Observations on the maintenance of Trichomonas vaginalis and Trichomonas foetus; the effects of cortisone and agar on enhancement of severity of subcutaneous lesions in mice

Abstract

Methods are described for the freezing of trichomonads, suspended in a liverinfusion broth, to – 20 °C and – 70 °C, and storage at these temperatures. After 9 months’ storage at – 70 °C, with dimethyl sulphoxide, six strains of T. vaginalis and three strains of T. foetus were alive; three trichomonas strains frozen earlier were also alive after 13 months. Four of the T. vaginalis strains survived equally well for 6 months at – 70 °C, in the presence of either dimethyl sulphoxide or glycerol.Although trichomonads may be maintained by subculture in a modified Diamond medium, storage at – 70 °C is more economical and less tedious; – 20 °C is not a satisfactory storage temperature for T. vaginalis or T. foetus, even in the presence of dimethyl sulphoxide or glycerol.The severity of subcutaneous lesions in mice produced by six freshly isolated strains of T. vaginalis, and two out of three stock culture strains of T. foetus, was enhanced by either the addition of agar to the suspending medium or administration of cortisone to the mice. One strain of T. foetus produced lesions only in the presence of agar, cortisone, on the dose schedule used, being ineffective.After storage at – 70 °C or cultivation in agar medium for 6 months, the virulence of trichomonads of either species was little affected, and lesions in mice were enhanced by agar in a similar degree to that found originally.For enhancing the severity of subcutaneous lesions in mice the agar method, involving only one injection, is simpler than the repeated administration of cortisone to infected mice; agar was shown to be more effective than cortisone with small infecting doses of one strain of T. vaginalis quantitatively tested.The subcutaneous injection of mice with trichomonads suspended in a liver-infusion broth containing 0·06% agar regularly produces lesions of the degree of severity required for the evaluation of compounds for therapeutic activity.We express our gratitude to Mr S. A. Price for constructive criticism and helpful suggestions during the preparation of the manuscript. Dr S. R. M. Bushby kindly provided details of the dimethyl sulphoxide method and apparatus in use in his laboratory for the low-temperature preservation of tumour cells and trichomonads. For supplying the trichomonas strains we are grateful to Dr L. P. Joyner, Miss M. Moore-Tucker and Mr C. S. Ledwich. We thank Mr W. A. Freeman for advice on the composition of the inoculum medium.