A Technique to Establish a Bacterial Infection Model in Insect Larvae

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 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 …

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 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

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A Technique to Establish a Bacterial Infection Model in Insect Larvae. J. Vis. Exp. (Pending Publication), e21706, doi: (2023).

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