The overall goal of this procedure is to assess the degree of squid colonization when the animals are inoculated with Vibrio fisher eye bacteria. This is accomplished by first preparing culture grown bacteria and collecting APO symbiotic squid hatchlings. Next, the squid hatchlings are inoculated with a specified concentration of the bacteria.
At the conclusion of the colonization, the luminescence of the animals is determined and the samples are surface sterilized by freezing. Finally, the samples are homogenized to release the symbiotic bacteria. Ultimately, results are obtained that show the median level of colonization for each bacterial strain or sample through dilution plating of the symbiotic homogenates.
This method can help answer key questions in the field of micro post interactions, such as How does host specificity develop among microbial strains To prepare bacterial inocular two days prior to squid inoculation plate, the relevant bacterial strains on LBS Auger incubate the bacteria at 25 to 28 degrees Celsius overnight the next day. For each strain under investigation, inoculate three milliliters of LBS medium in a glass culture tube with one colony of each V fisher eye strain for infection. Prepare duplicate tubes as a backup on the following day, subculture 37.5 microliters of the bacteria into a fresh tube of three milliliters of LBS and grow for one hour with aeration.
Measure the OD 600 of the culture and then determine the volume necessary for a target inoculum of three to five times 10 to the third CFU per milliliter by dividing 1.25 by the OD 600. Please see the written protocol for further details. To prepare plates for enumeration of the inocular, add five sterile plating beads to each LBS plate to collect squid juveniles.
First, use a refractometer to measure the salinity of instant ocean and adjust it to 35 parts per thousand using a filtration unit and an attached vacuum line or vacuum pump filter instant ocean to generate filter sterilized instant ocean oxygenate the water by swirling vigorously prior to each dispensing aliquot, 40 to 50 milliliters of FSIO into each of two disposable sample bowls. Label one as early and one as timely. Prepare an excess of plastic transfer pipettes for acquiring juvenile squid by cutting the pipette approximately one centimeter from the tip above the lowest ridges.
This facilitates a wider area through which the squid can pass upon collection, discard any pipettes with rough exposed surfaces using prepared transfer pipettes. Collect scalies that hatched overnight and transfer to the earlys bowl of FSIO. They have been in the egg system for over one hour and are susceptible to colonization by contaminating v Fisher eye.
Therefore, don't use earlys for sensitive colonization experiments. Check egg tanks every 30 to 45 minutes for new hatchlings and transfer them all into the timely bowl of FSIO. When the collection is finished, transfer the squid to the main laboratory where they should be colonized under uninterrupted light for a three hour inoculation.
After the incubation for each treatment, including an apo, symbiotic, or un colonized control, prepare a bowl of 40 milliliters of FSIO. Add the squid to the bowls. Using a P 10 pipette man, dispense the previously calculated aliquot of the prepared bacteria.
Start a three hour timer immediately after the first inoculation. 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 seconds. Thermal mixing of the inoculum with the squid is critical to the success of the experiment and is achieved by mixing in different directions and repeating Next plate, 50 microliters from each bowl onto a previously prepared LBS auger plate.
With plating beads incubate at 25 to 28 degrees Celsius overnight. Then prepare wash bowls by adding 100 milliliters of FSIO per treatment to a bowl. Prepare drosophila vials with four milliliters of FSIO for each squid.
After the three hour incubation, transfer the squid to their respective wash bowls. This stops the inoculation using new designated transfer pipettes for each treatment, transfer each squid to its own drosophila vial containing FSIO. Move the trays to the squid facility prior to dusk.
One day post inoculation. Transfer each squid to a new drosophila vial containing FSIO. Use a designated transfer pipette for each treatment just before dusk, two days post inoculation using a luminometer set for six second integration and auto read on lid closure.
Measure and record the luminescence of each squid. Measure a vial with FSIO only as a blank to control for background. After measuring luminescence, transfer each squid in a volume of about 700 microliters to a 1.5 milliliter micro fuge tube.
Place the tubes into a cardboard freezer box. Place the lid on the box and freeze at minus 80 degrees Celsius overnight. To prepare samples for measuring colonization levels, place pestles tip down in a 50 milliliter beaker filled with 95%ethanol to a height of about three centimeters.
Remove the pestles from the beaker and wipe the tips with a kim wipe. Then dip them back into the ethanol. Place them tip side up into a micro tube rack and allow them to completely air dry after thawing previously frozen squid and adjusting the volumes to 700 microliters.
Use a pestle to disrupt the animal tissue until the ink sac ruptures. Remove the pestle and ensure that all tissue remains in the tube. Vortex the tissue for 10 seconds and allow it to rest for 10 minutes so that the tissue settles.
After making serial dilutions of the remaining solution plate, 50 microliters of each dilution onto LBS auger plates. Incubate the plates overnight at 25 to 28 degrees Celsius. Proceed to data analysis as outlined in the written protocol.
Results from a colonization assay are shown here two strains of V fisher eye that exhibit. Different relative levels of luminescence were each inoculated into six squid and six squids served as apo, symbiotic, or un colonized controls. Similar inoculum levels led to 100%colonization within three hours at 48 hours.
The luminescence levels and CFU counts were determined to assess the colonization proficiency of the strain. Measuring the specific luminescence as shown here, allows for determination of the brightness of each bacterium during the symbiosis. After watching this video, you should have a good understanding of how to prepare bacteria and squid for the colonization assay, how to perform the colonization assay, and how to analyze the results of the assay to determine to what extent your bacteria colonizes eco.
