The anaerobic mesophilic cellulolytic bacteria.

the resazurin decolorizes. However, exposure to air during preparation and inoculation occasionally results in failure of some of the tubes to show reduction and no growth occurs. Also, when a large number of parallel subcultures are inoculated from a single tube, more reliable results are obtained if procedures to exclude oxygen are followed during the entire process. Criteria of Culture Purity. The following standard for culture purity has been used in isolating most of the bacteria described in the following sections. The organism must grow in solid cellulose medium and give isolated spots in which the cellulose is digested and in which the cellulose-digesting colony (or diffuse growth if no colony is formed) may be iden- tified and picked. Picked colonies must give growth in solid cellulose medium when inoculated into dilution series and the numbers of colonies obtained must be approximately proportional to the number expected. From cellulose, the organism must be inoculated into a solid sugar or other clear medium in which all types of colonies which develop can be seen. A single colony in a high dilution must be subcultured in a second clear dilution series and this must be continued until two successive series give colonies of only one sort, the uni-formity being determined not only macroscopically but also by microscopic examination. Any colony from this second sugar series must upon reinoculation into a solid cellulose medium give rise to cellulose-digesting colonies present in approximately the expected numbers in the various dilutions and containing cells microscopically similar to those in the sugar series. Cultures which successfully paws these tests under critical scrutiny are considered pure. In no case have any phenomena been observed which would suggest any impurity of cultures thus obtained, except that occasional chance contaminants have been encountered. These were eliminated by the procedure of the initial isolation. D-levulose, maltose, lactose, cellobiose, sucrose, raffinose, dextrin, soluble starch, hemicellulose from oat hulls, hemicellulose from black locust, and hemicellulose from white birch. Development was very rapid on these substrates and copious gas was produced after 24 hours of incubation. Acidity was tested after 4 days. the major part of the sugar was glucose. In this respect, the coccus resembles the thermophilic cellulolytic bacteria studied by McBee (22) which also produce glucose but are unable to ferment it. At the first subculture of this coccus a parallel cellobiose series was inoculated but the colonies developing were found to consist only of rods. At the second subculture a parallel glucose series showed colonies of rods and also smaller colonies of cocci, but these latter failed to grow upon subcultures into cellulose and glucose. At subsequent transfers a glucose dilution was always inoculated along with the cellulose series but on no occasion was any growth observed. This indicated that the culture was purer than before and the technique of diluting single colonies into solid medium probably gave a pure culture, but due to its failure to grow alone in the glucose medium the purity was not thoroughly established.