Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

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 showcases the in vivo bacterial burden measurement using Bacillus Calmette-Guérin, BCG, lux-infected insect larvae. The process involves larval homogenization, treatment with a luciferase substrate, and bioluminescence measurement to determine relative light units that correlate with bacterial burden.

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 Mycobacterium bovis Bacillus Calmette-Guérin (BCG) lux, the Montreal vaccine strain transformed with the sh…

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 safety cabinet	Any supplier	n/a
Erlenmeyer flask with vented cap (250 ml)	Corning	CLS40183
Ethanol (>99.7%)	VWR	208221.321
Galleria mellonella (250 per pk)	Livefood Direct UK	W250
Glycerol	Sigma-Aldrich	G5150
Homogenizer (FastPrep-24 5G )	MP Biomedicals	116005500
Hygromycin B	Corning	30-240CR
Luminometer (Autolumat LB 953)	Berthold	34622
Luminometer tubes	Corning	352054
Lysing matrix (S, 2.0ml)	MP Biomedicals	116925500
Micro syringe (25 µl, 25 ga)	SGE	3000
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
Small box	Any supplier	n/a	Dark vented or non-sealed box recommended
Tweezer	Any supplier	n/a	Short and narrow tipped/Blunt long tweezers
Petri dish (94/15mm)	Greiner	633181
Filter paper (94mm)	Any supplier	n/a	Cut to fit