Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva
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 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…
Declarações
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 | |
| 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 |
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Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva. J. Vis. Exp. (Pending Publication), e21707, doi: (2023).