Mycoplasma pneumoniae produces a soluble hemolysin active against guinea pig erythrocytes. This hemolysin appears to be a peroxide, since catalase or peroxidase inhibits its activity. The action of catalase and peroxidase is specific, since heating the enzymes abolishes their effect on the hemolysin. In addition, 3-amino- 1,2,4-triazole, a potent inhibitor of catalase, reverses the inhibitory effect of the enzyme. The hemolysin of M. laidlawii is also a peroxide. The hemolysins of M. pneumoniae and M. laidlawii seem unique for microbial organisms since the bacterial hemolysins which have been described have been protein or lipid in nature.

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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