M. bovis bacillus Calmette-Guérin (M. bovis BCG) Pasteur strain was cultured in 7H9 media (Sigma) plus OADC (Fisher) containing 0.05% Tween-80 (Sigma) at 37°C, shaking at ~50 r.p.m. M. bovis BCG bacilli were washed twice with sterile PBS and passed through a 40 μm nylon mesh strainer prior to use. M. bovis BCG concentration was determined by measuring absorbance at A600 and adjusted to appropriate concentrations in sterile PBS. Mice were anesthetized with isoflurane, and M. bovis BCG was administered via intranasal inoculation in 50 μL containing ~1.0 to 5.0 × 106 bacilli. Blood was collected from euthanized mice, and lungs and spleens were removed, weighed, and homogenized in sterile PBS. Homogenate dilutions were plated on 7H10 agar (Becton Dickinson) containing OADC and antimicrobials: polymyxin B sulfate (26 μg/mL, Sigma), trimethoprim lactate (20 μg/mL, Sigma), carbenicillin disodium (50 μg/mL, Sigma), and amphotericin B (2.5 μg/mL, Sigma). Plates were incubated at 37°C for 14–21 days prior to counting. Colony forming units (CFU) from the left lung, or spleen (spleen CFU were determine per gram of tissue as half of the spleen was used for additional assays) were determined. For histology, alternate lobes of the lungs were inflated with 10% formalin and processed for H & E staining.
7h9 media
Manufactured by Merck Group
Sourced in United States
7H9 media is a laboratory culture medium designed for the growth of mycobacteria, including Mycobacterium tuberculosis. It provides essential nutrients and growth factors required for the cultivation of these organisms in a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Tween 80, by Merck Group (2 mentions)
Trimethoprim lactate, by Merck Group (1 mentions)
Carbenicillin disodium, by Merck Group (1 mentions)
7h10 agar, by BD (1 mentions)
Polymyxin b sulfate, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Middlebrook s 7h11 selective media, by BD (1 mentions)
Oxytherm oxygen electrode, by Hansatech (1 mentions)
K2hpo4 kh2po4, by Merck Group (1 mentions)
Avanti j 25 centrifuge, by Beckman Coulter (1 mentions)
Oxytrace software, by Hansatech (1 mentions)
3 protocols using 7h9 media
1
Intranasal M. bovis BCG Infection in Mice
2
Cultivation and Enumeration of Mycobacterial Strains
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Virulent M. bovis and the BCG vaccine were grown separately in Middlebrook’s 7H9 media supplemented with 0.05% Tween 80 (Sigma Chemical Co., St. Louis, MO, USA) and 10% oleic acid–albumin–dextrose complex (OADC; Difco, Detroit, MI, USA) as previously described [55 (link),56 (link)]. At the mid-log-phase growth stage, bacilli were pelleted by centrifugation at 750× g, washed twice with PBS 0.01 M, (pH 7.2), and stored at −80 °C. Frozen stocks were warmed to room temperature and diluted to the appropriate cell density in PBS. Bacilli were enumerated by serial dilution plate counting on Middlebrook’s 7H11 selective media (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Respiration Inhibitor Evaluation in M. smegmatis
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M. smegmatis (ATCC 19420) cultures
were grown using 7H9 media (produced as suggested
by the manufacturer, Sigma-Aldrich) using a 1:100 inoculation every
3–4 days. Cells were incubated at 37 °C, 170 rpm. Cells
were prepared for the assay by harvesting after 72 h growth and centrifuging
at 5000g for 15 min at 4 °C (JLA 16.250 rotor,
Beckman Avanti J-25 Centrifuge). Centrifuged cells were washed with
filtered potassium phosphate (KPi) buffer (K2HPO4 + KH2PO4, Sigma-Aldrich) and then concentrated
to an OD600 = 3 by centrifugation and resuspension in fresh
7H9. Cells were stored at −20 °C until required. 1 mL
(CFU mL–1 = 7.5 × 106) assays were
conducted using a Hansatech Oxytherm+ oxygen electrode at 37 °C,
100% stirring. The 100% oxygen consumption rate was found for each
assay using the rate achieved after the addition of 10 mM glucose.
Once a steady oxygen rate was achieved, ETC inhibitors were sequentially
added every 3 min to achieve these ranges: CK-2-63 (20–200
μM), BDQ (1–56 nM), and Q203 (10–110 nM). Combinations
were assessed using the same ranges with the addition of CK-2-63 (3.5
μM) that was administered at the same time as the first drug
addition. Rates were recorded from 2 min after each drug addition
for 1 min. The results were analyzed using Hansatech Oxytrace software.
were grown using 7H9 media (produced as suggested
by the manufacturer, Sigma-Aldrich) using a 1:100 inoculation every
3–4 days. Cells were incubated at 37 °C, 170 rpm. Cells
were prepared for the assay by harvesting after 72 h growth and centrifuging
at 5000g for 15 min at 4 °C (JLA 16.250 rotor,
Beckman Avanti J-25 Centrifuge). Centrifuged cells were washed with
filtered potassium phosphate (KPi) buffer (K2HPO4 + KH2PO4, Sigma-Aldrich) and then concentrated
to an OD600 = 3 by centrifugation and resuspension in fresh
7H9. Cells were stored at −20 °C until required. 1 mL
(CFU mL–1 = 7.5 × 106) assays were
conducted using a Hansatech Oxytherm+ oxygen electrode at 37 °C,
100% stirring. The 100% oxygen consumption rate was found for each
assay using the rate achieved after the addition of 10 mM glucose.
Once a steady oxygen rate was achieved, ETC inhibitors were sequentially
added every 3 min to achieve these ranges: CK-2-63 (20–200
μM), BDQ (1–56 nM), and Q203 (10–110 nM). Combinations
were assessed using the same ranges with the addition of CK-2-63 (3.5
μM) that was administered at the same time as the first drug
addition. Rates were recorded from 2 min after each drug addition
for 1 min. The results were analyzed using Hansatech Oxytrace software.
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