SUMMARY: Phase-contrast microscopy enables germination to be studied in the spores of Bacillus cereus and B. subtilis a few minutes after the addition of adenosine; by this means adenosine can be identified in minute concentrations. Adenosine in high concentrations is, however, inhibitory to vegetative growth.Adenosine appears to act as an indicator of conditions suitable for vegetative existence, and induces, almost instantaneously, profound physical changes in spores. The analogy with hatching of eel-worm and amoebic cysts, also susceptible to specific excitants, is stressed.In recent papers Hills (1949a and b, 1950) reviewed the literature relating to spore germination and showed the primary importance of adenosine. He incubated spores in a chemically-defined medium, heated them for 15 min. a t 60°, and counted the spores remaining viable, When the viable count fell it was assumed that some spores had germinated in the test fluid. The results were submitted to statistical analysis, and indicated that adenosine was of primary importance for initiating germination. This interesting observation has been confirmed by the experiments here described, using a different technique, namely, examination of the germinating spores directly by phasecontrast microscopy.
TECHNIQUESpore suspensions of Bacillus cereus and B. subtilis isolated from a compost heap were prepared by surface growth at 37' on the medium of Knaysi & Baker (1947) for electron microscopy, containing glucose 0.2 g. and an equi-molar mixture of KH,PO, and KHPO,, 0.23 g. in 100 ml. of distilled water. This was modified by the addition of 0.5 g. NaCl and 1 yo New Zealand agar (Davis Gelatine Ltd., London). On this medium growth was profuse and spore formation excellent, and for practical purposes in a few days no vegetative forms remained. The spores were washed in saline, centrifuged and resuspended in small volumes of saline. After heating at 80" for 15 min. they were kept in a refrigerator until used. They did not germinate in the saline, and remained capable of germination in suitable media for at least three months.Slide cultures of the organisms were prepared by making streaks from 24-hr. cultures on nutrient agar on to a cover slip. After drying in air for 15 min. the melted agar medium of Knaysi & Baker at 50" was dropped on to the cover slip, which was mounted on a slide, and sealed with wax. Within a week in most cases very large numbers of spores had formed; those slides in which spore formation was poor were discarded (Pl. 1, fig. 1).