Remnant pellets of processed sputum samples were resuspended in 400 μl of Middlebrook 7H9 broth supplemented with OADC Enrichment (Becton, Dickinson and Company, Sparks, MD, United States) and PANTA (Becton, Dickinson and Company, Sparks, MD, United States) and divided in four 100 μl aliquots. In two aliquots, 4 μg/ml RIF was added and the other two were kept as control. Samples were recovered for 48 or 96 h and infected overnight with 5 μl of a stock of mCherrybombϕ (1010–1011 PFU/ml). After that, samples were fixed with 2% paraformaldehyde for 2 h at room temperature, centrifuged and the pellet was resuspended in 10 μl of phosphate buffered saline (PBS) and examined by fluorescence microscopy using 1,000× magnification. The criterion for detection was the presence of at least one fluorescent bacillus per 100 fields in the control samples and for determination of RIF resistance was the same in RIF treated samples. We also compared the results using the criterion of two fluorescent bacilli per 100 field but we did not observed significantly differences between both criteria so we opted for the first one. Detection results using mCherrybombϕ were compared with growth and colony counting (CFU) in LJ media and determination of RIF susceptibility with the proportion method (Canetti et al., 1963 (link)).
Panta
Manufactured by BD
Sourced in United States
PANTA is a multipurpose laboratory equipment designed for various applications in scientific research and analysis. It is a compact, versatile device that can perform a range of functions, including sample preparation, mixing, and incubation. The core function of PANTA is to provide a controlled environment for conducting experiments and procedures that require precise temperature and agitation control.
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Lab products found in correlation
Glycerol, by Wisent (2 mentions)
Omnitip plastic homogenizer probes, by Omni International (2 mentions)
7h9 broth, by BD (2 mentions)
Oadc enrichment, by BD (1 mentions)
Catalase, by BD (1 mentions)
0.45 μm nitrocellulose membrane, by Merck Group (1 mentions)
Middlebrook 7h10 medium, by BD (1 mentions)
Mgit 960, by BD (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Zeocin, by Merck Group (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Pbluescript 2 sk, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mtb h37rv, by American Type Culture Collection (1 mentions)
Middlebrook adc, by BD (1 mentions)
Middlebrook 7h11 agar plates, by BD (1 mentions)
15 protocols using panta
1
Rapid Mycobacterium Tuberculosis Phenotypic Drug Resistance Detection
2
Isolation of Mycobacteriaceae from Environmental Samples
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Each sample was totally concentrated under vacuum and filtered with a 0.45-μm nitrocellulose membrane (Millipore) for subsequent decontamination and culture of bacteria belonging to the family Mycobacteriaceae. The water samples from Lake70 were first filtered with a 20-μm mesh phytoplankton net and then with a 8-μm glass microfiber filter (AP20 Millipore), followed by filtration through a 5-μm cellulose ester membrane (Millipore) for the removal of microalgae [16 –18 ]. The crude and treated sewage samples were also previously pre-filtered due to a high concentration of organic matter.
After the sample concentration procedure, each membrane was transferred to a sterile tube with 10 mL of phosphate-buffered saline (PBS 1X, pH 7.4) and maintained under vigorous stirring for 30 min. The bacterial suspension was decontaminated with 0.05% cetylpyridinium chloride (CPC) and spread on Middlebrook 7H10 medium supplemented with 0.5% glycerol and 10% OADC-oleic acid, albumin, dextrose and catalase (Becton Dickinson) and 10% PANTA (40 U/mL polymyxin, 4 μg/mL amphotericin B, 16 μg/mL nalidixic acid, 4 μg/mL trimethoprim and 4 μg/mL azlocillin) (Becton Dickinson) according to the protocol described by Radomski et al., 2010 [19 (link)]. The cultures were incubated at 30°C and examined for 60 days.
After the sample concentration procedure, each membrane was transferred to a sterile tube with 10 mL of phosphate-buffered saline (PBS 1X, pH 7.4) and maintained under vigorous stirring for 30 min. The bacterial suspension was decontaminated with 0.05% cetylpyridinium chloride (CPC) and spread on Middlebrook 7H10 medium supplemented with 0.5% glycerol and 10% OADC-oleic acid, albumin, dextrose and catalase (Becton Dickinson) and 10% PANTA (40 U/mL polymyxin, 4 μg/mL amphotericin B, 16 μg/mL nalidixic acid, 4 μg/mL trimethoprim and 4 μg/mL azlocillin) (Becton Dickinson) according to the protocol described by Radomski et al., 2010 [19 (link)]. The cultures were incubated at 30°C and examined for 60 days.
3
Phenotypic Drug Susceptibility Testing for MTBC
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The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer.
Specimens were decontaminated using sodium hydroxide (NaOH) except sterile body fluids like cerebrospinal fluid. After concentration by centrifugation at 3000 g for 15 min, the sediment was resuspended in 1.5 mL of 0.5 M phosphate buffer (pH 6.8) and inoculated in MGIT-7H9 broth supplemented with oleic acid-albumin-dextrose-catalase (OADC) and PANTA (Becton Dickinson). This was incubated using MGIT 960 instrument (Becton-Dickinson and Company, Sparks MD, USA) as well as Lowenstein-Jensen (LJ) medium at 37°C. MTBC strains grown on MGIT medium were tested for drug susceptibility in MGIT 960.
Agar proportion method on 7H11 agar medium was used for the testing of the isolate which was determined resistant to rifampicin in Xpert MTB/RIF and susceptible in MGIT 960, according to standard procedures. The drug concentration used was rifampicin 1.0 μg/mL. The proportion of resistant organisms in the inoculum was calculated by comparing the number of colonies growing on the drug-free medium (minimum number required = 50) with the number growing on drug-containing medium. If > 1% of the inoculum was found to grow in the presence of the critical concentration used, the isolate/strain was regarded as drug resistant.
Specimens were decontaminated using sodium hydroxide (NaOH) except sterile body fluids like cerebrospinal fluid. After concentration by centrifugation at 3000 g for 15 min, the sediment was resuspended in 1.5 mL of 0.5 M phosphate buffer (pH 6.8) and inoculated in MGIT-7H9 broth supplemented with oleic acid-albumin-dextrose-catalase (OADC) and PANTA (Becton Dickinson). This was incubated using MGIT 960 instrument (Becton-Dickinson and Company, Sparks MD, USA) as well as Lowenstein-Jensen (LJ) medium at 37°C. MTBC strains grown on MGIT medium were tested for drug susceptibility in MGIT 960.
Agar proportion method on 7H11 agar medium was used for the testing of the isolate which was determined resistant to rifampicin in Xpert MTB/RIF and susceptible in MGIT 960, according to standard procedures. The drug concentration used was rifampicin 1.0 μg/mL. The proportion of resistant organisms in the inoculum was calculated by comparing the number of colonies growing on the drug-free medium (minimum number required = 50) with the number growing on drug-containing medium. If > 1% of the inoculum was found to grow in the presence of the critical concentration used, the isolate/strain was regarded as drug resistant.
4
Tuberculosis Diagnosis: Sputum Smear and Culture
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For direct smear microscopy, a loopful (5 mm diameter) of sputum sample was taken for smear preparation in an area of 2 by 3 cm, then air dried and heat fixed. The processed sediments were re-suspended in 300 μl of resuspension solution (0.05% Tween-80) of which 10% was used for smear preparation. All smear slides were stained with ZN stain, observed and graded as per the Revised National Tuberculosis Control Programme (RNTCP) guidelines [17 ]. For culture, 45% of the suspension (135 μl) was inoculated into 7H9 liquid medium supplemented with Albumin Dextrose complex and PANTA (polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin) supplement (Becton Dickinson, USA), and the tubes were incubated at 37°C for upto 8 weeks. The positivity in the 7H9 liquid culture was first identified on the basis of turbidity of the medium, which was then confirmed for presence of M. tuberculosis by ZN smear microscopy and IS6110 PCR (M. tuberculosis complex specific PCR) [18 (link)]. For DNA isolation, the remaining 45% of the suspension (135 μl) was pelleted and re-suspended in 250 μl lysis solution (10% Chelex-100, 0.03% Triton X-100 and 0.3% Tween-20), heated at 90°C for 40 min., and centrifuged at ~8800×g for 10 min. The supernatant was used directly for PCR.
5
Growth and Cloning of Mycobacterium and E. coli
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Escherichia coli DH5α and HB101 strains were used for cloning procedures in pBluescript II SK (+) (Invitrogen) vector or pJV53-zeo plasmid81 (link) propagation were cultured in Luria Bertani (LB) liquid or solid media. When required kanamycin (Sigma), zeocin (Invitrogen), hygromycin (Invitrogen) and ampicillin (Sigma) were added to a final concentration of 50 μg/ml (kanamycin), 25 μg/ml (zeocin), 100 μg/ml (hygromycin and ampicillin). Mtb reference strain H37Rv and Mtb clinical isolates were grown in Middlebrook 7H9 liquid medium, supplemented with 0.2% glycerol (w/v) and 10% enrichments containing BSA (Sigma) 5% (w/v), glucose (Sigma) 2% (w/v) and NaCl (Sigma) 0.85% (w/v). For growth on solid medium, Middlebrook 7H11 agar medium was used supplemented with glycerol 0.5%, and 10% oleic acid-albumin-dextrose-catalase enrichments (OADC). When needed, kanamycin, hygromycin or zeocin were added to a final concentration of 20 μg/ml, 100 μg/ml, 25 μg/ml, respectively. Plates used for CFU counting of lung homogenates contained a PANTA (Becton Dickinson) antibiotic mixture. Inspection of Middlebrook 7H11 agar plates for CFU counting of different bacterial strains in various experimental conditions was undertaken after 3 and 5 weeks of incubation.
6
Preparation of Modified Middlebrook 7H10 and LJ Media
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Standard Middlebrook 7H10 (containing 0.25 mg/L MG) was prepared (Atlas and Snyder 2006 ) and a series of modified Middlebrook 7H10 media was prepared with MG concentrations ranging from 2.5 to 2500 mg/L according to a standard protocol for Middlebrook 7H10 agar preparation (Atlas and Snyder 2006 ), except that 2% fresh MG (Krieg 1981 ) was added after the nutrients were autoclaved rather than being mixed with the nutrients before sterilization. The mixed medium was then poured into Petri dishes. PANTA-containing Middlebrook 7H10 agar was prepared similarly, except that MG was replaced with polymyxin B (40,000 U/L)–amphotericin B (4 mg/L)–nalidixic acid (16 mg/L)–trimethoprim (4 mg/L)–azlocillin (4 mg/L) (PANTA; Becton, Dickinson and Company, Spark, USA) reconstituted with oleic acid–albumin–dextrose–catalase (OADC; Becton, Dickinson and Company, Spark, USA) enrichment as recommended by the manufacturer. LJ was prepared according to a standard protocol (Atlas and Snyder 2006 ). PANTA-containing LJ was prepared similarly to LJ, except that PANTA reconstituted in sterile distilled water was added to the lysed eggs.
7
Bioaerosol Collection and Imaging
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The RASC is a purpose-built personal clean room equipped with a high efficiency bioaerosol collection system, which captures all exhaled particulate matter (bioaerosols) at 100–300 L/min in 15 mL of collection medium (sterilized phosphate-buffered saline supplemented with Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azilocillin (PANTA) [Becton Dickinson]). Bioaerosol samples were concentrated and stained with DMN-trehalose (Olilux Biosciences Inc.) overnight, as previously reported, before transfer to nanowell devices for imaging (Dinkele et al., 2021 (link)).
8
CFU Enumeration in Infected Cells and Tissues
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For CFU enumeration in in vitro infected cells, cells were lysed with 500μl sterile H2O for five minutes, followed by addition of 500μl PBS supplemented with 0.05% Tween80. For CFU enumeration in tissues, organs were homogenized in 1ml 7H9 broth (BD) supplemented with 0.2% glycerol (Wisent), 0.05% Tween80 (Fisher), and 10% ADC using OmniTip Plastic Homogenizer Probes (Omni International). Serial dilutions in PBS+0.05% Tween80 were plated on 7H10 agar plates with 10% OADC enrichment and PANTA (BD). Plates were then incubated at 37°C and counted after 21 days.
9
Murine Tuberculosis Infection Protocols
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Mtb Erdman (TMC 107, ATCC) or Mtb H37Rv (ATCC) were grown to log phase in 7H9 medium enriched with Middlebrook ADC (BD Pharmingen, San Diego, CA, USA) without Tween. Log-phase bacteria were aliquoted and stored at −80 °C. For in vivo infection, vials of Mtb Erdman or H37Rv were thawed and sonicated for 5 min in a water bath. Furthermore, bacterial clumps were removed by dispersing the suspensions through a syringe.
For i.v. infections, mice were infected with 5.0 × 104 CFU/mice in 100 µl injected into the tail vein. For aerosol infections, the inoculum was aerosolised for inhalation using a Biaera exposure system controlled via AeroMP software (Biaera, Hagerstown, MD, USA). The standard dose used in this study results in approximately 25–50 CFU/mouse.
The bacterial burden in lung was determined by homogenising the left lung lobe using M-tubes and a Miltenyi AutoMACS Dissociator (Miltenyi, Germany). The homogenate was serially diluted in 1x PBS containing panta (BD) and plated onto 7H11 MiddleBrook agar plates (BD). Colony forming units (CFU’s) were enumerated after 2 weeks of incubation at 37 °C, 5% CO2.
For i.v. infections, mice were infected with 5.0 × 104 CFU/mice in 100 µl injected into the tail vein. For aerosol infections, the inoculum was aerosolised for inhalation using a Biaera exposure system controlled via AeroMP software (Biaera, Hagerstown, MD, USA). The standard dose used in this study results in approximately 25–50 CFU/mouse.
The bacterial burden in lung was determined by homogenising the left lung lobe using M-tubes and a Miltenyi AutoMACS Dissociator (Miltenyi, Germany). The homogenate was serially diluted in 1x PBS containing panta (BD) and plated onto 7H11 MiddleBrook agar plates (BD). Colony forming units (CFU’s) were enumerated after 2 weeks of incubation at 37 °C, 5% CO2.
10
Mycobacterium tuberculosis Mouse Model
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C3HeB/FeJ female mice purchased from the Jackson Laboratory (USA, JAX stock #000658) were used in this study at 6 weeks of age. C3HeB/FeJ mice were infected with 1 × 105 CFU live Mtb strain H37Rv from glycerol stocks, stored at −80 °C. Mice were anaesthetised with 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane (isofluoran; Pharmachemie BV, The Netherlands) and intranasally (i.n.) infected. Six weeks post Mtb challenge, mice were euthanized with CO2 and lungs and splenocytes were aseptically removed. Organs were homogenised using 70 µM cell strainers (Corning, U.S.A.) and the amount of Mtb bacteria was determined by plating out serial dilutions of the homogenates on 7H11 plates (BD Bioscience, U.S.A), supplemented with BBL Middlebrook OADC enrichment (BD Bioscience, U.S.A) and PANTA (BD Bioscience, U.S.A). Colonies were counted after 3 weeks of incubation at 37 °C (Supplementary Data 2 ). For BCG immunisation, C3HeB/FeJ mice were injected subcutaneously in the right flank with 106 CFU BCG1331 (SSI, Denmark) from glycerol stocks, stored at −80 °C. Six weeks post BCG vaccination, mice were killed and splenocytes were aseptically removed. Naïve mice were included as control. All mice were daily monitored for ethical requirements, and weighed once a week according to the ethical regulations at the LUMC animal facility (DEC number 11183).
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