Various methods for the detection of hemolysin production by Mycoplasma species were compared. Inoculation of blood-agar by the push-block method and by use of concentrated mycoplasma cell suspensions was compared with the agaroverlay technique. The preferred method was direct surface inoculation of concentrated suspensions onto the blood-agar. Among the conditions tested, refrigeration of 48-hr cultures gave the best results. A wide variety of mycoplasma species were tested for hemolytic activity towards rabbit, sheep, guinea pig, duck, and chicken bloods. Guinea pig erythrocytes were found to be the most susceptible to lysis by mycoplasma, and rabbit erythrocytes were found to be the least susceptible. A sensitive technique for the detection of peroxide production by mycoplasma strains, employing agar containing benzidine and sheep blood, was used. With this method, peroxide production could be correlated with hemolysis on blood-agar. Peroxidase and catalase inhibited both the benzidine reaction and hemolysis. It was concluded that the major hemolysin of the Mycoplasma species examined is a peroxide. 157 GC2 M. fermentans G M. salivarium 156 04 and 013A M. orale types 1 and 2 Strain Washington Strain 4330 M. arthritidis 14152 (Campo) 158 (Campo) PB PN L4P (Preston) L4S 14124 El M. pulmonis Ti M. neurolyticum TA M. gallinarum M. gallisepticum Bit M. mycoides var. capri Strain tumor M. bovigenitalium 14173 Strain goat Strain bovine Cell culture strain Hela B Cell culture strain FL Cat strains Human genital tract Human genital tract Rat abscess Rat abscess Rat arthritis Rat arthritis Rat cancer tissue Inflammed rat eye Benign rat tumor Mouse brain Respiratory tract of fowl Turkey brain Goat Rat lymphosarcoma Bovine genital tract Cell cultures Cell cultures Cat conjunctiva Cat saliva Cat saliva

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Available evidence indicates that cycloserine acts by interfering with cell wall biosynthesis. Biochemical studies have shown that this antibiotic causes accumulation of Nacetylaminosugar ( 1 ) , inhibits incorporation of lysine into cell wall fractions(2), and inhibits alanine racemase and alanyl-alanine synthetase in Staphylococcus aureus (3,4,5). Nonetheless, cycloserine may affect mammalian cells as demonstrated by its clinical toxicity(6) in contrast to penicillin which clearly acts by specific inhibition of cell wall mucopeptide formation in growing bacteria. Michel and Hijmans were able to obtain L growth from a single strain of streptococcus by exposing the bacterium to cycloserine but stated that the L form was inhibited by low concentrations of this antibiotic( 7). Since L forms of bacteria represent the organisms which are capable of growth and reproduction yet lack a rigid cell wa11 (8,9,10), the L forms should be resistant to cycloserine action if indeed this antibiotic exerts a primary and specific action on cell wall formation. Therefore, we have studied the ability of cycloserine to induce L phase growth of bacteria. The cycloserine susceptibilities of L phase variants derived by exposing bacteria to penicillin have been compared with L phase variants obtained with cycloserine. Also the penicillin sensitivities of L forms derived with cycloserine were compared with those obtained with penicillin. The susceptibilities of the bacteria from which the L forms were iso-4 Supported by research grant from Nat. Inst. of lated were determined and compared with the sensitivities of the L forms. The sensitivities of selected strains of Mycoplasma to penlcillin and to cycloserine also have been compared.Materials and methods. Ninety-six bacterial strains isolated locally were studied (Table I ) . They were identified by the usual cultural and serologic technics. Nine strains of Mycoplasma were examined.+ The L phase variants were isolated on agar plates (Oxoid sensitivity test medium) containing 3% NaCl and 20% heated human ascitic fluid. Penicillin (1,OOO U/ml) or cycloserine (1,OOO pg/ml) was placed in wells on plates streaked with the test organism. The plates were incubated at 37°C semianaerobically by the Fortner method (1 1). L pbase growth was isolated from the zone of inhibited bacterial growth after 4-7 days incubation and was maintained by serial agar block transfer on agar plates containing penicillin (500 U/ml) or cycloserine (500 pg/ml). The Mycoplasma were maintained by serial transfer on PPLO agar (Difco) containing 20% heated human ascitic fluid.