To begin, fill a T75 flask with 30 milliliters of AC6 medium. Aseptically add the contents of a sample vial containing the Acanthamoeba into the flask. Incubate the culture for three to five days at 26 to 30 degrees Celsius until the flask reaches 50%to 80%confluence.
The day before the cells are required, shake and briskly strike the master culture flask twice to dislodge adherent trophozoites. Fill a new T75 flask with 30 milliliters of AC6 medium. Then transfer two milliliters of master culture into the flask.
Incubate the culture at 26 to 30 degrees Celsius for 18 to 24 hours. Prior to harvest, use a microscope set to 4x magnification to visually inspect the trophozoite population for adherence and uniformity. Briskly strike the flask twice to dislodge adherent trophozoites.
Then pour the contents into a 50 milliliter conical tube. Centrifuge the tube at 500 G for five minutes to pellet the amoeba. After discarding the supernatant, resuspend the palate in two to 10 milliliters of one fourth Ringer's solution.
Vortex the resuspended sample to ensure even mixing. Then transfer 10 microliters of the sample into a disposable hemocytometer. Count the colony forming units per milliliter.
Add Ringer's solution to dilute the amoeba pellet to the required concentration. Next, seed the amoeba on a well plate. To seed the amoeba into a sterile aluminum flow cell, slowly add four milliliters of the suspension through the sterile ports of the flow cell.
Then clamp the ports close.
