Penicillin-Lysozyme Conversion of Clostridium botulinum Types A and E into Protoplasts and Their Stabilization as L-Form Cultures

When logarithmically growing cultures of Clostridium botulinum types A and E are treated with penicillin in a liquid medium containing 8% polyethylene glycol, protoplast-like spherical bodies are formed. The penicillin effect shows a dose-response relationship; the largest yield of converted forms is obtained with 10,000 units/ml, but the treatment leaves many intact bacilli. Lower antibiotic concentrations produce smaller numbers of spherical bodies, but lysis of bacilli results in suspensions that are relatively free of rods. Cells grown under the same conditions and treated with 250 μg of lysozyme/ml do not form spherical bodies. However, a combination of 1,250 to 2,500 units of penicillin and 100 μg of lysozyme/ml yields suspensions which have sphere counts in excess of 1.0 × 10⁸/ml and only a few intact rods. The state of the culture at the time of addition of the antibiotic and enzyme is critical. Suspensions of these protoplasts can be adapted to grow as stable L-form cultures producing the same toxin type as the parent cultures.