Stock cultures of Mtb strains TB179, TB861, TB1430, TB1593, TB1659, TB1841, TB1945, and TB2666 were inoculated into mycobacterial growth indicator tubes and incubated until positive growth was detected using the Bactec 460 TB system (BD Biosciences). Approximately 0.2 mL was inoculated onto Löwenstein–Jensen medium and incubated over 6 weeks with weekly aeration until colony formation. Colonies were transferred into 20 mL of supplemented 7H9 Middlebrook medium (BD Biosciences) containing 0.2% (v/v) glycerol (Merck Laboratories), 0.1% Tween 80 (Merck Laboratories), and 10% dextrose, catalase. Once the culture reached an A600 of 0.9, 1 mL was inoculated into 80 mL of supplemented 7H9 Middlebrook medium and incubated until an A600 between 0.6 and 0.7 was reached. All steps were performed at 37°C until Mtb cells in mid-log growth phase were used for whole cell lysate protein extraction.
Bactec 460tb system
Manufactured by BD
Sourced in United States
The BACTEC 460TB system is a diagnostic instrument used for the detection and identification of mycobacterial species, including Mycobacterium tuberculosis, in clinical samples. The system utilizes radiometric technology to monitor the growth of mycobacteria in culture media.
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9 protocols using bactec 460tb system
1
Mycobacterium tuberculosis Strain Cultivation
2
Characterization of Drug-Resistant M. tuberculosis
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The 61 strains used in this analysis were obtained in 2006-2007 from patients hospitalized in the Center for Lung Diseases Treatment and Rehabilitation in Lodz, Poland. All strains were tested for susceptibility to isoniazid, rifampicin, pyrazinamide, streptomycin, and ethambutol using the Bactec 460 TB system (BD Diagnostic Systems, Sparks, MD, USA), as described previously [16 (link)]. This set of strains was previously characterized by IS6110-RFLP analysis, 15 locus MIRU-VNTR typing, and spoligotyping [16 (link)].
3
Long-term Tracking of M. tuberculosis Clone
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M. tuberculosis isolates were obtained from a previous collection of samples from 1 Swedish patient during 9 years (1991–1999) of pulmonary tuberculosis [20 (link)]. The high sequence similarity strongly suggests that the patient carried the same single clone during 9 years without reinfection. The samples (populations) were collected at 8 different time points before and during treatment. Each sample was grown on Löwenstein-Jensen slants at 37°C to subsequently isolate up to 10 single colonies. The isolates were analyzed for phenotypic drug susceptibility using the BACTEC 460TB system (Becton and Dickinson). Both populations and individual isolates were kept frozen at −70°C until used in this study.
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M. tuberculosis isolates were obtained from a previous collection of samples from 1 Swedish patient during 9 years (1991–1999) of pulmonary tuberculosis [20 (link)]. The high sequence similarity strongly suggests that the patient carried the same single clone during 9 years without reinfection. The samples (populations) were collected at 8 different time points before and during treatment. Each sample was grown on Löwenstein-Jensen slants at 37°C to subsequently isolate up to 10 single colonies. The isolates were analyzed for phenotypic drug susceptibility using the BACTEC 460TB system (Becton and Dickinson). Both populations and individual isolates were kept frozen at −70°C until used in this study.
4
Mycobacterial Culture and WGS Protocol
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Mycobacterial culture of samples from patients with suspected TB was done at Territory Pathology (Royal Darwin Hospital, Darwin), and positive cultures were referred to the Victorian Infectious Diseases Reference Laboratory (Doherty Institute, Melbourne) for identification and antimicrobial susceptibility testing. From 1989–2007 antimicrobial susceptibility testing was done using the radiometric BACTEC 460TB system (Becton Dickinson), and from 2007 onwards the BACTEC Mycobacterial Growth Indicator Tube 960 system (Becton Dickinson) was used. Between 1989–2007 DNA was extracted using phenol and chloroform,16 (link) and from 2010 by ethanol precipitation.17 (link) There were no samples available between 2008–2009. Whole genome sequencing was done at the Microbiological Diagnostic Unit Public Health Laboratory (Doherty Institute, Melbourne). Unique dual indexed libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina) and libraries were sequenced on the Illumina NextSeq 500/550 with 150-cycle paired end chemistry as outlined in the manufacturer's protocols.
5
Resistance Profiling of M. tuberculosis
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DNA was extracted from isolates cultured from sputum specimens as previously described [41 (link)]. RM-seq libraries were prepared as described above using the rmseq primers specific to pncA, ethA, and rpoB resistance determining regions (Additional file 1 : Table S1). Deep sequencing was performed on a Nextseq 500 platform using Reagent Kit v3 to produce 150 bp overlapping paired-end reads and analyses were performed using the RM-seq bioinformatics analysis pipeline. Primary M. tuberculosis culture and phenotypic susceptibility testing was performed using the radiometric BACTEC 460TB system (Becton Dickinson).
6
Amikacin Susceptibility Testing for Nontuberculous Mycobacteria
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Specimen culture was performed using the Bactec 460 TB system (Becton Dickinson Microbiology Systems, USA) until May 2000 and, thereafter, with the Bactec MGIT 960 system (Becton Dickinson Microbiology Systems). Specimens were processed and analyzed as described by Hanna et al. [15 (link)]. NTM cultures were speciated using DNA probes (AccuProbe, Gen-Probe, USA) for MAC and Mycobacterium gordonae, and by high-performance liquid chromatography for other species [16 (link)]. Drug Susceptibility Testing was performed using the CLSI-recommended method of broth microdilution, using commercially prepared microtiter dilution plates (Thermo Fisher, Cleveland, OH) [9 ]. Concentrations of amikacin tested ranged from ≤1 to 64 μg/ml. Isolates with a MIC ≤16 were considered susceptible, those with a MIC of 32 or 64 were considered intermediate and those with a MIC > 64 were considered resistant [8 (link), 16 (link)]. Since amikacin Drug Susceptibility Testing requires special request, it was not always requested in the early study period because of a lack of data regarding clinical correlation.
7
Diagnosis of Active Tuberculosis
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During the 25-month study period, 29 patients with a diagnosis of active tuberculosis, as
determined by radiologic, microbiologic, and clinical examinations, were evaluated. One
hundred healthy subjects with no respiratory symptoms comprised the control group. All
control subjects underwent chest X-ray examinations to rule out tuberculosis, and those
with suspicious lesions were excluded from the study. Written informed consent for
inclusion in the study was obtained from all participants in both the patient and control
groups.
Tuberculosis was diagnosed by clinical examination, identification of acid-fast bacilli
(AFB) in sputum, growth of M. tuberculosis on appropriate media, and
radiologic findings. Only patients with M. tuberculosis results obtained
by the automated BACTEC 460 TB system (Becton Dickinson, Franklin Lakes, NJ, USA) and
conventional methods were included in the study. Participants without microbiological
proof of M. tuberculosis were excluded.
determined by radiologic, microbiologic, and clinical examinations, were evaluated. One
hundred healthy subjects with no respiratory symptoms comprised the control group. All
control subjects underwent chest X-ray examinations to rule out tuberculosis, and those
with suspicious lesions were excluded from the study. Written informed consent for
inclusion in the study was obtained from all participants in both the patient and control
groups.
Tuberculosis was diagnosed by clinical examination, identification of acid-fast bacilli
(AFB) in sputum, growth of M. tuberculosis on appropriate media, and
radiologic findings. Only patients with M. tuberculosis results obtained
by the automated BACTEC 460 TB system (Becton Dickinson, Franklin Lakes, NJ, USA) and
conventional methods were included in the study. Participants without microbiological
proof of M. tuberculosis were excluded.
8
Comprehensive TB Detection Protocol
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Sample transport, processing, and direct detection of MTB by acid-fast bacillus (AFB) smear staining, as well as culture (LJ) were conducted based on the manual/WHO/2015 [12 ]. MTB isolates were identified using TB Ag MPT64 BIOEASY identification test (Standard Diagnostic, Republic of Korea). Drug susceptibility testing (DST) was conducted using BACTEC 460 TB system (Becton Dickinson Microbiology Systems, Sparks, Md), according to WHO recommendations [13 ].
Molecular Xpert MTB/RIF assay was performed with a fraction of the sample using on the GeneXpert® system (Cepheid, Sunnyvale, CA, USA) in accordance with the manufacturer’s instructions, and the Xpert MTB/RIF implementation manual/WHO/2014 [14 ,15 (link)]. Urine samples were processed as per the body fluids protocol; thus, 1 mL of the urine sample was mixed with 2 mL of Xpert sample reagent [16 (link)].
The AFB smear, LJ culture and Xpert assay test were performed at CHC/UFPR, and DST for M. tuberculosis was performed at the Reference Public Health Laboratory of Paraná (LACEN).
Molecular Xpert MTB/RIF assay was performed with a fraction of the sample using on the GeneXpert® system (Cepheid, Sunnyvale, CA, USA) in accordance with the manufacturer’s instructions, and the Xpert MTB/RIF implementation manual/WHO/2014 [14 ,15 (link)]. Urine samples were processed as per the body fluids protocol; thus, 1 mL of the urine sample was mixed with 2 mL of Xpert sample reagent [16 (link)].
The AFB smear, LJ culture and Xpert assay test were performed at CHC/UFPR, and DST for M. tuberculosis was performed at the Reference Public Health Laboratory of Paraná (LACEN).
9
Rapid Mycobacterium tuberculosis Growth Assay
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The BACTEC 460TB system (Becton Dickinson, USA) was employed to determine a growth index (GI) of the MTB. GI is the quantitative measure of 14 CO 2 liberated by metabolism of 14 C-labeled substrate in a medium and expressed in numbers on a scale from 0 to 999. Briefly, 0.1 ml of samples were transferred to 12B BACTEC vials, in duplicate for each sample/drug concentration, unless mentioned otherwise, and incubated at 37°C in 5% CO 2 atm. GI was calculated daily under aerobic condition until in 1:100 controls and a value greater than 30 was obtained. In order to determine the percent inhibition, undiluted control reading was used. Appropriate positive and negative controls were also included in the calculation. The growth inhibition was expressed as a ratio of GI of drug to the respective control vial. The percent growth inhibition was calculated for each drug concentration (24, 25) .
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