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 …

Divulgazioni

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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Citazione di questo articolo
Preparation and Quantification of Mycobacterium bovis BCG lux Inoculum for Infection. J. Vis. Exp. (Pending Publication), e21705, doi: (2023).

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