Jul 01, 2023
Version 2
Symbiotic Dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes V.2
Peer-reviewed method
- ard1,2,
- ejg1,2,
- agc1,2,
- Tim I Miyashiro1,2
- 1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA;
- 2The One Health Microbiome Center, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA
External link: https://doi.org/10.1371/journal.pone.0287519
Protocol Citation: ard, ejg, agc, Tim I Miyashiro 2023. Symbiotic Dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2155g3p/v2Version created by Tim I Miyashiro
Manuscript citation:
Donnelly AR, Giacobe EJ, Cook RA, Francis GM, Buddle GK, et al. (2023) Quantification of the capacity of vibrio fischeri to establish symbiosis with Euprymna scolopes. PLOS ONE 18(7): e0287519. https://doi.org/10.1371/journal.pone.0287519
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 30, 2023
Last Modified: July 01, 2023
Protocol Integer ID: 84312
Keywords: Preparation of V. fischeri Cultures, Preparation of Juvenile E. scolopes, Inoculation Phase, Measurement of Bioluminescence, Euthanasia and Storage of Animals, Scoring of Bioluminescence
Funders Acknowledgement:
National Institute of General Medical Sciences
Grant ID: R01 GM129133
Abstract
This protocol details symbiotic dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes.
Materials
Materials needed:
- Culture tubes
- LBS medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 2% (w/v) NaCl, 50 mM Tris–HCl (pH 7.5)], with 1.5% w/v agar for solid medium
- Shaking incubator at 28°C
- Spectrophotometer and cuvettes
- Plastic Tumblers, e.g., Fineline Mfr. #409-CL Savvi Serve 9 oz. Clear Hard Plastic
- Freshly hatched E. scolopes squid
- Transfer pipets, e.g., Fisherbrand Disposable Graduated Transfer Pipettes Catalog No. 13-711-9AM
- Filter-sterilized seawater (FSSW): Instant Ocean (Spectrum Brands, Blacksburg, VA) mixed according to instructions provided by manufacturer. Filter through 0.22-μm surfactant-free filter (Nalgene Rapid-Flow Sterile Disposable Filter Units with SFCA Membranes).
- Microfuge tubes
- 50-mL conical tubes
- Vials, e.g., VWR Drosophila vials narrow #75813-162
- Luminometer, e.g., GloMax 20/20 (Promega Corp., Madison, WI)
Preparation of V. fischeri Cultures
Preparation of V. fischeri Cultures
For each strain of interest, initiate a starter culture by inoculating 3 mL LBS with an isolated colony. Incubate starter cultures Overnight (~16 h) at 28 °C shaking at 200 rpm .
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.
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 200 rpm .
Selection and Preparation of Juvenile E. scolopes
Selection and Preparation of Juvenile E. scolopes
Using transfer pipet, collect freshly hatched juvenile squid into tumblers containing 100 mL FSSW, with no more than 50 squid/tumbler.
Prepare a new tumbler with 50 mL FSSW for each group.
Transfer animals from the 100 mL FSSW tumblers to the new tumblers individually .
Note
To minimize bias, add an animal to the tumbler of a different group with each transfer.
Preparation of Inoculums
Preparation of Inoculums
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.
Concentrate cells by centrifugation.
Concentrate cells by centrifugation at 5000 x g, 00:02:30 . Then, remove 0.9 mL supernatant, add 0.9 mL FSSW, and resuspend the pellet. (1/2)
2m 30s
Concentrate cells by centrifugation at 5000 x g, 00:02:30 . Then, remove 0.9 mL supernatant, add 0.9 mL FSSW, and resuspend the pellet. (1/2)
2m 30s
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.
Note
Note that three-fold dilutions can be used instead for greater resolution.
Prepare a control for an apo-symbiotic group by transferring 1 mL FSSW to a microfuge tube.
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.
Inoculation Phase
Inoculation Phase
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.
Sample tumblers by plating 100 µL onto solid LBS medium in triplicate and incubate the plates at 28 °C Overnight .
Note
Note that for high inoculum levels, a dilution may be necessary to obtain countable CFUs. For low inoculum levels, it may be preferable to use the known dilution factor from more concentrated inoculums to estimate the corresponding abundance of V. fischeri.
After 3.5 hours, wash the animals by serially transferring them as a group into a tumbler containing 100 mL FSSW twice, with 00:05:00 between transfers.
5m
Transfer animals into vials containing 4 mL FSSW, with one animal per vial.
Store animals in a room that has a 12-h day/12-h night light cycle.
Measurement of Bioluminescence
Measurement of Bioluminescence
After 16-18 h, transfer animals to clean vials containing 4 mL FSSW.
Using a luminometer, measure the luminescence emitted by each sample.
Euthanasia and Storage of Animals
Euthanasia and Storage of Animals
To initiate the anesthesia step, transfer each animal with seawater (total volume of 0.5 mL ) to a microfuge tube and place On ice .
After 00:05:00 , add 0.5 mL cold 6% ethanol/FSSW to each microfuge tube and keep On ice .
5m
After 00:15:00 , remove the liquid volume from the tube and store the anesthetized animal at -80 °C , thereby completing euthanasia.
15m
Scoring of Bioluminescence
Scoring of Bioluminescence
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.
Determining Inoculum Levels
Determining Inoculum Levels
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.
Calculation of SD50
Calculation of SD50
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
- animals that could be assumed to be symbiotic at higher inoculums and
- 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.