Viability and Virulence of Frozen and Dried Cultures of Meningococcus

Abstract

One of the difficulties and time-consuming labors in work with the meningococcus has been the maintenance of cultures. Even with Hitchen's semi-solid ascitic fluid agar it is advisable to transfer cultures every month and in no case can they be left much over 2 months. With other media the strains remain alive for much shorter periods. Bound up with this poor viability of meningococcus cultures in artificial media is the great difficulty of sending or transporting cultures on journeys requiring several days and exposure to varying temperatures. Cultures sent for example to Australia or India from this country almost never survive. At the same time it has been found that cultures lose certain of their characteristics on passage in artificial media. Thus the formation of the type-specific polysaccharide decreases 1 and, as the recent development of a mouse virulence test by Miller 2 has allowed one to show, the intraperitoneal virulence of a fresh culture may be rapidly lost during a few subcultures.

In order to obviate these difficulties, cultures of meningococci have been frozen and dried in our laboratory. Sixteen-hour cultures of the organisms on 10% rabbit's blood pneumococcus agar plates have been washed off with 10 cc. of hormone broth and distributed in 1 cc. amounts in soft glass tubes. These 1 cc. amounts have been rapidly frozen by immersion in a 95% alcohol-solid carbon dioxide snow mixture. The tubes are then placed in a high vacuum refrigerator and dried at —4°C. for 48 hours. At the end of this time the tubes are removed and placed in a desiccator at room temperature for 3 hours. They are then sealed off. It will be noted that as yet no attempt has been made to seal off in vacuo. This is shortly to be undertaken.