MINERAL DEFICIENCIES IN COMPLEX ORGANIC MEDIA AS LIMITING FACTORS IN THE SPORULATION OF AEROBIC BACILLI

When aerobic sporeforming bacteria are cultivated in a variety of the usual bacteriological organic media, good vegetative development is observed in all the cultures, but striking differences in degrees of sporulation may be observed. Table 1, dealing with Bacius cereus, is representative of the behavior of six different species of Bacillus. Spore counts were made by the malachite green, mercurochrome method as described by Wynne (1948). A point of special interest is that in each medium the vegetative growth was good, differences in cell yields between the various media being due to a difference of not more than a few generations, and often less than that. A detailed study of the negligible sporulation in "casamino acids" and "N-Zcase" media (asporogenic media) revealed the following points: (1) small proportions of yeast extract or Wilson liver "B" added to the asporogenic media induced good sporulation; (2) the effect was obtained by the ash of yeast extract or liver; and (3) the potassium ion was identified as the main factor critical for sporulation in the asporogenic media (table 2). Potassium, along with several other cations, has been shown by several authors to influence sporulation (Fabian and Bryant, 1933; Perdue, 1933), and spectrochemical analysis reveals its presence in aerobic spores (Curran et al., 1943). From table 2 one may compute that the addition of K+ led to about a 30 per cent augmentation in total cell yield (Petroff-Hauser count), but to upwards of a 1,000 per cent increase in spore yield, suggesting a definite role for potasium in the sporulation process. Also it appears from these and numerous other experiments on this subject that the sporogenicity of a medium may be independent of the amount of vegetative growth it supports. In sporulation work it is likely that certain standard media may, as this study shows, be deficient in the mineraLs essential for maximum sporulation but adequate for abundant vegetative development. Some evidence was obtained that certain other minerals, such as magnesium and iron, may also be deficient. The exact status of the deficiency would depend on the particular lot of medium employed. One may conclude that a salts mixture should be used in organic media employed in sporulation studies.