Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

This method can help answer key questions in the microbiology field about the dynamics of colonization and infection by microorganisms in the gastrointestinal tract. The main advantages of this technique are that it is more representative of food-borne infections in humans and that it reduces potential tissue damage in fish compared to oral gavage. Visual demonstration of this method is critical as the washing steps can be difficult to perform because of the highly motile nature of the paramecia.

From a live growing culture, add one milliliter of paramecium culture and one milliliter of an e-coli MG1655 culture to a ten milliliter tissue culture flask containing eight milliliters of E3 medium. Lightly swirl the flask before placing it at 22 degrees Celsius passaging one milliliter of the co-culture into a new ten milliliter tissue culture flask containing nine milliliters of fresh E3 medium, supplemented with 108 colony forming units per milliliter of e-coli MG1655, every two weeks. To determine the bacterial half-life within a paramecium count the number of paramecia from an optical density of 600 paramecia bacteria co-culture and add a 50 microliter aliquot of supernatant to a new 1.5 milliliter microcentrifuge tube every hour for six hours.

Add 950 microliters of a one percent non-ionic surfactant in PBS to the tubes. And lyse the paramecia in each sample with one minute of vortexing, then make one in ten dilutions of each sample in sterile PBS. And plate 100 microliters of each dilution onto selective plates for a 16 hour incubation at 37 degrees Celsius.

The next day, count only the isolated and distinct individual colonies to determine the number of bacterial colonies on the plate. And identify a plate with a dilution that yields 30-300 colony-forming units. On the day before the infection, collect one volume of bacteria from an optical density of 600 culture per treatment by centrifugation.

And re-suspend the pellets in one milliliter of E3 medium per tube. Next, add one microliter of an appropriate bacterial stain to each bacterial suspension. And protect the tubes photo bleaching with foil.

Before incubating the bacteria samples with end-over-end rotation for 15 minutes at room temperature. At the end of the incubation, wash the bacteria two times in a large volume of E3 medium to remove the excess dye. And re-suspend the pellets in one milliliter of fresh E3 medium per tube.

Then, add one milliliter of bacterial suspension to each of the two flasks of fresh paramecia per condition for a two hour incubation at room temperature. Pull the contents of both flasks per condition into individual 50 milliliter conical tubes. And pellet the samples by centrifugation.

Using a sterilogical pipette, replace approximately ten milliliters of the E3 supernatant in each tube with ten milliliters of fresh E3 medium for 3 washes by centrifugation. After the last wash, remove approximately ten milliliters of the E3 supernatants, taking care not to disrupt the pellets, and re-suspend the pellets in the remaining ten milliliters of E3 medium. Transfer 500 microliters of each suspension into individual 1.5 milliliter microcenrifuge tubes and pellet the paramecia by centrifugation.

Discard 400 microliters of the supernatants, and add 20 microliters of 36.5 percent formaldehyde solution to the remaining 100 microliters of paramecia with gentle pipetting. After five minutes at room temperature, measure the actual total volume in each tube by pipette before counting the number of dead paramecia per milliliter. For food-borne infection of the zebrafish, dilute the paramecia samples to a two times ten to the five paramecia per milliliter of E3 medium concentration.

And add 3 milliliters of each paramecia culture to one well of a 6 well plate per condition. Next, transfer ten anesthetized zebrafish to each well in a minimal volume of liquid. And incubate the co-cultures for two hours at 30 degrees Celsius in a diurnal incubator under daylight conditions.

To determine the preying rate, view the feeding zebrafish under a stereo microscope while acquiring video footage of the prey capture. Prey capture is characterized by the striking of a zebrafish toward the prey. Each strike is estimated to be one prey capture event.

At the end of the incubation, wash the zebrafish in 5 different wells containing 3 milliliters of fresh E3 medium supplemented with 100 milligrams per liter of tricaine per well. And embed each zebrafish in 3 milliliters of 1 percent low-melt agarose in a black welled six well plate. When all of the fish have been embedded, place the plate under a stereo microscope and use a clipped gel loading tip to make sure that the heads are on the left and the tails are on the right in each viewing field.

Wait for 5 minutes for the agarose to set, then overlay the embedded fish with fresh E3 medium supplemented with tricaine, and image the zebrafish on a fluorescent microscope to evaluate the progress of the bacterial infections. For pathogenic e-coli, the initial bacterial density is 790 bacteria per paramecium, and the bacteria are degraded within the vacuoles of each paramecium cut atom with a half-life of approximately 2.3 hours. Preying is accompanied by a characteristic striking behavior, and the determination of the preying rate is based on the assumption the each strike leads to the internalization of 1 paramecium.

Post-digestion, free bacteria move from the foregut to the mid and posterior intestine, where they are detected approximately 1 to 2 hours after the beginning of preying. S-enterica, for example, localizes primarily in the intestinal mucosae with some epithelial invasion leading to the infiltration of neutrophils into the epithelium. Don't forget that working with certain types of bacteria can be extremely hazardous, and that precautions such as wearing the proper personal protective equipment should always be taken while performing this procedure.