Counting Colony Forming Units (CFUs): Quantifying Bacteria from Fly Bodies

This protocol is an excerpt from Koyle et al., Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions, J. Vis. Exp. (2016).

1. Dechorionate Eggs and Transfer to Sterile Diet

1. Prepare the biosafety cabinet by spraying the inside (including sides) with 70% ethanol. Wipe the bottom with a lab tissue, and sterilize the hood with UV light for ~15 min. Sterilize all non-biological supplies (specimen cups, paintbrush, forceps, waste container, 400 ml sterilized water, and 100 ml of 0.6% sodium hypochlorite) by spraying with ethanol and immediately placing in the biosafety cabinet. Sterilize with UV light for 15 min.
2. Start the first of 2 sodium hypochlorite washes by placing the bushing with the eggs into a 120 ml specimen cup or other sterile container. Slowly pour ~90 ml of 0.6% sodium hypochlorite solution into the bushing until just below the rim.
3. Rinse eggs for 2.5 min. Periodically re-suspend the eggs by using forceps to move the bushing up and down in the hypochlorite solution.
4. Transfer the bushing directly into a second specimen cup, pre-filled with 90 ml bleach, inside the biosafety cabinet.
5. Repeat step 1.3 inside the biosafety cabinet. At the end of the second bleach treatment, the eggs should begin to adhere to the sides of the bushing.
6. Carry out steps 1.7-1.8 in the biosafety cabinet.
7. Discard the bleach and wash the bushing with sterile water 3 times. Re-suspend the eggs several times during each washing by moving the bushing with forceps. By the end of the third washing most eggs should be attached to the side of the bushing.
8. Using a paintbrush sterilized in ethanol, transfer eggs from the side of the bushing to the sterile diet. Transfer eggs individually or in small batches. Aim for 30-50 eggs per vial. Leave the caps loose to allow oxygen to enter the tube. If vials are to remain axenic, transfer to an insect incubator; otherwise, add bacteria as below.

2. Make Gnotobiotic Flies Using 4 Bacterial Species

1. Prepare Bacteria
   1. Prepare a sterilized biosafety cabinet with necessary supplies (pipettes, pipette tip boxes, sterilized centrifuge tubes, MRS broth, and test tube racks) as in step 1.1. Wipe the outside of test tubes with an ethanol-soaked laboratory wipe before placing in biosafety cabinet.
   2. Pellet the bacteria by first transferring 500 µl of overnight growth to a sterile microfuge tube. If bacterial density is low, add up to 1.5 ml to each tube or sequentially remove supernatant and add extra culture to the same tube. Remove samples from the biosafety cabinet and centrifuge for 10 min at 10,000 x g. Use filter tips to avoid contamination between samples.
   3. Determine the density of each culture by measuring OD₆₀₀. If using a multi-well plate reader, transfer 200 µl of each culture to a 96-well plate in 1-, 2-, and 4- fold dilutions.
   4. Determine the amount of mMRS in which to dilute each cell pellet (2.1.2) using a plate reading spectrophotometer and the following equations. Plan to add enough broth to inoculate 50 µl to each fly vial.
      1. Collect OD₆₀₀ readings for a 1:1, 1:2, and 1:4 dilution of each bacterial culture on a plate-reading spectrophotometer. Select the dilution for each bacterial strain that produces an OD₆₀₀ value between 0.1 and 0.2 and use this value and its corresponding dilution factor as 'O' and 'D' in the formulas given in 2.1.4.2 or 2.1.4.3.
      2. If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD₆₀₀ to CFU conversion determined previously in Newell and Douglas, Appl Environ Microbiol. (2014)) using this equation:
         E = ((O-B) x V x D)/C
         where E = volume to resuspend pellet in (µl), O = OD₆₀₀ bacteria, B = OD₆₀₀ blank media, D = fold-dilution, V = µl bacterial culture prior to centrifugation, C = OD₆₀₀ of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use "O" in place of "O-B".
         NOTE: The predetermined constants (units OD₆₀₀, normalized to 10⁷ CFU ml^-1, constants derived in Newell and Douglas, Appl Environ Microbiol. (2014)) are as follows: A. tropicalis (0.052), A. pomorum (0.038), L. brevis (0.056), L. plantarum (0.077).
      3. If using other bacterial species (no CFU/OD₆₀₀ constant is available), normalize density to OD₆₀₀ = 0.1 using this equation:
         E = ((O-B) x V x D)/0.1 OD₆₀₀
         NOTE: Units are the same as in step 2.1.4.2. See Supplemental Code File for examples of calculations using these equations.
   5. In the biosafety cabinet, remove supernatant with a pipet tip and resuspend the pellet in fresh mMRS or PBS as calculated in step 2.1.4.2.
2. Inoculate Bacteria
   1. Transfer 50 µl of the bacteria to the conical tubes with sterile diet and dechorionated eggs in biosafety cabinet. Add bacteria after egg transfer to prevent contamination between vials.
   2. Place inoculated tubes in an incubator at 25 °C.

3. Measure CFU Load/Test for Sterility

1. To measure the CFU load in whole body fly homogenates, transfer 5 flies (5-7 days post eclosion) to a 1.7 ml microfuge tube containing 125 µl of ceramic beads and 125 µl of mMRS broth. Homogenize flies using a tissue homogenizer for 30 sec at 4.0 M/sec.
   1. Alternatively, omit beads and hand homogenize in microcentrifuge tubes with plastic pestles for 1 min.
   2. If quantifying the gut microbiota, surface sterilize the flies to remove exogenous microbes. Transfer flies to a microcentrifuge tube containing 100 µl 70% ethanol for 1 min, aspirate ethanol, and transfer to a new microcentrifuge tube for homogenizations. If the DNA content of the gut will be measured, rinse for 1 min with 0.6% sodium hypochlorite before the ethanol wash.
2. Dilute the homogenate with 875 µl mMRS, vortex for 5 sec, and pipet 120 µl of homogenate into the first well of a microtiter plate.
3. Perform two sequential 1:8 dilutions using 10 µl homogenate and 70 µl MRS in the next two wells.
   1. Remove 10 µl from the first well and add it to the second well containing 70 µl MRS. Mix the contents of the second well thoroughly, transfer 10 µl from the second well to the third well containing 70 µl MRS, and mix thoroughly. This leads to 3 total concentrations of the original 1,000 µl homogenate: undiluted, 1:8, and 1:64.
4. Transfer 10 µl of each dilution to a mMRS plate (using a multi-channel pipet if desired). Slightly incline the dish to spread the dilution several millimeters down the agar surface and allow liquid to dry before moving the plate. The liquid dries on the plate quickly if the plates are 2 days old, reducing mixing of two neighboring droplets.
5. Incubate at 30 °C for 1-2 days. Remove plates from the incubator once distinct, individual colonies are visible, and count from a dilution with 10-100 isolated colonies.
6. Calculate CFU per fly using the equation E = C x D/P x V/F, where E = CFU per fly, C = number of colonies counted, D = dilution, P = µl plated, V = volume of fly homogenate, and F = number of flies homogenized.