A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Twenty different human rotavirus reassortants were characterized serologically by a plaque reduction assay as belonging to one of three distinct serotypes. Fourteen were similar if not identical to our prototype Wa strain; two were like the prototype DS-1 strain, and four belonged to a third serotype for which a prototype has not yet been selected. Hyperimmune sera raised against the three serotypes were required to distinguish among them, since postinfection sera had lower titers and were more cross-reactive than hyperimmune sera. These results confirmed the ability of a qualitative cytopathic neutralization test to predict correctly the Wa or DS-1 serotype. A strain of rhesus rotavirus (MMU 18006) was identified as belonging to the newly defined third serotype. Finally, an attempt was made to correlate previously published serotype analysis by neutralization of fluorescent cell-forming units with the results determined by the plaque reduction neutralization assay.

IntroductionBiochemical and biophysical studies of M . pneumoniae have been hampered by contamination of suspensions of organisms with constituents from the relatively complex medium required for growth. In previous studies,1.2 organisms were concentrated from a broth medium by centrifugation, a procedure which often resulted in concurrent concentration of denatured proteins and other constituents from the growth medium. We and others have also experienced difficulty in preparing complement-fixing antigens from M . pneumoniae broth cultures because of the development of high levels of anti-complementary activity during growth of the organism.Recently, we have developed a method for cultivating M . pneumoniae which in large measure obviates the difficulties just described. This method of cultivation was based upon the observation that the organism can attach to a glass surface and grow there to form a confluent layer of colonies which do not detach during multiple washings. This technique for cultivation of M . pneumoniae and its a p plication to the preparation of concentrated suspensions of organisms forms the subject matter of this report.The FH strain of M . pneumoniae was originally isolated from material supplied by LiuSs The organisms were grown on a medium of the same formulation as originally used in the isolation of M . pne~moniae,~ but modified by the addition of 1.0% glucose and 0.002% phenol red. We used laboratory-adapted organisms that were in the 15th and 325th passage on artificial medium. Adhesiveness of Mycoplasma to GlassDuring growth in broth, M . pneumoniae forms macroscopic spherical colonies. This phenomenon was first described by Kenny.2 Recently, we observed that a significant number of the colonies adhered to the surface of Povitsky bottles used for large-scale cultivation of the organism. Within 48 hours, the colonies formed a confluent layer which covered the glass surface beneath the broth medium (FIGURE 1). This layer has a mosaic appearance with colonies growing in close continguity (FIGURE 2). With further incubation the layer of organisms became thicker.When the broth was removed and the layer of organisms on glass washed repeatedly with saline, the colonies adhered tenaciously to the surface.The colonies could be removed, however, by scraping or treatment with 2.5% trypsin (detached more slowly with lesser concentrations of the enzyme). We attempted to detach the colonies by adding EDTA, but the organisms remained on the glass. This finding suggested that divalent cations were probably not involved in attachment.

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