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

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 containing fly homogenate and dispense into the second row.
      4. Pipette up and down at least 10 times to thoroughly mix, and then take 10 µl and transfer to the third row. Repeat this procedure using the remaining rows.
      5. Starting from the bottom row (most dilute bacteria suspension), use the multichannel pipette to take 10 µl from each well and deposit on an LB plate, taking care that the samples are dispensed as discrete spots that do not touch each other. Repeat until all wells have been sampled from each row, dispensing them in descending order of dilution on the LB plate.
      6. Leave the plate at RT until the spots have completely soaked into the LB plate.
         NOTE: Drying the LB plate for a few days at RT prior to use is recommended to ensure that the liquid is readily absorbed, minimizing the chances of accidental contact between sample spots.
   4. Incubate the plates O/N, taking care not to overgrow the plates so colonies remain small and discrete.
      NOTE: Depending on the bacterial growth medium and incubation temperature used, colonies derived from the fly's endogenous gut microbiota may eventually appear on the plate. However, most bacteria used for pathogenic infection grow much faster than the gut microbiota on LB agar at 37 °C.
   5. Remove plates from the incubator when the experimental bacteria have grown visible colonies (typically 8 – 12 hr), but before Drosophila gut microbiota colonies appear (approximately 36 hr).  For slow-growing experimental bacteria, use a selective antibiotic in the LB agar to remove any colonies from the gut microbiota.
   6. Count the number of colonies for each homogenate.
      1. For spiral plates, count the colonies that grow using an automated colony counter that can estimate the bacterial load per ml of homogenate based on the number and position of the colonies on the plate.
         NOTE: Spiral counts are most accurate when the bacterial concentration is in the range of 5 x 10² to 5 x 10⁴ bacteria per ml.
      2. For spot plates, manually count the colonies for each fly from whichever dilution contains 30 – 300 colonies and calculate the number of bacteria per ml of original homogenate.
2. Analyze the data.
   1. If the bacterial load is non-normal, either transform the data to better approximate a normal distribution or analyze the data using non-parametric statistical analysis (e.g., Mann-Whitney U test). Use a Box-Cox analysis to find the optimal transformation; natural log or base -10 log transformations are often effective.
   2. If the data are sufficiently normally distributed and there are only two treatments being contrasted, perform a t-test to determine whether the two treatments result in different mean bacterial loads. If there are multiple experimental variables or other potentially predictive factors, use Analysis of Variance (ANOVA) to determine which factors are significant predictors of bacterial load.