The sputum was harvested and stored at 4°C in a 50 mL container. After purification with 4% NaOH, the bacteria were cultured in 2% Kudo medium. Subsequently, the purified colonies of M tuberculosis transferred in Middlebrook 7H9 broth medium (Becton Dickinson, Sparks, MD, USA) for the collection of the bacterial suspension, followed by the detection of the purified bacilli with the Capilia TB (TOUNS, Numazu, Japan) which was employed for identifying M tuberculosis complex (MTC). The drug susceptibility testing (DST) was carried out on Lowenstein–Jensen (L–J) medium containing Isoniazid (0.2 μg/mL). Bacilli diluted 100-fold were seeded on drug-free L–J medium as a control. The initial colony count started after 4 weeks and was finally completed at 6 weeks. Resistance was shown if colonies were grown on the drug-containing medium comparatively to the control.
Middlebrook 7h9 broth medium
Manufactured by BD
Sourced in United States
Middlebrook 7H9 broth medium is a microbiological culture medium used for the growth and isolation of mycobacteria, including Mycobacterium tuberculosis. It provides the necessary nutrients and growth factors required for the cultivation of these bacteria.
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Lab products found in correlation
Middlebrook 7h10 agar, by BD (5 mentions)
Glycerol, by Merck Group (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Tween 80, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Luria bertani medium, by Merck Group (1 mentions)
7h10 agar, by BD (1 mentions)
Middle brook 7h11 plate, by BD (1 mentions)
Tween 80, by Avantor (1 mentions)
Glycerol, by Maclin Biochemical Technology (1 mentions)
Glycerol, by BD (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Oleic albumin dextrose catalase oadc, by BD (1 mentions)
M bovis bcg, by American Type Culture Collection (1 mentions)
M smegmatis, by American Type Culture Collection (1 mentions)
Recombinant mouse il 1β, by Thermo Fisher Scientific (1 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
21 protocols using middlebrook 7h9 broth medium
1
Tuberculosis Diagnostic and Drug Susceptibility
2
Antimycobacterial Activity Evaluation
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The assay
was performed by the twofold serial microdilution method (in 96-well
microtiter plates) using a Middlebrook 7H9 Broth medium (Beckton Dickinson)
containing 10% of OADC (Beckton Dickinson). The inoculum was prepared
from fresh LJ culture in the Middlebrook 7H9 Broth medium with OADC,
adjusted to a no. 0.5 McFarland tube, and diluted 1:20. The stock
solution of a tested molecule was prepared in water and diluted in
the Middlebrook 7H9 Broth medium with OADC by fourfold the final highest
concentration to be tested. Compounds were diluted serially in sterile
96-well microtiter plates using 100 μL of the Middlebrook 7H9
Broth medium with OADC. Concentrations of tested agents ranged from
0.25 to 512 μg·mL–1. A growth control
containing no antibiotic and a sterile control without inoculation
were also prepared on each plate. After incubation at 37 °C for
21 days, the MICs were visually assessed as the lowest concentration
showing complete growth inhibition of the reference microbial strains.
Isoniazid (INH) and rifampicin (RMP) were used as reference drugs.
was performed by the twofold serial microdilution method (in 96-well
microtiter plates) using a Middlebrook 7H9 Broth medium (Beckton Dickinson)
containing 10% of OADC (Beckton Dickinson). The inoculum was prepared
from fresh LJ culture in the Middlebrook 7H9 Broth medium with OADC,
adjusted to a no. 0.5 McFarland tube, and diluted 1:20. The stock
solution of a tested molecule was prepared in water and diluted in
the Middlebrook 7H9 Broth medium with OADC by fourfold the final highest
concentration to be tested. Compounds were diluted serially in sterile
96-well microtiter plates using 100 μL of the Middlebrook 7H9
Broth medium with OADC. Concentrations of tested agents ranged from
0.25 to 512 μg·mL–1. A growth control
containing no antibiotic and a sterile control without inoculation
were also prepared on each plate. After incubation at 37 °C for
21 days, the MICs were visually assessed as the lowest concentration
showing complete growth inhibition of the reference microbial strains.
Isoniazid (INH) and rifampicin (RMP) were used as reference drugs.
3
Culturing Murine Cells and Mycobacteria
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Female C57BL/6 mice, aged 6–8 weeks, were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China) and maintained under specific pathogen-free conditions at Soochow University. All experimental animal procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals (Ministry of Health, China, 1998). The guidelines were approved by the ethics committee of Soochow University. HEK293T cells and the murine macrophage cell line, RAW264.7, were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), supplemented with 1% penicillin-streptomycin (Invitrogen) and 10% fetal bovine serum (FBS, Hyclone). Human monocytic THP-1 cells were cultured in Roswell Park Memorial Institute medium (RPMI, Hyclone) supplemented with 1% penicillin-streptomycin and 10% FBS. For THP-1-derived macrophages, 40 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) was used to stimulate THP-1 cells for three days. Cells were cultured in a humidified incubator at 37°C and 5% CO2. The M. smegmatis (MS) strain mc2155 was grown in Luria-Bertani medium supplemented with 0.05% Tween-80 (Sigma). H37Rv and H37RvΔRv0790c were purchased from Gene Optimal Inc., Shanghai, and grown in Middlebrook 7H9 broth medium (Becton Dickinson) supplemented with 10% ADC (Becton Dickinson), 0.5% glycerol (Sigma), and 0.05% Tween-80 at 37°C.
4
Mycobacterial Strains Preparation and Isolation
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ML (viable and lethally-irradiated) derived from the footpads of athymic nu/nu mice was kindly provided by Patrícia Samarco Rosa (Lauro Souza Lima Institute). M. bovis BCG Pasteur (ATCC35734) and M. smegmatis (mc2 (link) 155) strains were grown at 37 °C in Middlebrook 7H9 broth medium (Becton Dickinson, Sparks, MD, USA) supplemented with 10% OADC (v/v) and 0.05% Tween 80 (v/v) under constant agitation on a magnetic plate until the exponential phase, counted according to Shepard and McRae52 , and kept frozen at −70 °C until use. Addicionally, ML was isolated from the skin nodules of LL patients as detailed below.
5
Mycobacterium marinum Culture and Quantification
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We used the “M” strain (ATCC BAA-535) of Mycobacterium marinum. The bacteria were grown at 30°C with slow agitation in Middlebrook 7H9 broth medium (Becton Dickinson), enriched with 0.5% albumin (SIGMA), 0.5% glycerol (SIGMA), 0.4% glucose (Biopack), and 0.25% Tween 80 (SIGMA) and was used at the mid‐ to late‐log phase (OD = 1.0). The bacteria were kindly provided to us by Professor Maria I. Colombo (IHEM-CONICET, Mendoza, Argentina) (9 (link)). Cultured bacteria were washed twice with phosphate‐buffered saline (PBS) before use. For disrupting bacterial clumps, suspensions were passed 5–10 times through a 200 μL-micropipette tip and centrifuged at 1500 rpm for 1 min. To verify the bacterial dose, M. marinum suspension was diluted in serial dilutions and plated on 7H10 agar (Becton Dickinson) enriched with 0.5% albumin (Sigma), 0.5% glycerol (Sigma) and 0.4% glucose (Biopack) for counting bacterial colonies.
6
Isolation and Characterization of M. ulcerans
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M. ulcerans 1059 is an isolate originating from a clinical specimen from a patient in Ghana and the autoluminescent M. ulcerans (AlMu) was created based on this isolate12 . The TB47-resistant M. ulcerans mutants were obtained from AlMu in one or two steps. A uniform homogenous suspension was prepared from the colonies by suspending them in sterile phosphate buffered saline (PBS, GENOME, Hangzhou, China) and vortexing them with sterile glass beads before injection into the mouse footpads. All M. ulcerans strains were grown in Middlebrook 7H9 broth medium (Becton, Dickinson and Company, New Jersey, USA) with 10% 2-oxo-acid dehydrogenase complexes (OADC, Becton, Dickinson and Company)+0.2% glycerol (Shanghai Macklin Biochemical, Shanghai, China)+0.05% Tween 80 (Amresco, USA) for culture or on Middlebrook 7H11 plates (Becton, Dickinson and Company) supplemented with 0.2% glycerol and 10% OADC. Plates were incubated at 30 °C for 12 weeks before counting colony-forming units (CFUs).
7
Antimicrobial Activity Evaluation of Compounds
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The assay was performed by the two-fold serial microdilution method (in 96-well microliter plates) using Middlebrook 7H9 Broth medium (Beckton Dickinson) containing 10% of OADC (Beckton Dickinson). The inoculum was prepared from fresh LJ culture in Middlebrook 7H9 Broth medium with OADC, adjusted to a no. 0.5 McFarland tube, and diluted to 1 : 20. The stock solution of a tested molecule was prepared in water and diluted in Middlebrook 7H9 Broth medium with OADC by four-fold the final highest concentration to be tested. Compounds were diluted serially in sterile 96-well microtiter plates using 100 μl Middlebrook 7H9 Broth medium with OADC. Concentrations of the tested agents ranged from 0.25 to 512 μg mL−1. A growth control containing no antibiotic and a sterile control without inoculation were also prepared on each plate. After incubation, (Mycobacterium avium for 7 days, and Mycobacterium terrae, Mycobacterium tuberculosis strains at 37 °C for 21 days), the MICs were visually assessed as the lowest concentration showing complete growth inhibition of the reference microbial strains. Isoniazid (INH) and Rifampicin (RMP) were used as the reference drugs.
8
Infection Kinetics of M. tuberculosis
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HEK293T cells were cultured in DMEM medium supplemented with 10% FBS. RAW264.7 cells purchased from the American Type Culture Collection (ATCC) were maintained in RPMI1640 medium supplemented with 10% FBS. All cells were maintained at 37 °C and 5% CO2 in a humidified incubator. M. tuberculosis strain H37Ra (ATCC 25177) was grown in Middlebrook 7H9 broth medium supplemented with 10% OADC (Becton, Dickinson and Company, Franklin Lakes, NJ). Cells were infected at a multiplicity of infection of 5 bacteria per cell (MOI 5:1). After 6 h of incubation, the infected cells were washed 6 times with RPMI1640 to remove any extracellular bacteria, and then incubated in fresh medium for a further 18 hours.
9
Microbial Growth and Viability Assay
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The Mueller-Hinton Broth (MHB), and Mueller-Hinton Agar (MHA) were obtained from Biolab (Midrand, South Africa). Middlebrook 7H9 broth medium (Becton Dickinson GmbH, Heidelberg, Germany). The LIVE/DEAD BacLight Bacterial Viability Kit was purchased from Molecular Probes (Eugene, OR, USA). All other chemicals and reagents were purchased from Sigma-Aldrich Co. unless otherwise stated.
10
Mycobacterium tuberculosis Strain Preparation
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The M. tuberculosis strain H37Ra (ATCC # 25177, Manassas, VA) was prepared in Middlebrook 7H9 broth medium (Difco Laboratories, Detroit, MI) supplemented with 10% albumin-dextrose-catalase (ADC, Difco Laboratories) and 0.2% glycerol. The mycobacteria were incubated for 21 days at 37°C with 5% CO2 on an orbital shaking incubator, and the M. tuberculosis suspension was harvested, centrifuged (1972 g, 15 min, 25 °C), resuspended in fresh 7H9 medium and aliquoted into one-millilitre samples that were stored at −70 °C until use. The mycobacteria stock concentration was determined by colony forming units (CFU) counts from ten-fold serial dilutions in 7H9 medium after 21 days of incubation on 7H10 agar. For infection assays, M. tuberculosis stock was thawed and centrifuged (5220 g, 8 min, 25 °C), and the supernatant was replaced by complete medium (supplemented medium with 10% non-heat-inactivated human pool serum). Then, mycobacteria were declumped by mixing and repeatedly forcing the sample out of a 27-G syringe needle, followed by a 1-min sonication (Untrastonic 28X, Ney Dental International, Yacaipa, CA) and centrifugation (82 g, 2 min, 25 °C). The supernatant containing disaggregated mycobacteria was recovered and used in infection assays.
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