Measuring E. coli Bacterial Load in Drosophila melanogaster following E. coli Infection

Abstract

Source: Khalil, S., et al. Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster. J. Vis. Exp.  (2015)

In this video, we demonstrate a technique for measuring the E. coli bacterial load after injecting E. coli into the thorax of Drosophila melanogaster.

Protocol

1. Assay Bacterial Load Post-infection

1. Measure bacterial load.
   1. At a prescribed time post-injection, place each fly into a microcentrifuge tube on ice.
   2. Add 250 µl PBS to each tube and homogenize the flies either using a pestle or a bead beater.
   3. Plate the homogenate on an LB agar plate, either using a spiral plater or a serial dilution.
      1. To plate using serial dilution, transfer each fly homogenate to the first row of a 96-well plate.
      2. Fill each well of the remaining rows with 90 µl of PBS.
      3. Using a multichannel pipette, take 10 µl from the first row containin....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri Dishes with Clear Lids, Raised Ridge; 100 x 15 mm;	VWR international	25384-302
LB Agar, Miller	Difco	244520
Inoculating Loop	VWR international	80094-488
1.5mL Microcentrifuge tubes; Seal Rite	USA Scientific Inc.	1615-5500
Motorized Pestle; Talboys Laboratory Stirrer	Troemner	103
Talboys High Throughput Homogenizer	OPS Diagnostics	930145
5/32'' Grinding Balls	OPS Diagnostics	GBSS 156-5000-01
Vortex Genie	Scientific indurstries inc.	G560
Multichannel Pipettor (10uL-300uL)	Sartorius	730360
WASP2 Whitley Automated Spiral Plater	Microbiology International
ProtoCOL automated colony counter / plate counter/ plate reader	Microbiology International
Incubator	Powers Scientific, Inc	DROS52SD