BACTEC MGIT960 system, oscillator, centrifuge, biosafety cabinet, mycobacterial growth indicator tube (MGIT), oleic acid, albumin, dextrose, and catalase medium, PANTA bacteria inhibitor (an antibiotic mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), and drug sensitive kit were all provided by Becton, Dickinson and Company, United States. It was reported that the positive coincidence rate of the BACTEC MGIT960 system is about 95% to 100%,[8 (link)] and it is a more commonly used clinical detection method. Nosova et al believe that the BACTEC MGIT 960 system is fast effective and reliable when it comes to detecting MDR-TB.[9 (link)]
Bactec mgit 960 system
Manufactured by BD
Sourced in United States, Cameroon, China, Germany
The BACTEC MGIT 960 system is a fully automated mycobacterial growth indicator tube (MGIT) system designed for the detection and identification of mycobacteria in clinical specimens. The system utilizes fluorescent technology to continuously monitor for bacterial growth in liquid culture media.
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Lab products found in correlation
Mgit tube, by BD (3 mentions)
Bactec mgit 960 growth supplement, by BD (2 mentions)
Genexpert mtb rif, by Cepheid (2 mentions)
Middlebrook 7h11 agar, by BD (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Oleic acid, by BD (1 mentions)
Advia centaur xp, by Siemens (1 mentions)
Advia 2400, by Siemens (1 mentions)
Myco f lytic, by BD (1 mentions)
Bactec mgit 960 tube, by BD (1 mentions)
Middlebrook 7h9 broth, by BD (1 mentions)
Bactec 460tb system, by BD (1 mentions)
Xe 5000, by Sysmex (1 mentions)
Immage 800, by Beckman Coulter (1 mentions)
Eb buffer, by Qiagen (1 mentions)
Te buffer, by Merck Group (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Xpert mtb rif testing, by Cepheid (1 mentions)
Genotype mtbc assay, by Hain Lifescience (1 mentions)
117 protocols using bactec mgit 960 system
1
Rapid and Reliable MDR-TB Detection
2
Mycobacterium Isolation and Drug Susceptibility
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Isolation of Mycobacterium strains and susceptibility tests for the TB drugs were carried out according to the MGIT manual by FIND (Geneva, Switzerland). Briefly, sputum samples (kept at 4 °C for 1–2 months) were processed with NaOH-NALC procedure (at final concentration of NaOH 1.25–2.16%), inoculated onto Middlebrook 7H11 Agar (Beckton Dickinson, Difco) or into BACTEC MGIT tube (with PANTA) with the BACTEC MGIT 960 System (Becton Dickinson, Sparks, Md., USA) and cultured at 37 °C for up to 42 days. Bacterial isolates were passage into a fresh MGIT tube (without PANTA) and drug susceptibility test was carried out according to the manufacturer’s instruction of BACTEC SIRE kit, including isoniazid (INH, 0.1 μg/ml), rifampin (RIF, 1.0 μg/ml), ethambutol (EMB, 5.0 μg/ml) and streptomycin (STR, 1.0 μg/ml).
3
Mycobacterium tuberculosis Culture and Identification
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Sputum samples digested and decontaminated by the N-acetyl l -cysteine (NALC) (Sigma–Aldrich, Germany) and 3% sodium hydroxide (NaOH) (VWR, Belgium) method were processed for mycobacterial culture using the Bactec™ MGIT™ 960 system (Becton Dickinson, USA) as previously described [24] (link). Days to positivity (DTP), defined as the time it took for MGIT cultures to become positive were used as an inverse measure of the bacillary load in the sputum sample. Smears were prepared from positive MGIT cultures, stained with ZN stain and examined for AFB by light microscopy. The presence of Mtb in all cultures that were positive for AFBs was confirmed using the MPT64 antigen test (Becton Dickinson, USA) according to the manufacturer's instructions. MGIT cultures were positive for Mtb if both smear microscopy for AFB and MPT64 antigen test results were positive. They were reported negative if there was no growth after 42 days incubation.
4
Linezolid MIC Determination for M. tuberculosis
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Sputum samples were collected at each visit and were sent to the prefectural TB reference laboratory for the microbiological examination. The BACTEC MGIT 960 system (Becton Dickinson, Franklin Lakes, NJ, United States) was used for bacterial culture of the M. tb isolates, phenotypic drug susceptibility testing and MIC determination for linezolid (Springer et al., 2009 (link)). All suspension from the growth in the plain MGIT medium were used within 3 days after found positive in MGIT incubator. The growth control tube containing 1:100 diluted bacterial suspension, was inoculated in the MGIT 960 instrument as well. The range of concentrations for MIC testing was .06–1 mg/L for linezolid. The MIC was defined as the lowest concentration of a drug that inhibited the bacterial growth. M. tb H37Rv (ATCC 27294) was used as the reference strain for the quality control.
5
Identification of Nontuberculous Mycobacteria
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The BACTEC MGIT960 system (Becton Dickinson & Co., Franklin Lakes, NJ, USA) was used for Mycobacterium culture. Pulmonary samples were collected by expectoration, gastric aspiration, and sputum induction. Extrapulmonary samples (pleural fluid, spinal fluid, and lymph nodes) were collected by lumbar puncture, pleural tap, fine needle aspiration, lymph node biopsy, and other techniques. The participants were defined as HIV-infected patients with positive NTM culture results. Their NTM isolates were further grown on the Löwenstein-Jensen (L-J) medium for up to 4 weeks. The p-nitrobenzoic acid and 2-thiophenecarboxylic acid hydrazide were used for NTM identification at first. All collected NTM strains were frozen in the strain bank of the hospital for backup, and the control strain H37Rv was monitored.
6
Mycobacterial Growth Measurement Protocol
7
Identification of NTM Species Infections
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Acid-fast bacilli smears and mycobacterial cultures were performed following the recent guidelines [11 (link)]. All cultures were grown in both solid Ogawa media (Shinyang Diagnostics, Seoul, South Korea) and the BACTEC MGIT 960 system (Becton-Dickinson and Co., Sparks, MD, US). NTM species were identified by sequencing of the 16S ribosomal RNA gene using the algorithm specified in the Clinical and Laboratory Standards Institute guidelines [13 (link)], as well as sequencing of the rpoB gene [21 (link)]. rpoB gene sequencing and patterns of clarithromycin resistance were checked to differentiate between M. abscessus subspecies abscessus and M. abscessus subspecies massiliense [22 (link)]. M. abscessus subspecies taxonomy was in accordance with recent suggestions [23 (link)]. Patients were considered to have mixed NTM species infections if an NTM species other than the original species was isolated at least twice during the follow-up period.
8
Sputum Culture Protocol for Mycobacterium Tuberculosis
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Both the BACTEC MGIT 960 system (Becton Dickinson, Sparks, MD, USA) (liquid media) and Löwenstein-Jensen agar were systematically used for sputum culture samples, irrespective of smear microscopy result. Positive growth without contaminant was confirmed by Ziehl-Neelsen staining and by the rapid test confirmation kit (i.e. MPT64 protein detection-based immunochomatographic test; SD Bioline Kit, Standard Diagnostics, Yongin-si, Korea) [11] (link). The interpretation of results was based on the manufacturer's instructions. Positive results were considered as belonging to M. tuberculosis complex. Negative results were regarded as atypical mycobacteria and were not taken into account in this study.
9
Tuberculosis Culture and Drug Susceptibility
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A total of 465 randomly selected clinical samples from different patients (Table 1 ) mostly came from Chongqing local and surrounding areas between April 2014 and August 2018 in Chongqing Public Health Medical Center (Chongqing, China) were cultured in BACTEC MGIT 960 system (Becton Dickinson, Sparks, MD, United States), according to manufacturer’s instructions (Kruuner et al., 2006 (link)). The flagged positive samples were subjected to DST using proportion method on Lowenstein-Jensen (L-J) solid medium (Encode, Zhuhai, China) containing MDR- and XDR-related drugs, isoniazid, rifampicin, ofloxacin, levofloxacin, moxifloxacin, amikacin, and capreomycin. The Mycobacterium species identification were assigned based on sequence polymorphisms in 16S rRNA, hsp65, and rpoB (Kim and Shin, 2018 (link)), and on the results of 5 μg/ml TCH and 500 μg/ml PNB on L-J solid medium. Isolates were frozen in 25% glycerol at −70°C refrigerator until use.
10
Fluorescent Oxygen Consumption Assay
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A fluorescent compound is embedded in silicone on the bottom of 16 × 100 mm round bottom tubes. The fluorescent compound is sensitive to the presence of oxygen dissolved in the broth. Initially, the large amount of dissolved oxygen quenches emissions from the compound and little fluorescence can be detected. Later, actively respiring microorganisms consume the oxygen and allow the fluorescence to be detected.
Tubes are filled with samples in the broth and continuously incubated at 37 °C. The tubes are monitored for increasing fluorescence to determine whether the tube is instrument positive, that is, the test sample contains viable organisms. Fluorescence can be recorded by automated instruments such as Becton Dickinson’s BACTEC MGIT 960 System, or manually using Wood’s lamp or other long-wave UV light source.
Tubes are filled with samples in the broth and continuously incubated at 37 °C. The tubes are monitored for increasing fluorescence to determine whether the tube is instrument positive, that is, the test sample contains viable organisms. Fluorescence can be recorded by automated instruments such as Becton Dickinson’s BACTEC MGIT 960 System, or manually using Wood’s lamp or other long-wave UV light source.
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