At 12 weeks after boosting with MVA vectors, mice were challenged with 50–100 CFU of aerosolized M. tuberculosis H3Rv strain (ATCC No. 27294, Rockville, MD) in a BSL-3 laboratory using a GlasCol aerosol infection system (Terre Haute, IN), as described elsewhere [17 (link), 19 (link)]. Six weeks later, the mice were euthanized for harvest of lungs and spleens. To determine bacterial loads, harvested organs were homogenized, serially diluted, and plated on Middlebrook 7H10 agar plates containing 10% oleic-acid albumin-dextrose-catalase (OADC – BD, Sparks, MD) and 2 μg/mL of 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich, St. Louis, MO) to selectively inhibit the growth of residual BCG. The plates were incubated at 37°C for 21 days before colonies were counted.
Oleic acid albumin dextrose catalase
Manufactured by BD
Sourced in United States
Oleic acid-albumin-dextrose-catalase is a laboratory reagent that serves as a growth supplement for culturing various microorganisms. It provides a source of fatty acids, carbohydrates, and enzymes to support the nutritional needs of the target organisms during cultivation.
Automatically generated - may contain errors
Lab products found in correlation
Tween 80, by Merck Group (17 mentions)
Middlebrook 7h9 broth, by BD (14 mentions)
Albumin dextrose catalase, by BD (10 mentions)
Glycerol, by Merck Group (8 mentions)
Middlebrook 7h10 agar, by BD (6 mentions)
Middlebrook 7h9 medium, by BD (5 mentions)
Glycerol, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Middlebrook s 7h9 broth medium, by BD (3 mentions)
Isoniazid, by Merck Group (3 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (2 mentions)
Middlebrook 7h11 agar, by BD (2 mentions)
Rifampicin, by Merck Group (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Glycerol, by Thermo Fisher Scientific (2 mentions)
Dna polymerase, by Takara Bio (2 mentions)
2 thiophenecarboxylic acid hydrazide, by Merck Group (1 mentions)
Luria bertani broth or agar, by BD (1 mentions)
Oleic acid albumin dextrose catalase supplement, by BD (1 mentions)
Casamino acid, by BD (1 mentions)
51 protocols using oleic acid albumin dextrose catalase
1
Aerosol Tuberculosis Challenge in Mice
2
In vitro Macrophage Infection Assay for Mycobacterium tuberculosis
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For in vitro macrophage assays, a log-phase culture of M. tuberculosis was pelleted and resuspended in macrophage culture medium. Bacterial single-cell suspensions were prepared by low speed centrifugation (60g for 8 minutes). The number of M. tuberculosis in the resulting supernatant was estimated by measuring absorbance at 600 nm, followed by infection of macrophages at a MOI of 1, 5, or 10. The infectious dose administered was calculated by plating CFUs from an aliquot of the bacterial suspension. After 4 hours, macrophages were washed 3 times with warm media to remove extracellular bacteria. To estimate intracellular M. tuberculosis growth, infected macrophages were lysed in 0.06% sodium dodecylsulfate (SDS) solution at the indicated time points, and serial dilutions of the lysates were plated on 7H10 agar plates (catalog 283810; BD Biosciences) containing glycerol and Middlebrook OADC enrichment (oleic acid-albumin-dextrose-catalase, catalog 212351; BD Biosciences). The number of CFUs was calculated 14 to 21 days later.
3
Cultivation of Mycobacterial Species
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M. tuberculosis mc26206 (an avirulent ΔpanCDΔleuCD mutant of M. tuberculosis H37Rv; kind gift from Dr. W. R. Jacobs Jr., Albert Einstein College of Medicine, New York) and Mycobacterium smegmatis mc2155 were grown in Middlebrook 7H9 broth with 10% oleic acid-albumin-dextrose-catalase supplement (BD Biosciences), 0.5% glycerol, and 0.05% Tween 80 or on Middlebrook 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase (BD Biosciences) and 0.5% glycerol. All media used to grow M. tuberculosis mc26206 were supplemented with 0.2% casamino acids, 48 μg/ml pantothenate, and 20 μg/ml l -leucine. Escherichia coli DH5α, the strain used for cloning, was grown in Luria-Bertani (LB) broth or agar (BD Biosciences). Kanamycin (20–50 μg/ml), hygromycin (50–150 μg/ml), ampicillin (100 μg/ml), and 2% sucrose were added to the culture media when needed.
4
Mycobacterial Culture Protocols
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Mycobacterial species were cultured in either Middlebrook 7H9 broth or Middlebrook 7H10 agar media supplemented with albumin–dextrose–catalase (ADC) or oleic acid–albumin–dextrose–catalase (OADC) enrichments and 0.2% glycerol, 0.2% casamino acids, 24 μg/mL pantothenate, 1 μg/mL penicillin G, 10 μg/mL cyclohexamide, purchased from BD Biosciences. All reagents were purchased from Sigma-Aldrich, Gillingham, UK unless stated otherwise.
5
Mycobacterium tuberculosis Strain Characterization
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M.tb 1,458 strain (GenBank accession no. NZ_CP013475) is a strain of bovine origin isolated by the National Animal Tuberculosis Para-Reference Laboratory (Wuhan) at Huazhong agricultural university, Wuhan, China (Xiong et al., 2017 (link)). BCG Tokyo 172 (ATCC 35737, GenBank accession no.GCA_000010685.1) was a kind gift from Prof. Junyan Liu (School of Basic Medicine Sciences, Wuhan University, China). Both strains were grown in broth medium Middlebrook 7H9 with 10% of oleic acid–albumin–dextrose–catalase (BD, Franklin, NJ, United States), 0.5% glycerol (Sigma–Aldrich), and 0.05% Tween 80 (Sigma–Aldrich) at 37°C. The human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, United States) containing 10% fetal bovine serum (Gibco, United States) with 5% CO2 at 37°C.
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M.tb 1,458 strain (GenBank accession no. NZ_CP013475) is a strain of bovine origin isolated by the National Animal Tuberculosis Para-Reference Laboratory (Wuhan) at Huazhong agricultural university, Wuhan, China (Xiong et al., 2017 (link)). BCG Tokyo 172 (ATCC 35737, GenBank accession no.GCA_000010685.1) was a kind gift from Prof. Junyan Liu (School of Basic Medicine Sciences, Wuhan University, China). Both strains were grown in broth medium Middlebrook 7H9 with 10% of oleic acid–albumin–dextrose–catalase (BD, Franklin, NJ, United States), 0.5% glycerol (Sigma–Aldrich), and 0.05% Tween 80 (Sigma–Aldrich) at 37°C. The human acute monocytic leukemia cell line THP-1 (ATCC TIB-202) was cultured in RPMI 1640 medium (HyClone, United States) containing 10% fetal bovine serum (Gibco, United States) with 5% CO2 at 37°C.
6
Culturing Mycobacterium tuberculosis H37Rv
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M. tuberculosis strain H37Rv was grown to mid-log phase in 5 ml of Middlebrook 7H9 (Difco) liquid culture medium supplemented with 0.5% glycerol, 0.15% Tween 80, and 10% oleic acid-albumin-dextrose-catalase (BD Biosciences). Bacteria were then washed twice with 45 ml phosphate-buffered saline (PBS; Gibco). Bacterial density was measured with a spectrophotometer at an absorbance of 600 nm.
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7
Standardized Cultivation of M. abscessus
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For all experiments, M. abscessus ATCC 19977 (American Type Culture Collection) was used. Stock cultures were stored at −80°C in Middlebrook 7H9 broth (BD Difco) supplemented with 10% oleic acid albumin dextrose catalase (BD Biosciences) and 20% glycerol. With each experiment, a fresh vial of bacteria was thawed, centrifuged at 8,000 rpm for 5 min, and the bacterial pellet was resuspended in fresh media. Bacteria were incubated at 37°C for ~24 hours until at logarithmic growth phase prior to diluting for initial inoculum. With the exception of glycerol stocks, all bacterial culture was carried out in Cation-adjusted Mueller-Hinton II broth (MH2; Sigma-Aldrich or VWR). Amikacin sulfate powder was purchased from Sigma-Aldrich. Stock amikacin was stored at 25 mg/mL in sterile ddH2O at −20°C.
8
Genetically Modified Mycobacterium marinum Strains
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Mycobacterium marinum M. marinum ,2011 ). M. marinum
Strains were routinely grown at 30 °C on Middlebrook 7H10 agar plates (Difco) supplemented with 10% oleic acid‐albumin‐dextrose‐catalase (BD Bioscience) or in Middlebrook 7H9 broth (Difco) with 10% Middlebrook albumin‐dextrose‐catalase (BD Bioscience), 0.05% Tween‐80, and the appropriate antibiotic selection marker.
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Strains were routinely grown at 30 °C on Middlebrook 7H10 agar plates (Difco) supplemented with 10% oleic acid‐albumin‐dextrose‐catalase (BD Bioscience) or in Middlebrook 7H9 broth (Difco) with 10% Middlebrook albumin‐dextrose‐catalase (BD Bioscience), 0.05% Tween‐80, and the appropriate antibiotic selection marker.
9
Infection of U937 cells with Mycobacterium tuberculosis
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We grew human embryonic kidney 293T cells (ATCC, Manassas, VA), the human monocytic U937 and T-lymphocytic Jurkat cells (ATCC, Manassas, VA), and the chronically infected U1 and J1.1 cells (AIDS Research and Reference Reagent Program, National Institutes of Health) in culture medium as recommended by the ATCC. For differentiation and HIV-1 activation, U937 and U1 cells were treated with 5 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) for 12 h. The bacterial strains used in this study were as follows: M. tuberculosis H37Rv and the field isolates Jal 2287 and MYC 431 (kind gift from Dr. Kanury V.S. Rao, ICGEB, New Delhi, India). Bacteria were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) oleic acid albumin dextrose catalase (BD Biosciences), 0.1% (v/v) glycerol, and 0.1% (v/v) Tween 80 until the mid-log phase (A600nm of 0.8). The PMA-differentiated U937 cells were seeded at 2 × 105 cells per well in 24-well plates and infected with M. tuberculosis strains H37Rv, Jal 2287, and MYC 431 at a multiplicity of infection (m.o.i.) of 10 for 4 h. Extracellular bacteria were removed by washing twice with 1× PBS.
10
Preparation of Mycobacterium Reference Strains
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Thirteen reference strains from Mycobacterium and three non-mycobacterial bacteria reference strains were used for testing the effect of MTB binding peptide and the new detection method for MTB (Table 1 ). Briefly, the mycobacteria were incubated in Middlebrook 7H9 broth plus 10% oleic acid-albumin-dextrose-catalase (OADC) (BD Biosciences, San Jose, CA, USA) (Middlebrook 7H9/OADC broth) to logarithmic phase at 37°C, collected by centrifugation, and washed with phosphate-buffered saline (PBS) twice. Cultures were thoroughly declumped by vortex thrice with several 3 mm glass beads for 2 minutes for the purpose of breaking large clumps and left to set down for 5 minutes. The amount of bacteria in the suspension was tested with the help of the PhoenixSpec NepheloMeter (BD Biosciences) and the accurate count (CFU/mL) was determined by spreading diluted suspensions onto the Middlebrook 7H10/OADC agar plates before detection and incubating for a suitable time at an appropriate temperature, depending on the bacteria species. Cells were then subjected to a water bath at 80°C for 30 minutes to kill the bacteria and were then stored at −20°C. Colonies of three non-mycobacterial bacteria were scraped from the blood plate and processed like mycobacterium.
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