Quantifying Bacterial Shedding to Evaluate Infection Transmission Potential in Drosophila

Abstract

Source: Siva-Jothy, J. A., et al., Oral Bacterial Infection and Shedding in Drosophila melanogaster. J. Vis. Exp. (2018)

This video demonstrates the procedure for monitoring bacterial shedding in Drosophila by transferring an infected fly across multiple food-containing tubes, recovering shed bacteria, and quantifying viable colonies through serial dilution and plating.

Protocol

1. Measure Bacterial Shedding
   1. Measure the shedding alongside the internal load.
   2. After infection, transfer single flies to 1.5 mL microcentrifuge tubes containing ~50 µL of Lewis medium for 24 h.
   3. Remove the flies for the internal load measurement and wash the tubes with 100 µL of 1x PBS (phosphate buffered saline) by vortexing heavily for 3 s.
   4. Measure the CFUs (colony-forming units) in this wash by plating on LB (Luria-Bertani) nutrient agar.
   5. After infection, transfer single flies to 1.5 mL microcentrifuge tubes containing ~50 µL of Lewis medium for 24 h.
   6. Transfer the flies to new microcentrifuge tubes containing ~50 µL of Lewis medium for a further 24 h. Wash the contaminated tubes with 100 µL of 1x PBS by vortexing heavily for 3 s.
   7. Measure the CFUs in this wash by plating on LB nutrient agar.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vials	Sarstedt Ltd.	58.49	Polystyrene flat base tube 75mm x 23.5mm, 23ml
Agar	Sigma-Aldrich	A7002	Agar, ash 2.0-4.5%
Brown sugar	Bidvest	66032	Light brown soft sugar
Maize	Dove's Farm		Organic maize flour
Fermipan yeast	Bidvest	96360	Dry, instant yeast
Petri dishes	Fisher Scientific UK Ltd.	15788517	X600 Petri Dish 90 X 16.2MM Sterile Triple Vent
Eppendorf tube (1.5ml)	Sarstedt Ltd.	72.690.001	Sarstedt Micro Tube Conical Base Push Cap 1.5ml Standard Neutral
Ethanol	VWR International Ltd.	20821.33	ETHANOL ABSOLUTE ANALAR NP ACS/R.PE - Analytical Grade
LB broth, Miller	Fisher Scientific UK Ltd.	BP1426	Contains 10g tryptone, 5g yeast extract, 10g sodium chloride per litre
96-well microplate	Scientific Laboratory Supplies Ltd.	353072	Falcon 96 Well Clear Flat Bottom TC-Treated Polystyrene Cell Culture Microplate with Lid Sterile
Vortex	Fisherbrand		Whirlimixer 75W 50-6-Hz 220-240V
10 µL Pipette tips	Greiner Bio-one Inc	741015	Easlyload gilson-style. Graduated, clear, refill, 960 pcs
200 µL Pipette tips	Greiner Bio-one Inc	741065	Easlyload gilson-style. Graduated, clear, refill, 960 pcs
1000 µL Pipette tips	Greiner Bio-one Inc	741045	Easlyload gilson-style. Graduated, clear, refill, 960 pcs
20 µL Filtered pipette tips	Greiner Bio-one Inc	774288	Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 96pcs
200 µL Filtered pipette tips	Greiner Bio-one Inc	739288	Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 96pcs
1000 µL Filtered pipette tips	Greiner Bio-one Inc	740288	Filter tip gilson-style. Clear, rack blue, single packed, R/Dnase free, 10 racks of 60pcs