The Mycobacterium peregrinum reference strain (ATCC 14467) was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The two clinical strains (KMRC-A9381 and KMRC-B0585) were obtained from The Korean Mycobacterium Resource Center (KMRC) of the Korean Institute of Tuberculosis (Osong, Republic of Korea). These strains were cultured in Middlebrook 7H9 Broth medium (Difco Laboratories, Detroit, MI, USA) supplemented with 10% oleic albumin dextrose catalase (OADC: Becton Dickinson, Sparks, MD, USA) for 5–7 days at 37 °C.
Oleic albumin dextrose catalase oadc
Manufactured by BD
Sourced in United States, Germany
Oleic albumin dextrose catalase (OADC) is a nutrient supplement used in the cultivation of microorganisms, particularly mycobacteria. It contains oleic acid, albumin, dextrose, and catalase, which provide essential nutrients and growth factors for the growth and maintenance of these organisms in laboratory settings.
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Lab products found in correlation
Genotype ntm dr ver 1, by Hain Lifescience (2 mentions)
Vitek ms, by bioMérieux (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Middlebrook 7h9 broth medium, by BD (1 mentions)
Middlebrook 7h9, by BD (1 mentions)
Thp 1 cells, by Leibniz Institute DSMZ (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Difco middlebrook 7h9, by BD (1 mentions)
Difco middlebrook 7h10 agar, by BD (1 mentions)
Middlebrook 7h9 medium, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Middlebrook 7h9 medium, by BD (1 mentions)
Middlebrook 7h11 agar, by Merck Group (1 mentions)
Lb agar, by Merck Group (1 mentions)
Middlebrook 7h10 mb 7h10 agar, by BD (1 mentions)
Glycerol, by Avantor (1 mentions)
Tween 80, by Avantor (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Merck Group (1 mentions)
12 protocols using oleic albumin dextrose catalase oadc
1
Culturing Mycobacterium peregrinum Strains
2
Intracellular Growth of M. avium hominissuis
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The Mycobacterium avium subsp. hominissuis ATCC 700898 reference strain was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in Middlebrook 7H9 supplemented with 10% oleic albumin dextrose catalase (OADC; Beckton-Dickinson, Vianen, the Netherlands) for 5 days prior to the experiment. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany; ACC 16 Lot 32) and cultured in RPMI (Roswell Park Memorial Institute medium) 1640 with 20% heat-inactivated fetal bovine serum (FBS; Life Technologies Limited, Paisley, UK) for the first three passages and with 10% FBS afterward at 36°C and 5% CO2.
AZT, EMB, RIF, and CFZ were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). Stock solutions of the compounds were prepared in ethanol, Milli-Q water, dimethyl sulfoxide (DMSO) and DMSO, respectively, and stored at −20°C until use. Syringe solutions of AZT (40%/60% (vol/vol) ethanol/Milli-Q water) and EMB (Milli-Q water) were prepared every 3 days. RIF (in 0.08%/99.2% (vol/vol) DMSO/Milli-Q water) was replaced every 2 days due to drug instability. CFZ bolus solutions at 0.1 mg/mL were prepared daily in 10% DMSO, 0.5% Tween 80 in RPMI-2% FBS.
AZT, EMB, RIF, and CFZ were purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). Stock solutions of the compounds were prepared in ethanol, Milli-Q water, dimethyl sulfoxide (DMSO) and DMSO, respectively, and stored at −20°C until use. Syringe solutions of AZT (40%/60% (vol/vol) ethanol/Milli-Q water) and EMB (Milli-Q water) were prepared every 3 days. RIF (in 0.08%/99.2% (vol/vol) DMSO/Milli-Q water) was replaced every 2 days due to drug instability. CFZ bolus solutions at 0.1 mg/mL were prepared daily in 10% DMSO, 0.5% Tween 80 in RPMI-2% FBS.
3
Genomic Analysis of M. abscessus in German CF Patients
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A representative set of M. abscessus isolates has been collected from 14 German CF centers (located in the cities of Essen, Oldenburg, Cologne, Munich, Berlin, Münster, Dresden, Erlangen, Gießen, Tübingen, Würzburg, Hamburg, Heidelberg, and Frankfurt) for the time period of 2004 to 2020. We recorded the date of cultivation, patient age, type of specimen (sputum, bronchoalveolar lavage, endotracheal swabs) and if isolates were primary or sequential isolates. In addition, the duration between the first and last positive culture was recorded. From Frankfurt University Hospital sequential isolates were included, if available. Bacterial culture was performed on Middlebrook 7H10 agar with oleic albumin dextrose catalase (OADC, Becton, Dickinson, Heidelberg, Germany) at 37°C until visible growth could be detected. M. abscessus species identification was verified by Matrix-assisted-laser desorption ionization-time of flight analysis (MALDI-TOF; Vitek MS; bioMérieux, Nürtingen, Germany) and in case of no identification with the GenoType NTM-DR VER 1.0 (Hain Lifescience, Nehren, Germany). Bacterial cultures were then transferred to the German Reference Center for Mycobacteria at Research Center Borstel for DNA extraction and whole-genome sequencing. Furthermore, 14 M. abscessus isolates from non-CF patients were included as controls.
4
Mycobacterial Culture and Handling
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M. smegmatis mc2155 is a laboratory stock strain and was grown as previously described (21 (link)). M. tuberculosis strains were obtained from Sebastien Gagneux Swiss Tropical and Public Health Institute. Liquid cultures were grown by inoculating isolated colonies in 10 ml Middlebrook 7H9 media with oleic albumin dextrose catalase (OADC) (Becton, Dickinson) and 0.05% Tween 80 until visibly dispersed (10 days to 3 weeks) at 37°C with shaking. Lineage 5 and 6 strains were further supplemented with 40 mM sodium pyruvate (Sigma). Strains were grown on solid Middlebrook 7H11 agar (Difco, Remel) supplemented with OADC and 1 mM CaCl2 for 2 to 6 weeks at 37°C .
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5
Genomic Analysis of M. abscessus in German CF Patients
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A representative set of M. abscessus isolates has been collected from 14 German CF centers (located in the cities of Essen, Oldenburg, Cologne, Munich, Berlin, Münster, Dresden, Erlangen, Gießen, Tübingen, Würzburg, Hamburg, Heidelberg, and Frankfurt) for the time period of 2004 to 2020. We recorded the date of cultivation, patient age, type of specimen (sputum, bronchoalveolar lavage, endotracheal swabs) and if isolates were primary or sequential isolates. In addition, the duration between the first and last positive culture was recorded. From Frankfurt University Hospital sequential isolates were included, if available. Bacterial culture was performed on Middlebrook 7H10 agar with oleic albumin dextrose catalase (OADC, Becton, Dickinson, Heidelberg, Germany) at 37°C until visible growth could be detected. M. abscessus species identification was verified by Matrix-assisted-laser desorption ionization-time of flight analysis (MALDI-TOF; Vitek MS; bioMérieux, Nürtingen, Germany) and in case of no identification with the GenoType NTM-DR VER 1.0 (Hain Lifescience, Nehren, Germany). Bacterial cultures were then transferred to the German Reference Center for Mycobacteria at Research Center Borstel for DNA extraction and whole-genome sequencing. Furthermore, 14 M. abscessus isolates from non-CF patients were included as controls.
6
Mycobacterial and E. coli Culture Protocols
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M. tuberculosis H37Rv, M. bovis BCG China (purchased from China National Institutes Food and Drug Control), and rBCG strains were cultured on Difco™ Middlebrook 7H9 (BD, NJ, USA) or Difco™ Middlebrook 7H10 agar (BD, NJ, USA), supplemented with 10% oleic-albumin-dextrose-catalase (OADC) (BD, NJ, USA), 0.5% glycerol, and 0.05% Tween 80. Escherichia coli DH5α, and BL21(DE3) were cultured in Luria-Bertani medium or agar and used for cloning and expression. Kanamycin was used at a concentration of 25 μg/mL and ampicillin used at a concentration of 100 μg/mL.
7
Mycobacterial Culture and Characterization
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Mycobacterial cultures were performed as described previously12 (link). Mtb H37Rv was provided by Dr. R.L. Friedmann (University of Arizona, Tucson, AZ, USA). Mycobacteria were grown in Middlebrook 7H9 medium (Difco, Detroit, MI, USA, 271310) supplemented with 10% oleic albumin dextrose catalase (OADC; BD Biosciences, Franklin Lakes, NJ, USA, 212240), 5% glycerol, and 0.05% Tween 80 (Sigma, P1754). The Mtb strains expressing ERFP were described previously53 (link). The E. coli Mtb shuttle plasmid pMV262-RFP harboring ERFP under the control of the HSP60 promoter was used. Mtb harboring the ERFP gene was cultivated in 7H9 medium supplemented with kanamycin (Sigma, 60615). For D. melanogaster infection, the M. marinum strain Aronson (ATCC 927, fish isolate) was cultured in 7H9 medium with OADC and 0.2% Tween 80 at 30 °C for 8 weeks in the dark without agitation. Single-cell suspensions of Mtb, Mtb-ERFP, and M. marinum were aliquoted and stored at −80 °C. Mid-logarithmic-phase bacteria (OD, 0.6) were used in all assays. The CFUs were enumerated on Middlebrook 7H10 agar (Difco, 262710).
8
Cultivation and Characterization of Mycobacterium
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Mtb H37Rv was kindly provided by Dr. R. L. Friedman (University of Arizona, Tucson, AZ, USA). Mtb was grown at 37 °C with shaking in Middlebrook 7H9 broth (Difco, Paris, France) supplemented with 0.5% glycerol, 0.05% Tween-80 (Sigma-Aldrich), and oleic albumin dextrose catalase (OADC; BD Biosciences). Mtb-expressing enhanced red fluorescent protein (Mtb-ERFP) and recombinant M. smegmatis strains were grown in Middlebrook 7H9 medium supplemented with OADC and 50 μg/ml kanamycin (Sigma-Aldrich). Bacterial strains were then harvested by centrifugation at 3000 rates per min for 30 min and the pellets were resuspended in ice-cold phosphate-buffered saline (PBS). All mycobacterial suspensions were aliquoted and stored at −80 °C until just before use. For all experiments, mid-log-phase bacteria (O.D = 0.6) were used. The number of CFUs of the inoculum was verified by serially diluting and plating on Middlebrook 7H10 agar (Difco).
9
Mycobacterium mucogenicum Strain Cultivation
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The Mmuc reference strain (ATCC49650) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The two clinical strains (KMRC 00136-76002 and KMRC 00136-76003) were obtained from The Korean Mycobacteria Resource Center (KMRC) of the Korean Institute of Tuberculosis (Osong, Korea). Mmuc strains were grown in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with 10% oleic albumin dextrose catalase (OADC; BD Biosciences, San Diego, CA, USA) and 0.5% glycerol at 37 °C for 7 days. The number of colony-forming units (CFU)/mL was determined on 7H10 agar supplemented with OADC at 37 °C.
10
Mycobacterial Cultivation and Strain Preparation
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M. abscessus, M. bolletii, and M. massiliense clinical isolates were a kind gift from the National Mycobacterium Reference Laboratory, Borstel, Germany. An auxotrophic mutant strain of M. tuberculosis (H37Rv ΔpanCD ΔleuCD) was used as a control (Vilchèze et al., 2018 (link)). Mycobacterial strains were grown on Middlebrook 7H11 agar (Sigma-Aldrich) supplemented with 0.5% glycerol and 10% oleic albumin dextrose catalase (OADC) (Becton Dickinson (BD), Oxford, UK), and incubated at 37°C for 3 to 5 days for MABC strains and, 14 to 21 days for M. tuberculosis. E. coli BW25141 was grown on LB agar (Sigma-Aldrich) and used as a further control.
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M. abscessus, M. bolletii, and M. massiliense clinical isolates were a kind gift from the National Mycobacterium Reference Laboratory, Borstel, Germany. An auxotrophic mutant strain of M. tuberculosis (H37Rv ΔpanCD ΔleuCD) was used as a control (Vilchèze et al., 2018 (link)). Mycobacterial strains were grown on Middlebrook 7H11 agar (Sigma-Aldrich) supplemented with 0.5% glycerol and 10% oleic albumin dextrose catalase (OADC) (Becton Dickinson (BD), Oxford, UK), and incubated at 37°C for 3 to 5 days for MABC strains and, 14 to 21 days for M. tuberculosis. E. coli BW25141 was grown on LB agar (Sigma-Aldrich) and used as a further control.
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