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Competitive squid colonization assay

This protocol is a variation of the squid colonization protocol that has been modified for how to perform a competition between 2 bacterial strains. Steps bolded below highlight the differences between protocols.

Supplies and Considerations

Make sure one strain is labeled so strains can be differentiated from each other. This protocol specifically reviews marking one strain with the LacZ containing plasmid (pVSV103). LBS-Xgal plates will be needed to do blue/white screen to differentiate colonies.

Alternatively strains could be differentiated with resistance markers or fluorescence for microscopy. Make sure proper plates are prepared depending on the conditions needed to differentiate the strains.

Protocol

1. Preparation of Bacterial Inocula
  1. Day 0 - Two days prior to squid inoculation, plate the relevant bacterial strains on LBS agar.
  2. Incubate bacteria at 25-28°C overnight.
  3. 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. Use selective media (LBS-Kan) for the strain containing pVSV103
  4. Day 2 - (Coordinate with squid steps) 1 h prior to inoculation, subculture bacteria 1:80 (37.5 μl) into 3 ml LBS in a glass culture tube and grow for 1 h with aeration.
  5. Measure the OD600 of the sample prior to inoculation. Typical measurements are 0.15-0.4 depending on the strain.
  6. For a target inoculum of a total 3-5 x 103 CFU/ml with a 50/50 mix calculate the inoculum volume as follows: Inoculum volume (μl) = 1.25 / OD600. Multiply this number by 50. For each competition add (1.25 / OD600) * 50 = ul of each strain to the tube. Vortex well to mix. Divide the total volume in the tube by 100 and add this amount directly to the seawater containing the squid in Step 4.1. This calculation may need to be adjusted for different strains of V. fischeri or for inoculation at lower or higher levels than those specified here.

Example calculation:

Strain OD 1.25/OD ul to add
Strain A 0.25 5 ul 250 ul
Strain B 0.30 4.2 ul 210 ul

Total ul in tube = 460 ul

Mix together 250 ul Strain A and 210 ul of Strain B in a microtube. Add 4.6 ul (460 ul / 100) of this mix to the seawater of the squid.

2. Preparation of Agar Plates for Enumeration of the Inocula
  1. For each treatment, label LBS-Xgal plates (2 per treatment) to plate samples of the inoculum in Step 4.1. If LBS-Xgal plates are not available, spread 100ul of 20 mg/ml Xgal solution onto each plate as directed here.
  2. Add 5 sterile plating beads per plate.
3. Collection of Squid Juveniles
  1. Measure the salinity of Instant Ocean using the refractometer and adjust to 35‰.
  2. Filter 1 L of Instant Ocean using the filtration unit and an attached vacuum line or vacuum pump, to generate filter-sterilized Instant Ocean (FSIO). Oxygenate the water by swirling vigorously prior to each dispensing. The filter unit can be reused for 2 days.
  3. Aliquot 40-50 ml of FSIO into each of two (2) disposable sample bowls. Label one as earlies and one as timelies.
  4. Prepare an excess of plastic transfer pipettes for acquiring juvenile squid by cutting the pipette approximately 1 cm from the tip, above the lowest ridges (see Figure 3). This facilitates a wider area through which the squid can pass upon collection. Discard any transfer pipettes in which there is a rough exposed surface.
  5. Using prepared transfer pipettes, collect E. scolopes that hatched overnight and transfer to the earlies bowl of FSIO. Early hatchlings have been in the egg system for over 1 h and are susceptible to colonization by contaminating V. fischeri in the egg system. Do not use earlies for sensitive colonization experiments.
  6. Check egg tanks every 30-45 min for new hatchlings. Ensure that all hatchlings are cleared during each check. Remove hatchlings with a transfer pipette, and deposit into the timelies bowl of FSIO. Animals collected in a timely fashion are available for colonization experiments
  7. When the collection has finished (~ 45 min after dusk), transfer the squid to the main laboratory. Empirically it is advantageous to colonize the animals under uninterrupted laboratory light conditions for 3 h inoculations.
  8. For each treatment, prepare a bowl with 40 ml FSIO. Add squid to the bowls for the assay (maximum n=40 per bowl).
  9. Prepare an additional bowl as an aposymbiotic (negative) control.
  10. Prepare an additional competition that competes the marked strain against a version of itself without the marker as a control. For example, ES114 vs. ES114 pVSV103.
  11. Prepare a dedicated transfer pipette for each treatment.
  12. Euthanize extra squid in 2% ethanol.
4. Squid Colonization
  1. Day 2 - Using a P10 Pipetman, dispense the calculated aliquot of bacteria (Step 1.5) into each squid bowl (Step 3.8) for each treatment. Start a 3 h timer immediately after the first inoculation.
  2. 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.
  3. Plate 50 μl from each bowl onto an LBS-Xgal agar plate from Step 2.2 (for technical replicates, plate two 50 μl plates per treatment). Incubate at 25-28°C overnight.
  4. Prepare wash bowls (100 ml FSIO/ea) for each treatment.
  5. Prepare overnight bowl with 40 ml FSIO/each for each treatment.
  6. After exactly 3 h, transfer the squid to their respective wash bowls (complete for all treatment). This stops the inoculation.
  7. Proceed to transfer squid into their respective overnight bowls.
  8. Place bowls on "E" tray and move to squid facility to return to the day/night light cycle the animals experienced during embryogenesis.
  9. Day 3 - Prepare new overnight bowls for each treatment.
  10. Prior to dusk at 22-24 h post-inoculation, transfer each squid to their new respective overnight bowl. Use a designated transfer pipette for each treatment.
  11. Day 4 - Prepare labelled 1.5 ml microcentrifuge tubes (1/squid).
  12. Prior to dusk at 46-48 h post-inoculation, transfer each squid in a volume of approximately 700 μl to a 1.5 ml microcentrifuge tube from Step 4.12.
  13. Measure and record the luminescence of each squid in the microtube (luminometer set for 6 s integration and auto-read on lid closure).
  14. As a negative control for background luminescence, measure a microtube with FSIO that does not contain any squid.
  15. Move to a cardboard freezer box. Once the lid is placed on the box, do not remove it as the light cues for bacteria expulsion are not well-understood.
  16. Freeze microcentrifuge tubes at -80°C overnight.
5. Determination of Colonization Levels
  1. For each squid, prepare two (2) microcentrifuge tubes, each with 475 μl FSIO (or autoclaved 70% Instant Ocean).
  2. Prepare pestles by first using a Kimwipe to clean the pestle and remove gross debris and/or tissue.
  3. Place pestles tip-down in a 50 ml beaker containing 95% ethanol. Ethanol should be added to a height of approximately 3 cm.
  4. For each pestle, remove from the beaker and wipe the tip with a Kimwipe.
  5. Dip pestle back into the ethanol bath, remove and insert (tip up) into an microcentrifuge tube rack and allow to air dry completely for approximately 15 minutes.
  6. Thaw squid in a microcentrifuge tube rack (maximum n=8).
  7. If necessary, adjust the volume to 700 μl.
  8. Using a pestle from Step 5.5, disrupt the animal tissue until the ink sac ruptures (the water will turn a murky grey color).
  9. Remove the pestle and ensure all tissue remains in the tube.
  10. Vortex the tissue briefly for exactly 10 seconds (use a timer).
  11. Allow the tissue to rest for 10 min. The tissue will settle and the bacteria and ink remain in solution For the calculations that follow, the bacteria/ink solution is the [A] dilution (i.e. the E. scolopes light organ homogenate in 700 μl). Serial 1:20 dilutions ([B], [C]) are described below.
  12. For the [B] dilution, add 25 μl [A] to one of the microcentrifuge tubes prepared in Step 5.1. Vortex.
  13. For the [C] dilution, add 25 μl [B] to one of the microcentrifuge tubes prepared in Step 5.1. Vortex.
  14. Plate 50 μl of each dilution onto LBS-Xgal agar, 2 replicates per treatment.
  15. Incubate the plates at 25-28°C for 36-48 hours to allow blue color to develop in pVSV103 containing bacteria. If blue colonies are still hard to distinguish, placing plates at 4°C may help color develop further.

Note: If using antibiotics to mark different strains, plate dilutions from squid onto LBS first. Then patch 100 colonies onto LBS and selective media. Some strains do not grow well on selective media when immediately coming from the squid.

6. Data Analysis

  1. To calculate the Competitive Index, count colonies on the dilution plates for each treatment in which 10-400 colonies are present. Note the number of blue vs white colonies.

  2. Calculate the competitive index

    competitive index = Log10 ((White/Blue)output / (White/Blue)inoculum )

    Notes on equation:

    • (White/Blue)inoculum = number of white colonies divided by blue colonies for the inoculum in step 4.3
    • (White/Blue)output = number of white colonies divided by blue colonies for the output plates in step 6.1
    • Taking the Log10 transforms the data into competitive index for better visualization. If the white strain wins, the competitive index will be positive. If the blue strain wins the index will be negative. Values close to zero indicate equal competitive fitness.

  3. Plot individual data points and medians, centering 0 as the middle of the axis.

  4. The data are often not normally distributed, with different variances, and the outliers may contain biologically meaningfully information. Therefore, non-parametric tests provide a useful method to determine whether the treatments differ significantly.

  5. Use GraphPad Prism software or Python for statistical analysis. For two treatments, use the Wilcoxon Rank Sum test. For comparisons among greater than two treatments, use the Kruskal-Wallis test with appropriate post-tests.

Citations:
Naughton LM, Mandel MJ. 2012. Colonization of Euprymna scolopes squid by Vibrio fischeri. J Vis Exp 61:e3758, doi: 10.3791/3758.
Dunn AK, Millikan DS, Adin DM, Bose JL, Stabb EV. 2006. New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol. 72(1):802-10, doi: 10.1128/AEM.72.1.802-810.2006