Cryogenics

Cryopreservation of Phytophthora

1. Introduction

Cryopreservation of Phytophthora cultures in a liquid nitrogen atmosphere, at or close to –196 C, allows their long­term storage in a genetically stable state. As with other organisms and biological tissues, the key features of this technology are the use of controlled rate freezing to avoid ice crystal formation within the tissues and immersion of the samples in a cryoprotectant solution such as dimethyl sulfoxide (DMSO), glycerol or skim milk/glycerol. Major collections of this species, such as the World Phytophthora Genetic Resource Collection (WPC, part of the World Oomycete Genetic Resource Collection, WOC) at the University of California, Riverside and ATCC, use this method.

2. Controlled Freezing

Control of the rate of freezing is critical to optimize the ice nucleation process and minimize cell injury. Fast freezing results in intracellular ice nucleation. This is lethal to the cell or tissue. At very slow freezing rates cell death is also excessive due the ‘pickling’ effects of long periods of exposure to hypertonic solutions. At optimal rates of freezing the exposure time to hypertonic conditions is minimized while intracellular ice formation is prevented. Finally, as most physical parameters which cells are exposed to vary in a non­linear fashion with respect to temperature, ideally the freezing process should take this into account.

The basic process involves the following steps:

  1. Equilibration of the sample with the cryoprotectant (see section 3 below) at room temperature to permit uptake of the solution
  2. Cooling of the samples at ~ 1 to 2 C per min to 0 C, then 10 min at O C, followed by ~1 C per min down to –10 C
  3. Following thermal equilibration of the samples prior to ice crystal growth, the temperature is then dropped further to ­44 C
  4. Finally, the samples are cooled very rapidly from ­44 C to – 120 C in ~10 min

The frozen samples are then deposited under liquid nitrogen at –196 C.

Samples can be withdrawn from the cryogenic facility and brought to room temperature by immersion in a water bath at 20 C, or simply by placing them on a solid surface at room temperature. They are then plated out, following removal of surplus cryogenic fluid using an absorbent surface such as sterile paper towels, in or on a suitable nutrient medium, either liquid or agar based.

Specific Protocol used at UCR

The following actual protocol (based on that originally devised by Hohl and Iselin, 1987) is used with a programmable freezer at UC Riverside:

  1. ambient (~24 C) to 0 C in 20 min
  2. hold at 0 C for 10 min
  3. 0 C to –10 C in 6.3 min
  4. immediately to –40 C
  5. –40 C to –37 C in 0.3 min
  6. hold for 1.5 min at –37 C
  7. –37 C to –19 C in 3.4 min
  8. –19 C to –44 C in 16 min
  9. –44 C to –120 C in 10 min
  10. hold for 20 min at –120 C
  11. transfer to liquid nitrogen at –196 C

This protocol has been used at UCR since 1986 with consistently good results.

3. Cryoprotectants

Cryoprotective additives suitable for Phytophthora include skim milk/glycerol, dimethyl sulfoxide (dimethyl sulphoxide, DMSO), and 1,2­-propanediol. Cell membranes are permeable to these compounds. They reduce damage following freezing and thawing, by increasing the unfrozen fraction and thereby reduce the ionic composition of the tissues. There can be significant differences in the protective efficiency of different cryoprotectants. This may be the result of differing cellular toxicities and/or permeability to specific compounds. In our own experience at UCR we have noticed no difference in recovery with either a skim­milk/glycerol mixture or DMSO used as cryoprotectants. In limited trials using 15% 1,2-propanediol it also gave good results.

Skim milk/glycerol

Aqueous 20% glycerol and 17% Difco Bacto skim milk are autoclaved at 121 C for 10 min. A 1:1 mixture of the two sterilized solutions is prepared.

DMSO

A 15% aqueous solution of DMSO is autoclaved at 121 C for 15 min.

Paul Tooley has provided a different protocol for DMSO:

Make up some 10% DMSO in the hood (DMSO is toxic) and filter sterilize into a sterile test tube, or beaker. Pipette 1 ml of sterile 10% DMSO into screw­cap cryogenic storage vials. Label the vials with the isolate number and date.

Use 0.2-­micron solvent­resistant membranes to filter­sterilize the DMSO. We use Teflon membranes in 25 mm disposable syringe filters made by Schleicher and Schuell (Spartan­-T membranes). DMSO will melt ordinary membranes. Alternatively, buy presterilized DMSO (Sigma D­-2650 vials, etc.) and dilute it to 10% using sterile water, in sterile test tubes. Keep at room temp covered with aluminum foil.

3­-18­-93, updated 3­-12­-01

Paul Tooley

4. Additional Factors

Isolates are grown at their optimal temperatures (16 to 24 C) for 4 to 10 days dependent on growth rate on a nutrient rich medium such as V8 juice or Rye seed agar. From the growing margins of these cultures, ~0.5 to 0.8 cm agar culture disks are punched with a sterile cork borer. These are then placed in a cryo tube (such as Nunc polypropylene tube with polyethylene cap, radiation sterilized). Cryoprotectant is added to cover the agar disks. We place 5 agar disks in 1 ml of cryoprotectant in 2 ml volume cryo tubes and add 1 ml of cryoprotectant solution.

Additionally, the prepared cryotubes are routinely stored at 4 to 5 C for 1­-2 days to facilitate cold hardening. This step is probably not necessary.

It is very important to ensure that cultures are completely free from bacterial or other microbial contaminants prior to cryopreservation. In our experience contaminated cultures can be extremely difficult to ‘clean up’ following this procedure, particularly if they are inherently very slow growing. A simple method to test that the agar­grown culture is bacteria­free is to incubate sample agar culture disks in LB broth overnight.

5. References

Dahmen, H., Th. Staub and F.J. Schwinn. 1983. Technique for the long­term preservation of phytopathogenic fungi in liquid nitrogen. Phytopathology 73: 241­246.

Hohl, H.R. and K. Iselin. 1987. Liquid nitrogen preservation of zoosporic fungi. In Zoosporic fungi in teaching and research. Pages 143­145. Edited by M.S. Fuller and Alan Jaworski.

Jong, S.C. and W.B. Atkins. 1985. Conservation, collection, and distribution of cultures. In Fungi pathogenic for humans and animals. Vol. B, Part 2. Edited by D.H. Howard.

Smith, D. 1982. Liquid nitrogen storage of fungi. Trans. Br. Mycol. Soc. 79: 415­421.

Smith, D., G.E. Coulson and G.J. Morris. 1986. A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing. J. Gen. Microbiol. 132: 2013­2021.

Tooley, P.W. 1988. Using uncontrolled freezing for liquid nitrogen storage of Phytophthora species. Plant Dis. 72: 680­682.

Michael David Coffey
22 July 2008 (update 21 October 2010)