formation by Bacillus popilliae in liquid medium containing activated carbon. J. Bacteriol. 91:2270-2274. 1966.-Heretofore, it has not been found possible to evoke sporulation of Bacillus popilliae in liquid culture. We have discovered that sporulation will occur in tryptone-glucose-yeast extract broth shaken cultures if activated carbon (charcoal) is present during growth. The spores so engendered have survived drying in air and subsequent storage for several months as dry films and also in dry soil, sand, and a mixture of powdered calcium carbonate and talc. Furthermore, the longevity of cultures, even when spores are absent, is extended, in cultures containing activated carbon, to several weeks at a population of millions of cells per milliliter. This extension of life is the result of a marked change from rapid decline in numbers to an almost stationary population. Although Bacillus popilliae Dutky and B. lentimorbus Dutky sporulate in the hemocoele of Popillia japonica Newman (Japanese beetle) and Amphimallon majalis (Razoumowsky; European chafer) larvae, they sporulate sparsely on solid laboratory media (7, 10) and not at all in liquid media. In searching for a means to evoke the formation of spores in liquid media, we became aware of the work of Foster, Hardwick, and Guirard (2). They experienced difficulty in causing B. larvae White, another insect pathogen, to sporulate. Sporulation of this species was greatly enhanced in media they treated with activated charcoal. Perhaps it was their success that induced Steinkraus (9) in 1957 to add activated charcoal to the solid medium on which he found B. popilliae to produce spores. It seemed unlikely to us that a practical method for the production of milky disease bacteria spores for use in controlling insect hosts could be developed on the basis of spores formed on solid media. Therefore, we continued to work with liquid media and included the use of activated carbon. MATERIALS AND METHODS Microorganism. B. popilliae Dutky NRRL B-2309S was used exclusively. It is a substrain of NRRL B-2309 that was previously selected because of its ability to form spores on solid media (7). Both the parent and the substrain were able to infect Japanese beetle larvae. Medium. The growth medium described by St. Julian et al. (11) [tryptone, 0.5%;yeast extract, 1.5%; K2HPO4, 0.3%; glucose, 0.2% (sterilized separately); distilled water; pH 7.3 to 7.5], designated J medium for brevity in this report, was used. Activated carbon. In the past, powdered carbon has been used to stimulate growth of microorganisms. The nature of milky disease bacteria makes it convenient to work with clear media in which their characteristic birefringent turbidity may be observed. Powdered carbons convert clear media into murky, black ones. Furthermore, phase microscopy is hampered in cultures containing powdered carbon, because microscope fields are cluttered with innumerable refractile particles which make observation difficult, if not impossible. We chose, therefore, to use pelleted and granular carbon...
