Nov 14, 2023
Storage & Revival of Oomycetes
- 1ICMP - Landcare Research
- Diana Lee: https://orcid.org/0000-0002-7861-3128
Protocol Citation: Diana Lee 2023. Storage & Revival of Oomycetes. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmqw7l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2023
Last Modified: November 14, 2023
Protocol Integer ID: 90683
Keywords: Oomycetes, Oomycetes cryopreservation, Oomycetes Liquid Nitrogen, Phytophthora, Pythium, Cryopreservation, Phytophthora long term storage, Phytophthora cryopreservation
Abstract
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
Materials
- Rye broth
Protocol
NAME
Rye Broth RecipeCREATED BY
Diana Lee
- Carbon Filter Paper cut into strips
Equipment
Whatman® application specific filter, activated carbon loaded paper, Grade 72
NAME
Whatman
BRAND
WHA1872-050
SKU
LINK
- Mr Frosty
Equipment
Mr. Frosty Freezing Container
NAME
Thermo Scientific
BRAND
5100-0001
SKU
LINK
- Glycerol solution (10%-20%)
- Petri dishes, non-vented
- Cryovials
- -80 °C Freezer
- Liquid Nitrogen Dewar
Safety warnings
Follow safety protocols required for handling of liquid nitrogen.
Preparation of Oomycetes for Storage
Preparation of Oomycetes for Storage
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.
1w
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.
1d
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.
Revival
Revival
2w
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.
2w
Subculture the culture onto 10% V8 agar or PDA to check for contamination and morphology.