TITLE: Storage &amp; Revival of Oomycetes
AUTHORS: Diana Lee

[DESCRIPTION]
Cryopreservation of oomycetes such as Phytophthora and Pythium species is essential for the long-term maintenance of these organisms. In this method we present an improved cryopreservation protocol for oomycete cultures, using carbon filter paper, to enhance their viability during liquid nitrogen storage with consistency. Our method benefits researchers working with these organisms by long-term, who do not have access to a programmable freezing equipment. 

[GUIDELINES]
Here are some additional tips for storing and reviving chromists:
 
Use young, actively growing cultures for storage.
 
It is highly important to ensure hyphae does not get damaged during storage (steps 7-10)
 
Ensure the cultured carbon filter strips are fully immersed in 10%-20% glycerol for 1-3 hours as some may display hydrophobicity. 
 
If the culture does not revive after the first attempt, try thawing another straw and using a different medium. Some species may prefer a solid medium such as Rye B agar, or a different broth such as V8. Addition of B-sitosterol (0.05g/L dissolved in small amount of dichloromethane) to rye broth may help with revival. 

If the culture still does not revive, store the culture in 20% glycerol.

If liquid nitrogen is not available, this method should also work for long term storage of oomycetes at -80 °C 

[STEPS]
SECTION: Preparation of Oomycetes for Storage
1. Inoculate a small agar plug of the oomycete culture into a thin film of rye broth in a petri dish. 



SECTION: Preparation of Oomycetes for Storage
2. Incubate at 20 °C  until mycelium has grown about 4-5cm in diameter.

SECTION: Preparation of Oomycetes for Storage
3. Tear the mycelium mat and place the small pieces onto carbon filter paper strips laid out on rye agar (or V8, or PDA). 
SECTION: Preparation of Oomycetes for Storage
4. Incubate at 20 °C  until dense aerial mycelium is observed growing on the strips, usually in 3-7 days.

SECTION: Preparation of Oomycetes for Storage
5. 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.
SECTION: Preparation of Oomycetes for Storage
6. 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. 
SECTION: Preparation of Oomycetes for Storage
7. 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.
SECTION: Preparation of Oomycetes for Storage
8. 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.
SECTION: Preparation of Oomycetes for Storage
9. Dab as much liquid off from the strips onto sterile filer paper laid out onto an empty petri dish.
SECTION: Preparation of Oomycetes for Storage
10. Place the strip into a cryovial using sterile equipment and fill the vial with 10% glycerol, ensuring the entire filter strip is immersed.
SECTION: Preparation of Oomycetes for Storage
11. 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. 

SECTION: Revival
12. Immediately thaw the cryovial in water warmed to 30-40 °C

SECTION: Revival
13. Place the culture strip into a petri dish containing rye broth.
SECTION: Revival
14. Incubate at 20 °C for 7-14 days and watch for growth.

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