Symbiotic Dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes

For each strain of interest, initiate a starter culture by inoculating 3mL LBS with an isolated colony. Incubate starter cultures (~16 h) at 28°C shaking at 200rpm.

Measure the OD₆₀₀ of each starter culture. In a microfuge tube, normalize each starter culture by diluting it to an OD₆₀₀ of 1.0 in fresh LBS to a final volume of 1.0mL. Vortex briefly.

Initiate an intermediate culture by inoculating 3mL LBS in a fresh culture tube with 30µL of the normalized cell suspension. Incubate at 28°C shaking at 200rpm.

Using transfer pipet, collect freshly hatched juvenile squid into tumblers containing 100mL FSSW, with no more than 50 squid/tumbler.

Prepare a new tumbler with 50mL FSSW for each group.

Transfer animals from the 100 mL FSSW tumblers to the new tumblers individually .

For each strain, when the turbidity of culture is OD₆₀₀ = 0.8-1.0, transfer culture volume equivalent to 1mL of OD₆₀₀ = 1.0 to a microfuge tube.

Concentrate cells by centrifugation.

Concentrate cells by centrifugation at 5000x g. Then, remove 0.9mL supernatant, add 0.9mL FSSW, and resuspend the pellet. (1/2)

Concentrate cells by centrifugation at 5000x g. Then, remove 0.9mL supernatant, add 0.9mL FSSW, and resuspend the pellet. (1/2)

Prepare a serial dilution by transferring 100µL of the cell suspension described in Step 8 into 0.9mL FSSW in a microfuge tube (10-1 dilution). Then, continue ten-fold dilutions until the desired dilution range has been achieved.

Prepare a control for an apo-symbiotic group by transferring 1mL FSSW to a microfuge tube.

For each group, transfer 100µL from the corresponding microfuge tube into a 50-mL conical tube containing 50mL FSSW and invert several times to mix.

To initiate the inoculation phase, pour the cell suspension into the corresponding tumbler to bring the total volume to 100mL. Repeat for the control described in Step 10.

Sample tumblers by plating 100µL onto solid LBS medium in triplicate and incubate the plates at 28°C .

After 3.5 hours, wash the animals by serially transferring them as a group into a tumbler containing 100mL FSSW twice, with 0h 5m 0s between transfers.

Transfer animals into vials containing 4mL FSSW, with one animal per vial.

Store animals in a room that has a 12-h day/12-h night light cycle.

After 16-18 h, transfer animals to clean vials containing 4mL FSSW.

Using a luminometer, measure the luminescence emitted by each sample.

To initiate the anesthesia step, transfer each animal with seawater (total volume of 0.5mL) to a microfuge tube and place On ice.

After 0h 5m 0s, add 0.5mL cold 6% ethanol/FSSW to each microfuge tube and keep On ice.

After 0h 15m 0s, remove the liquid volume from the tube and store the anesthetized animal at -80°C, thereby completing euthanasia.

Use the luminescence measurements of the apo-symbiotic group to determine the 99.9th percentile, above which animals are considered to be bioluminescent.

Score each animal as symbiotic or non-symbiotic by comparing the corresponding luminescence measurement with the bioluminescence cutoff defined in Step 22.

Count CFU on the inoculum plates generated in Step 13. Also verify that no CFU are present on the apo-symbiotic control plates.

Calculate the concentration of CFUs in each inoculum cell suspension described in Step 9 by dividing the CFU counts by the volume plated (in mL) and multiplying by the dilution factor, if any.

For each strain, generate a table with the number of symbiotic and non-symbiotic animals at each inoculum concentration, with rows arranged in order of highest to lowest concentration.

Prepare two additional columns containing adjusted counts for

1. animals that could be assumed to be symbiotic at higher inoculums and
2. animals that could be assumed to be non-symbiotic at lower inoculums.

Calculate the adjusted percent of symbiotic animals at each inoculum by dividing the adjusted counts of symbiotic animals by the total adjusted animal counts in the corresponding row.

Calculate the SD₅₀ using the equation:

SD₅₀ = 10^[log(DF^X) + log(c)], where

• X = [(50%-a)/(b-a)] and
• a = the adjusted percent symbiotic below 50% closest to 50%.
• b = the adjusted percent symbiotic above 50% closest to 50%.
• c = the inoculum concentration of the adjusted percent colonized below 50% closest to 50%.
• DF = the dilution factor or fold-change difference between groups in the experiment.