A Technique to Establish a Bacterial Infection Model in Insect Larvae

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 a method to establish a Mycobacterium bovis infection model in the larvae of the greater wax moth, Galleria mellonella. A bioluminescent strain of Mycobacterium bovis BCG is injected into the hemolymph of the larva to establish infection, and the bioluminescence is measured at predefined intervals to study the progress of infection.

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 …

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
Hygromycin B	Corning	30-240CR
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