Chemical and Morphological Bacterial Spore Formation

From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers--cortex, coat, and exosporium--in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is 1/~ fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whi ten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions. I N T R O D U C T I O N The high refractility of the bacterial spore as seen in the light and dark phase contrast microscope is a characteristic which develops during the very late stages of spore formation. Preliminary evidence has associated the development of refractility in time with the laying down of those structures of the mature spore-the cortex, spore coat, and exosporium--which lie peripheral to the membrane-enclosed spore body (23). Chemically, these advanced stages of spore development have also been associated with the synthesis of dipicolinic acid (8, 17), the uptake of calcium (17, 21) and a change in the distribution of hexosamine-containing peptides (17). In an at tempt to understand better the development of refractility we have correlated the structural with some chemical changes which occurred in populations of Bacillus cells during the late stages of reasonably synchronous sporulation. We have also extended these studies to cells sporulating in media varying in calcium concentration. M A T E R I A L S A N D M E T H O D S C u l t u r i n g Techn iques Two strains, A + (crystal-forming) and A(noncrystal-forming), of Bacillus cereus var. alesti were the organisms studied. Their origins and characteristics have been described previously (6). 115 on Jne 7, 2017 D ow nladed fom Published January 1, 1962