Observations on the structure of Bacillus spores.

Abstract

SUMMARY: An internal organization is revealed in resting spores of Bacillus spp. by brief treatment at room temperature with x-HNO3 or, more effectively, a mixture of n/3-HNO3 (or HCI) with 0·1 % potassium permanganate. Two distinct steps are involved: the first initiates the reaction without visible changes in the spore; the second is an instantaneous process which abolishes the refractility of the spore and allows staining of the contents. These differentiating procedures have no observable effect on vegetative cells, and the spore's response to them is abruptly abolished with the onset of germination. They are ineffective on heat-killed spores and are inhibited, under certain conditions irreversibly, by 0·5-1 % sodium fluoride. Differentiating reagents probably act indirectly by stimulating abnormal chemical activity of the spore. Three structures, interpreted as the outer spore membrane, nucleus and cytoplasm, can be distinguished in treated spores. Differentiation does not alter the size or the shape of the spore but shrinks the cytoplasm away from the membrane and, in permanganate-containing mixtures, changes the configuration of the nucleus. The extent of these rearrangements varies with the temperature at which differentiation is carried out. The nucleus is on the outer surface of the cytoplasm and connected with it by a short narrow stalk. The nucleus gives a positive Feulgen reaction. Nucleus and cytoplasm of spores differentiated with n/3-HNO3 or HCI containing 0·1% of KMnO4 are soluble in phosphate buffers at pH 6·3-7·6 and m/16 sodium acetate at pH 7·4. Nucleus and cytoplasm are insoluble in these solutions after differentiation with n-HNO3, but the nucleus of spores treated in this way is destroyed by trypsin in phosphate butter. The existence of a second, inner, spore membrane, jointly surrounding nucleus and cytoplasm, is postulated. This layer is believed to be the seat of the spore's bright retractility and imperviousness to stains. The huge spores of Metobacterium polyspora react like Bacillus spores to differentiating reagents and permit direct observation of the act of transformation from the featureless to the differentiated aspect of the spore.