grown in accordance with a previously published in vitro test model for implant associated infections [23 (link)].
4
Quantifying Bacterial Burden in Lung Tissues
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Bronchoalveolar lavage (BAL) was performed as described elsewhere [31 (link)]. Lungs were removed and homogenized in 500 µL PBS. BAL fluid, blood and lung tissue homogenate were serially diluted, plated on Columbia-Agar plates (BD Bioscience, Heidelberg, Germany), incubated at 37 °C for 18 h and bacterial colonies were counted to calculate the CFUs per ml tissue/liquid.
5
Spore Preparation for Bacillus and Geobacillus
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Bacillus anthracis Sterne 1043, Bacillus subtilis ATCC 19659, and Bacillus atrophaeus ATCC 51189 were maintained on Columbia agar plates (BD Diagnostic Systems) and grown at 37°C. Spore suspensions were prepared using the method of Leighton and Doi (1971 ). Spore stock solutions were quantified by serial dilutions and viable plating. Stocks typically yielded between 1 × 108–1 × 109 CFU/ml. Suspensions were stored at 4°C until use. Geobacillus stearothermophilus ATCC 12980 was maintained on Columbia agar. Spore suspensions were created by growing lawns of G. stearothermophilus on nutrient agar supplemented with Mg+2, Mn+2, Ca+2, K+, and Fe+2 ions at 55°C for 8–10 days until sporulation was greater than 90%. Spores were harvested by adding cold PSS +0.01% Tween 80 (PSST) to plates, scraping off the spores, and centrifugation at 4000 × g for 15 min to pellet the spores. Washing by centrifugation and resuspension in PSST was repeated 5 times, and suspensions were checked for purity via phase‐contrast microscopy before storage at 4°C until use.
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Bacillus anthracis Sterne 1043, Bacillus subtilis ATCC 19659, and Bacillus atrophaeus ATCC 51189 were maintained on Columbia agar plates (BD Diagnostic Systems) and grown at 37°C. Spore suspensions were prepared using the method of Leighton and Doi (
6
Oral Administration of Anaerobic Bacterial Consortia
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Frozen stocks of Clostridium scindens (ATCC 35704) or engineered Bacteroides strains were streaked on Columbia agar plates (BD) and grown at 37°C inside an anaerobic chamber. Bacteria were scraped from agar plates with bacteriological loops into sterile anaerobic PBS. Consortia were assembled and transported in airtight tubes to the animal facility and administered to experimental animals by oral gavage. Mice were housed in flexible PVC isolators (Park Bioservices) or Sentry Sealed positive pressure cages (SPP, Allentown Inc.) for the duration of the experiments. For experiments in SPP cages, animals were manipulated with sterile gloves using aseptic techniques inside biosafety cabinets.
7
Bacterial Isolation and DNA Extraction
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All bacterial isolates used in this study are listed in Table S2 in the supplemental material. B. pseudomallei K96243 was used as the reference strain. Isolates were stored at −80°C with 20% (vol/vol) glycerol in tryptic soy broth (TSB), were streaked onto Columbia agar plates supplemented with 5% sheep blood (Becton Dickinson, Heidelberg, Germany), and were incubated overnight under aerobic conditions at 37°C. Fresh cultures grown overnight in Lennox broth or tryptic soy broth (TSB) were harvested for bacterial DNA extraction using a QIAamp DNA purification kit (Qiagen, Hildesheim, Germany).
8
Isolation and Identification of Helicobacter pylori
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Each biopsy transported in BHI broth with 10 % glycerol was macerated with a sterile wood applicator. In total, 50 µl of the homogenates were cultivated on Columbia Agar plates (Becton Dickinson, NC, USA) added with 10 % ram blood, IsoVitaleX Enrichment and Helicobacter pylori selective supplement Dent (10 mg/L of vancomycin, 5 mg/L of trimethroprim, 5 mg/L of cefsulodin, 5 mg/L of amphotericin B) (Oxoid, Basingstoke, UK) at pH 6.8–7.0. The homogenates were distributed on the culture medium by isolation strip. The inoculated plates were incubated under microaerophilic conditions with 5 % O2, and 5 % CO2 at 37 °C in GasPak jars for 3–7 days. H. pylori was identified by colony morphology (small, transparent colonies, 1 mm in diameter), Gram staining and biochemical tests (urease, catalase and oxidase positive). H. pylori strain ATCC 43504 was used as a positive control.
9
Isolation and Identification of Helicobacter pylori
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Each biopsy transported in BHI broth with 10% glycerol was macerated with a sterile wood applicator. Fifty microliters of the homogenates were cultivated on Columbia Agar plates (Becton–Dickinson, North Carolina, USA) supplemented with 10% ram blood, IsoVitaleX Enrichment and Helicobacter pylori selective supplement Dent (10 mg/L of vancomycin, 5 mg/L of trimethoprim, 5 mg/L of cefsulodin, 5 mg/L of amphotericin B) (Oxoid, Basingstoke, UK) at pH 6.8 to 7.0. The homogenates were distributed on the culture medium by isolation strip. The inoculated plates were incubated under microaerophilic conditions with 5% O2 and 5% CO2 at 37 °C in GasPak jars for 3–7 days. H. pylori was identified by colony morphology (small, transparent colonies, 1 mm in diameter), Gram staining and biochemical tests (urease, catalase and oxidase positive). H. pylori strain ATCC 43504 was used as positive control.
10
Gentamicin Susceptibility Testing of Staphylococci
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The 24 h cultures of staphylococci strains on Columbia agar plates (Becton, Dickinson and Company, Heidelberg, Germany) were used for the method purpose. Firstly, the bacterial suspension at a density of 0.5 McFarland (densitometer Densitomat II, BioMerieux, Warsaw, Poland) was prepared in sterile 0.9% NaCl (Chempur, Piekary Slaskie, Poland). Next, using a sterile swab stick, the inoculum was seeded on the on the Mueller–Hinton agar (Biomaxima, Lublin, Poland) by streaking the swab three times over the agar surface. A disc impregnated with 10 µg gentamicin (Becton, Dickinson and Company, Sparks, MD, USA) was placed on the inoculated agar plate and incubated for 18 h at 35 °C. After the time period, the inhibition zones were measured and interpreted with the European Committee on Antimicrobial Susceptibility Testing breaking points table [78 ].
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