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With the introduction of the InPouch test for Trichomonas vaginalis,1T gallinae, and T foetus, it was desirable to have a procedure available for maintaining extended culture viability. The three trichomonads are viable after 8 days by subculture in the InPouch at 33°C. Extended viable storage of these three trichomonads is the subject of this letter.
We have evaluated various procedures involving freezing 24 hour InPouch cultures at −70°C. We now report a procedure that has demonstrated storage of viable trichomonad cultures for more than 2 years.
The freshly subcultured trichomonads are incubated at 35°C for 24 hours, which should produce a viable count of approximately 1.0 × 105/ml. It is important to note that subsequent subculture will require an adequate nutrient available for growth in the pouch. Then 0.1 ml of pure sterile glycerol is added to the medium in the pouch and thoroughly mixed employing the “shoe-shine” technique. It is important to immediately place the pouch in a −70°C freezer.
After freezing most of the trichomonads in the pouch are non-viable, but successful subculture is routinely achieved upon thawing. When the pouch is removed from the freezer, it should be immediately placed in an incubator at 35–37°C. After 3 days a few viable trichomonads will be observed, and after 4 days it may be subcultured.
This procedure has been effective for T vaginalis, T gallinae, and T foetus.
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