Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection
Published: September 29, 2023
Abstract
Source: Asai, M. et al., Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex. J. Vis. Exp. (2019)
This video demonstrates the preparation and quantification of Mycobacterium bovis Bacillus Calmette-Guérin lux inoculum using bioluminescence assay and the enumeration of colony-forming units. The prepared inoculum can be used for infection studies.
Protocol
NOTE: All work described below is to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines. 1. Preparation of M. bovis BCG lux for Infection Defrost a frozen 1.2 mL glycerol (15%) stock of M. bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi …
Divulgaciones
The authors have nothing to disclose.
Materials
| 1.5ml reaction tube (Eppendorf) | Eppendorf | 22431021 | |
| 20, 200 and 1000 µl pipette and filtered tips | Any supplier | n/a | |
| 24-well culture plate | Greiner | 662160 | |
| 25 ml pipettes and pipette boy | Any supplier | n/a | |
| 3 compartment Petri dish (94/15mm) | Greiner | 637102 | |
| Centrifuge | Any supplier | n/a | |
| Class II saftey cabinet | Any supplier | n/a | |
| Ethanol (>99.7%) | VWR | 208221.321 | |
| Glycerol | Sigma-Aldrich | G5150 | |
| Hygromycin B | Corning | 30-240CR | |
| Luminometer (Autolumat LB 953) | Berthold | 34622 | |
| Luminometer tubes | Corning | 352054 | |
| Microcentrifuge | Any supplier | n/a | |
| Middlebrook 7H11 agar | BD Bioscience | 283810 | |
| Middlebrook 7H9 broth | BD Bioscience | 271310 | |
| Middlebrook ADC enrichment | BD Bioscience | 212352 | |
| Middlebrook OADC enrichment | BD Bioscience | 212240 | |
| Mycobacterium bovis BCG lux | Various | n/a | |
| n-decyl aldehyde | Sigma-Aldrich | D7384-100G | |
| Orbital shaking incubator | Any supplier | n/a | |
| Phosphate-buffered saline | Sigma-Aldrich | P4417-100TAB | |
| Polysorbate 80 (Tween-80) | Sigma-Aldrich | P8074-500ml | |
| Erlenmeyer flask with vented cap (250 ml) | Corning | CLS40183 | |
| Winterm (V1.08) | Berthold | n/a | Program LB953.TTB |
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Citar este artículo
Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection. J. Vis. Exp. (Pending Publication), e21705, doi: (2023).