TITLE: Symbiotic Dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes
AUTHORS: ard, ejg, agc, Tim I Miyashiro

[DESCRIPTION]
This protocol details symbiotic dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes.

[STEPS]
SECTION: Preparation of V. fischeri Cultures
1. For each strain of interest, initiate a starter culture by inoculating 3 mL LBS with an isolated colony. Incubate starter cultures   (~16 h) at 28 °C shaking at  .

SECTION: Preparation of V. fischeri Cultures
2. 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 1.0 mL.  Vortex briefly.
SECTION: Preparation of V. fischeri Cultures
3. Initiate an intermediate culture by inoculating 3 mL LBS in a fresh culture tube with 30 µL of the normalized cell suspension.  Incubate at 28 °C shaking at  .

SECTION: Selection and Preparation of Juvenile E. scolopes
4. Collect freshly hatched juvenile squid into tumblers containing 100 mL FSSW, with no more than 50 squid/tumbler.

SECTION: Selection and Preparation of Juvenile E. scolopes
5. For each group, add 50 mL FSSW to a tumbler.
SECTION: Selection and Preparation of Juvenile E. scolopes
6. Transfer animals individually to each tumbler.  
 

SECTION: Preparation of Inoculums
7. For each strain, when the turbidity of culture is OD600 = 0.8-1.0, transfer culture volume equivalent to 1 mL of OD600 = 1.0 to a microfuge tube.
SECTION: Preparation of Inoculums
8. Concentrate cells by centrifugation.
SECTION: Preparation of Inoculums
9. Prepare a serial dilution by transferring 100 µL of the cell suspension described in Step 8 into 0.9 mL FSSW in a microfuge tube (10-1 dilution).  Then, continue ten-fold dilutions until the desired dilution range has been achieved.  
 

SECTION: Preparation of Inoculums
10. Prepare a control for an apo-symbiotic group by transferring 1 mL FSSW to a microfuge tube.

SECTION: Preparation of Inoculums
11. For each group, transfer 100 µL from the corresponding microfuge tube into a 50-mL conical tube containing 50 mL FSSW and invert several times to mix.
SECTION: Inoculation Phase
12. To initiate the inoculation phase, pour the cell suspension into the corresponding tumbler to bring the total volume to 100 mL.  Repeat for the control described in Step 10.

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

SECTION: Inoculation Phase
14. After 3.5 hours, wash the animals by serially transferring them as a group into a tumbler containing 100 mL FSSW twice, with 5 min between transfers.
SECTION: Inoculation Phase
15. Transfer animals into vials containing 4 mL FSSW, with one animal per vial.

SECTION: Inoculation Phase
16. Store animals in a room that has a 12-h day/12-h night light cycle.
SECTION: Measurement of Bioluminescence
17. After 16-18 h, transfer animals to clean vials containing 4 mL FSSW.
SECTION: Measurement of Bioluminescence
18. Using a luminometer, measure the luminescence emitted by each sample.
SECTION: Euthanasia and Storage of Animals
19. To initiate the anesthesia step, transfer each animal with seawater (total volume of 0.5 mL) to a microfuge tube and place on ice.
SECTION: Euthanasia and Storage of Animals
20. After 5 min, add 0.5 mL cold 6% ethanol/FSSW to each microfuge tube and keep on ice.

SECTION: Euthanasia and Storage of Animals
21. After 15 min, remove the liquid volume from the tube and store the anesthetized animal at -80 °C, thereby completing euthanasia.
SECTION: Scoring of Bioluminescence
22. Use the luminescence measurements of the apo-symbiotic group to determine the 99.9th percentile, above which animals are considered to be bioluminescent.
SECTION: Scoring of Bioluminescence
23. Score each animal as symbiotic or non-symbiotic by comparing the corresponding luminescence measurement with the bioluminescence cutoff defined in Step 22.
SECTION: Determining Inoculum Levels
24. Count CFU on the inoculum plates generated in Step 13.  Also verify that no CFU are present on the apo-symbiotic control plates.
SECTION: Determining Inoculum Levels
25. 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.
SECTION: Calculation of SD50
26. 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.
SECTION: Calculation of SD50
27. Prepare two additional columns containing adjusted counts for 
animals that could be assumed to be symbiotic at higher inoculums and
animals that could be assumed to be non-symbiotic at lower inoculums.
SECTION: Calculation of SD50
28. 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.
SECTION: Calculation of SD50
29. 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.
SECTION: Preparation of Inoculums
8.1. Concentrate cells by centrifugation at 5000 x g, 150 s.  Then, remove 0.9 mL supernatant, add 0.9 mL FSSW, and resuspend the pellet.  (1/2)
SECTION: Preparation of Inoculums
8.2. Concentrate cells by centrifugation at 5000 x g, 150 s.  Then, remove 0.9 mL supernatant, add 0.9 mL FSSW, and resuspend the pellet.  (1/2)