Storage of viable protozoa in samples of tissue

[Adrian.Philbey at SMTPGWY.AGRIC.NSW.GOV.AU]

          I am involved in a small project to isolate Australian
          strains of the protozoan Neospora, which causes abortion and
          perinatal mortality in cattle. One component of the project
          involves inoculating cell cultures with tissue homogenates
          from fresh affected foetuses. Immunohistochemistry and PCR
          is also being done by others in the project for detection of
          Neospora in fixed tissues.

          I have a need to store portions of tissues containing viable
          protozoal cysts that can be thawed at a later stage in an
          attempt to culture Neospora. For example, we will be
          collecting tissues from many sites of an adult cow suspected
          to be a carrier of Neospora. Following immunohistochemical
          or PCR detection of the organism in specific tissues, it is
          envisaged that I could go back to stored viable samples from
          the same tissue site and attempt to isolate Neospora in
          cell culture. Otherwise the number of cell cultures would
          be overwhelming.

          So far, I have only had experience freezing viable cultured
          cells. What are some suitable methods for preparing tissue
          containing protozoa that could be stored in a viable state?
          I have access to liquid nitrogen and -80 degrees Celsius
          freezer storage space and am assuming that the former would
          be necessary, but would appreciate any advice to the
          contrary (for example, we store our virus stocks at -80
          degrees Celsius). Would it be necessary to add storage
          medium to the samples? Should the samples be homogenised
          before freezing down and, if so, should they be homogenised
          "dry" (without added medium) or in storage medium? What are
          appropriate freezing down rates for such samples?

          Thanks for your assistance.

          Adrian W Philbey
          Veterinary Research Officer
          Elizabeth Macarthur Agricultural Institute
          Private Mail Bag 8
          Camden  NSW  2570
          Australia

          Telephone: 61-46-293332
          Facsimile: 61-46-293429
          email: philbea at agric.nsw.gov.au