Measuring aggregation of bacterial cells in the squid host requires higher inocula (105-107 CFU/ml) than the typical colonization protocol.
- Day 0 - Two days prior to squid inoculation, plate the relevant bacterial strains on LBS agar.
- Incubate bacteria at 25-28°C overnight.
- Day 1 - Inoculate 3 ml LBS medium in a glass culture tube with one colony of each V. fischeri strain for infection. Prepare duplicate tubes as backup. Squid will be inoculated directly from the overnight culture.
- For each sample, label 6 LBS plates on which to plate samples of the inoculum labeled:
- sample #, replicate 1, 10-1
- sample #, replicate 1, 10-2
- sample #, replicate 1, 10-3
- sample #, replicate 2, 10-1
- sample #, replicate 2, 10-2
- sample #, replicate 2, 10-3
- For apo prepare two plates labeled "aposymbiotic 100"
- Add 5 sterile plating beads per plate.
- For each sample, prepare 6 microfuge tubes with 900 μl autoclaved 70% FSIO labeled as above.
See this section in the standard colonization protocol.
- Day 2 - Prepare bowls of squid containing 40 ml FSIO and animals. Inoculate with the relevant overnight culture by adding 40 μl of the overnight culture directly to the bowl.
- For each treatment, create a "vortex" in the bowl with the dedicated transfer pipette by placing the pipette near the edge of the bowl and pipetting up and down repeatedly to mix the water and squid for approximately 10 sec. Thorough mixing is critical.
- To determine the inoculum levels, add 100 μl from the sample bowl to the microfuge tube labeled replicate 1, 10-1. Repeat into the replicate 2, 10-1 tube. Conduct serial dilutions of each to 10-2, 10-3 and plate all 6 onto the plates prepared above. Incubate at 25-28°C overnight.
If you plan to fix or stain animals, proceed with the fixation and staining protocol at this point.
- Anesthetize animals in 2% EtOH/FSIO and mix gently (i.e., in a conical tube).
- Transfer the animal to a depression well slide dissect off the mantle and funnel, and image.