The BALF samples were quantitatively cultured for bacteria by inoculation of 100 μL onto a medium for the growth of fastidious organisms with clearly visible hemolytic reactions (Thermo Scientific Columbia agar plate with 5% sheep bloodPLUS, Wesel, Germany). Subsequently, samples were serially diluted in sterile water to 10−1, 10−3, and 10−5. One hundred microliter of each dilution then was inoculated onto a Columbia agar plate with 5% sheep bloodPLUS (Thermo Scientific, Wesel, Germany). Plates were incubated at 37°C for at least 24 hours. Plates were examined for growth after both 24 and 48 hours of incubation. When growth was observed, the number of colony forming units (cfu/mL) was calculated by multiplying the number of colonies by 10, and applying the dilution factor. Bacterial identification and antibiotic susceptibility testing also were performed (VITEK‐2 analyzer, Biomerieux, Craponne, France). At the discretion of the clinician, quantitative polymerase chain reaction (qPCR) was performed for Mycoplasma sp. detection. Specific mycoplasma cultures were not performed. Finally, when >1 bacterial species were retrieved from BALF culture, the species with the highest cfu/mL was used for statistical analysis.
Columbia agar plate
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Germany
Columbia agar plates are a type of microbiological culture media used for the growth and isolation of a wide range of bacterial species. The plates are comprised of Columbia agar, a nutritious growth medium that supports the cultivation of diverse bacterial populations. These plates provide a standardized and consistent platform for laboratory procedures such as bacterial identification, antimicrobial susceptibility testing, and other microbiological applications.
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Lab products found in correlation
Sheep blood, by Thermo Fisher Scientific (9 mentions)
Brucella broth, by BD (3 mentions)
Oxoid campygen, by Thermo Fisher Scientific (3 mentions)
Dneasy kit, by Qiagen (2 mentions)
Wilkins chalgren broth, by Thermo Fisher Scientific (2 mentions)
Bca protein quantitative method, by Beyotime (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Todd hewitt broth, by Thermo Fisher Scientific (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Hemin, by Merck Group (1 mentions)
Chocolate agar plate, by Thermo Fisher Scientific (1 mentions)
Columbia agar base, by BD (1 mentions)
Gc agar plates, by Thermo Fisher Scientific (1 mentions)
Vitox, by Thermo Fisher Scientific (1 mentions)
Tryptone soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Cellulose acetate filter, by Merck Group (1 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Campygen gas pack, by Thermo Fisher Scientific (1 mentions)
48 protocols using columbia agar plate
1
BALF Bacterial Quantification and Identification
2
Selective Agar Plates for Bacterial Isolation
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We used plates containing Columbia agar base (Becton Dickinson Norway AS) supplemented with 15 mg/L NAD (BioNor Laboratories AS, Norway) and 15 mg/L hemin (Sigma Aldrich Norway AS), as well as chocolate agar plates (Oxoid).
Selective agar plates were Columbia agar plates containing rifampicin (10 mg/L) (rif plates) and rifampicin (10 mg/L) + azithromycin (30 mg/L) (rif+azm plates). The plates were incubated at 35°C, 5% CO2 for 24 or 48 h before colony counting.
Selective agar plates were Columbia agar plates containing rifampicin (10 mg/L) (rif plates) and rifampicin (10 mg/L) + azithromycin (30 mg/L) (rif+azm plates). The plates were incubated at 35°C, 5% CO2 for 24 or 48 h before colony counting.
3
Extracellular DNA extraction from bacterial strains
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Extracellular DNA was extracted from the matrix of Escherichia coli ATCC25922, E. coli ATCC 472217, E. coli G39, E. coli dPHF, E. coli MUP6, Porphyromonas gingivalis ATCC BAA-308, Burkholderia burgdorferi ATCC35210, and Tetzosporium hominimis VT-49. All bacterial strains were subcultured from freezer stocks onto Columbia agar plates (Oxoid, Hampshire, UK) and were incubated at 37 °C for 48 h. Human genomic DNA (0.2 g/L in 10 mM tris-HCl, 1 mM EDTA, pH 8.0, Cat. No. 11691112001) was purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA) and consisted of high molecular weight (>50,000 bp) genomic DNA isolated from human blood.
4
Bacterial Culture and Antibiotic Conditions
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Escherichia coli and N. meningitidis strains, plasmids and PCR primers used in this study are listed in Table 1 . DH5α Escherichia coli was grown in Luria-Bertani (LB) broth or on LB-agar plates at 37°C. N. meningitidis was grown at 37°C in a humidified 5% CO2 atmosphere on GC agar plates (Oxoid) supplemented with VitoX (2% v/v, Oxoid SR0090A) or, for genetic manipulations, on Columbia Agar plates supplemented with horse blood (Oxoid) for serum bactericidal assay (SBA). Tryptone Soy Broth (Oxoid) was used for liquid cultures of N. meningitidis. When required, media were supplemented with antibiotics (Sigma-Aldrich): ampicillin (amp) 100 μg/ml, kanamycin (kan) 50 μg/ml for E. coli and 100 μg/ml for N. meningitidis, erythromycin (ery) 300 μg/ml for E. coli and 5 μg/ml for N. meningitidis, tetracycline (tet) 5 μg/ml for E. coli and 2 μg/ml for N. meningitidis. Normal human serum (NHS) obtained from a healthy adult human volunteer with no previous history of meningococcal disease or immunization was used as complement source for SBA and hfH binding ELISA.
5
Helicobacter pylori Infection Protocol
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H. pylori was cultured on Columbia agar plates (Oxoid, Basingstoke Hampshire, UK) containing 5% sheep blood, at 37 °C under microaerophilic conditions. Colonies were identified as H. pylori by morphology, Gram staining, positive reactions to oxidase, catalase, and urease activities. H. pylori were suspended in phosphate buffered saline (PBS) to estimate the concentration by spectrophotometry (OD600 nm, A600 = 1 × 108 CFU/mL). According to the concentration proportion of H. pylori:cell = 100:1 (antibiotic-free medium with 10% fetal bovine serum was used to dilute H. pylori), and the H. pylori solutions were added to infect MKN45 or SGC-7901 cells.
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H. pylori was cultured on Columbia agar plates (Oxoid, Basingstoke Hampshire, UK) containing 5% sheep blood, at 37 °C under microaerophilic conditions. Colonies were identified as H. pylori by morphology, Gram staining, positive reactions to oxidase, catalase, and urease activities. H. pylori were suspended in phosphate buffered saline (PBS) to estimate the concentration by spectrophotometry (OD600 nm, A600 = 1 × 108 CFU/mL). According to the concentration proportion of H. pylori:cell = 100:1 (antibiotic-free medium with 10% fetal bovine serum was used to dilute H. pylori), and the H. pylori solutions were added to infect MKN45 or SGC-7901 cells.
6
Isolation and Identification of H. pylori
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Fragments of gastric mucosa were homogenized in 200 μL of sterile saline solution 0.89%. The homogenate was seeded in columbia agar plates (Oxoid, Basingstoke, Hampshire, England) with defibrillated lamb blood to 7% plus selective supplement for H. pylori (Dent) and incubated in microaerophilic conditions (6% O2, 6% CO2, 88% N2 using CampyPak Plus Envelop, BBL, Nashville, TN United States) at 37 °C during 4 to 8 d[4 (link)]. The compatible colonies with H. pylori were transferred to columbia agar defibrillated lamb blood to 10%, to purify and identify them by the tests of urease, catalase, oxidase, Gram staining. A PCR of the ureA gene was used to confirm the species[14 (link)].
7
Extracellular DNA Extraction and DNase Treatment
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Extracellular DNA was extracted from the matrix of P. aeruginosa ATCC 27853, E. coli ATCC 25922, Escherichia coli 472217, Porphyromonas gingivalis, Borrelia burgdorferi.
Tetzerella alzheimeri VT-16-1752, Tetzosporium hominis and Candida albicans.
All bacterial strains were subcultured from freezer stocks onto Columbia agar plates (Oxoid, UK) and incubated at 37 °C for 48 h, fungal strain was subcultured from freezer stocks onto Sabouraud dextrose agar (Oxoid, UK) and incubated at 30 °C for 48 h.
To extract the extracellular DNA, bacterial and fungal cells were separated from the matrix by centrifugation at 5000 g for 10 min at 4 °C. The supernatant was aspirated and filtered through a 0.2-μm-pore-size cellulose acetate filter (Millipore Corporation, USA). Extracellular DNA was extracted by using a DNeasy kit (Qiagen). Human genomic DNA (Roche Cat#11691112001) was purchased from Sigma (Sigma-Aldrich).
A part of the DNA probes were treated with 100 units of DNase I (Sigma- Aldrich, USA) for 20 minutes at 37 °C in order to degrade DNA in the probes.
Tetzerella alzheimeri VT-16-1752, Tetzosporium hominis and Candida albicans.
All bacterial strains were subcultured from freezer stocks onto Columbia agar plates (Oxoid, UK) and incubated at 37 °C for 48 h, fungal strain was subcultured from freezer stocks onto Sabouraud dextrose agar (Oxoid, UK) and incubated at 30 °C for 48 h.
To extract the extracellular DNA, bacterial and fungal cells were separated from the matrix by centrifugation at 5000 g for 10 min at 4 °C. The supernatant was aspirated and filtered through a 0.2-μm-pore-size cellulose acetate filter (Millipore Corporation, USA). Extracellular DNA was extracted by using a DNeasy kit (Qiagen). Human genomic DNA (Roche Cat#11691112001) was purchased from Sigma (Sigma-Aldrich).
A part of the DNA probes were treated with 100 units of DNase I (Sigma- Aldrich, USA) for 20 minutes at 37 °C in order to degrade DNA in the probes.
8
Microaerobic Campylobacter Growth
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Bacteria were grown microaerobically (Oxoid Campygen, Thermo Fisher Scientific, Waltham, USA) at 42°C and routinely resuscitated from frozen stocks by first growing overnight on blood agar (Columbia agar plates supplemented with 5% horse blood; Oxoid, Basingstoke, UK) followed by overnight incubation in Brucella broth (Becton, Dickinson and Company, Franklin Lakes, USA). To collect bacteria, cultures were centrifuged at 8000g for 5–10 min.
9
Quantification of C. coli Gut Burdens
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For quantification of the gastrointestinal C. coli burdens, fecal and luminal stomach, duodenum, ileum, and colon samples were homogenized and serial dilutions streaked onto Columbia agar plates supplemented with 5% sheep blood and onto selective Karmali plates (both from Oxoid, Wesel, Germany). The inoculated plates were incubated in a jar containing CampyGen gas packs (Oxoid, Wesel, Germany) under microaerophilic conditions (37 °C, 48 h). C. coli were identified by the distinct macroscopic morphotypes, microscopic appearance following Gram-staining, and oxidase-positive reaction.
10
Biofilm Formation on Nephrostomy Catheters
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Sterile 4 cm segments of 16-gauge percutaneous nephrostomy polyurethane and silicone catheters were immersed in TSB containing 106 CFUml− 1 of C. striatum and incubated at 37 °C for 48 h then Maki’s semi-quantitative roll plate technique were performed. Basically, after washing (three times) with phosphate buffered saline (PBS) 0.1 M pH 7.2, contaminated abiotic substrates were rolled up on Columbia agar plates supplemented with 5% sheep blood (Oxoid, Germany) for 48 h at 37 °C were analyzed presence of bacterial carpet [23 , 35 (link)].
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