Phytophthora europaea Hansen et Jung
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
1. To thaw a frozen ampoule, place in a 2530 °C water bath, until just thawed (approximately 5 minutes). Immerse the ampoule just sufficient to cover the frozen material. Do not agitate the ampoule.
2. Immediately after thawing, wipe down ampoule with 70% ethanol and aseptically transfer 10 microliter (or any amount desired up to all) of the content onto a plate or broth with medium recommended.
3. Incubate the inoculum/strain at the temperature and conditions recommended.
4. Inspect for growth of the inoculum/strain regularly. The sign of viability is noticeable typically after 1-2 days of incubation. However, the time necessary for significant growth will vary from strain to strain.
Many Phytophthora strains have very low viability post-freezing and cryopreservation. For some recalcitrant strains, it often takes more than five days to show viable sign even under the optimal revival condition. It is often beneficial for them, upon thawing, to be cultured in liquid YM (yeast mold medium) supplemented with 0.1% biotin and 0.1% thiamine overnight (or until showing viability sign) and then transfer to other recommended media for continued growth and characterization. Freshly made, moist, appropriate medium is the key factor for their revival, in addition to thawing temperature (25°C for approximately 4 min, or if thawing at 55C until just thawed, be very attentive and not over stay at this temperature). Many Phytophthora strains are unable to grow at a temperature above 22C and the temperature in most incubators can fluctuate 2 degrees C around the set temperature.
In general, richer media like V-8 Juice Rye Agar (ATCC medium 1970) and PYG (peptone, yeast extract, and glucose, ATCC medium 663) are better than some of the recommended media (oatmeal medium, rye medium or cornmeal medium) in reviving frozen Phytophthora cultures. Media such as oatmeal medium, rye medium, cornmeal medium, lima bean medium are mainly for examining some characteristics of the strain developed during its life cycle, thus they are not necessarily the best recovery media for cryopreserved cultures. So it is a common practice that one inoculates the frozen culture sample on at least two media (one is a 5-mL liquid medium) to increase the chance of post-thawing revival.
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The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
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Everett M Hansen, personal communication