Storage & Revival of Oomycetes

Inoculate a small agar plug of the oomycete culture into a thin film of rye broth in a petri dish.

Incubate at 20°C until mycelium has grown about 4-5cm in diameter.

Tear the mycelium mat and place the small pieces onto carbon filter paper strips laid out on rye agar (or V8, or PDA).

Incubate at 20°C until dense aerial mycelium is observed growing on the strips, usually in 3-7 days.

Tear off the cultured carbon filter strips from the agar and place 4-5 strips into a petri dish containing a thin film of rye broth.

Leave the strips in the broth for 16-24 hours, or longer if the culture is slow growing. This process allows the hyphae to repair any damage caused by being torn off the agar.

Rinse the strips in two changes of 10-20% glycerol to remove as much rye broth as possible. You can do this by gently swirling the strips in glycerol solution inside a petri dish.

Place the strips in a petri dish of fresh 10% glycerol solution and leave it sitting for 1-3 hours (at least 3 hours if the culture is slow growing) for the glycerol to be up taken into the cells.

Dab as much liquid off from the strips onto sterile filer paper laid out onto an empty petri dish.

Place the strip into a cryovial using sterile equipment and fill the vial with 10% glycerol, ensuring the entire filter strip is immersed.

Freeze at -80°C overnight then place into liquid nitrogen storage. It is best to slowly freeze the culture inside something similar to a Mr. Frosty that has a cooling rate of -1°C per minute., but not necessary.

Immediately thaw the cryovial in water warmed to 30-40°C

Place the culture strip into a petri dish containing rye broth.

Incubate at 20°C for 7-14 days and watch for growth.

Subculture the culture onto 10% V8 agar or PDA to check for contamination and morphology.