S U M M A R YMycoplasmas which metabolized urea and produced small colonies on agar medium were isolated from the genital tracts of dogs and from the throats of squirrel monkeys. Attempts to isolate similar organisms from baboons, cats, horses, pigs and rabbits were not successful. The biological and physical properties of the mycoplasmas isolated from dogs and monkeys were closely similar to those of T-mycoplasmas isolated previously from man and cattle, so that the canine and simian strains could be regarded undoubtedly as members of the T-mycoplasma group. The metabolic-inhibition technique was used to show that the T-mycoplasmas of the four 'host' species were not related to known large colony-forming mycoplasmas of these species. The same technique revealed that some of the Tmycoplasma strains isolated from a particular species were different from one another, except the simian strains which appeared to be the same as each other. In addition, the results of tests with a single or a few strains isolated from each of the four species showed that the strains of one species were not serologically related to those of another. On the other hand, polyacrylamide gel electrophoresis studies, in which at least 1 1 lines were observed with T-mycoplasma antigens, indicated that the structural proteins of strains isolated from the various species were closely similar. The fact that all the strains examined could be regarded as belonging to one closely related group is discussed in the context of the eventual nomenclature for these organisms.

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IT is known that mycoplasmas may be preserved in the frozen state, although there is only one report (Kelton, 1964) concerning the viability of a variety of mycoplasmas after their long-term storage. In this laboratory, cultures of many prototype strains and other strains of mycoplasmas have been prepared for routine use. These have been stored at -70"C, and some have been stored also at -50°C and -30°C. In order to store numerous stock cultures economically, a micro-system with storage in plastic trays has been used. The viability of some mycoplasmas stored in this way has been compared with their viability after storage in glass vials. Some cultures of mycoplasmas have also been lyophilised in order to facilitate transport to other laboratories. At various intervals over a period of at least 2 yr, samples of all these frozen and lyophilised cultures have been titrated for their content of viable organisms and the results are presented in this communication. MATERIALS AND METHODSMycoplasmas. The organisms studied in most detail were recognised strains of mycoplasmas isolated from both avian and mammalian sources. Their origin and other details have been described previously (Manchee and Taylor-Robinson, 1968 ; Taylor-Robinson, Williams and Haig, 1968).Media. The mycoplasmas were grown in a liquid medium composed of 70 ml Difco " PPLO " broth, 20 ml unheated Burroughs Wellcome horse serum no. 6, 10 ml of a 2.5 per cent. (wfv) aqueous extract of dried yeast (Distillers Co. Ltd), 2 ml of a 2.5 per cent. aqueous solution of thallium acetate (1 ml only for T-strain mycoplasmas), 1 ml of a solution containing 100,000 units of penicillin G , and 2 ml of a 0.1 per cent. aqueous solution of phenol red. Depending upon the mycoplasmas under study, glucose, arginine or urea was added to this basic medium to a final concentration of 0-1 per cent. Medium containing glucose was adjusted to pH 7.8 and media containing arginine or urea to pH 7.0. Solid medium was prepared by the addition of 1 per cent. agar (Oxoid Ionagar no. 2 or Agar no. 1) to the PPLO broth before the other ingredients were added.Titration of viabZe organisms. Serial 1 0-fold dilutions were made in medium contained in vials of c. 2 ml with screw caps and rubber liners (Anchor Glass Co. Ltd, London). Each of the mycoplasmas metabolises glucose, arginine or urea so that growth in medium with the appropriate substrate alters the p H and this changes the colour of the phenol red in the medium. Vials were incubated at 36°C until colour changes were complete. The highest dilution at which a colour change occurred was the end-point of the titration. Sometimes the test organism was seeded on solid medium and incubated in a humidified atmosphere of

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