A simple in vitro system has been used to culture 34 species of microorganisms derived from human calculus. A confluent growth on the surface of an agar dise, was subjected to a daily 4 hr incubation in culture medium followed by 20 hr in a metastable solution of calcium phosphate, in a simplified organ culture vessel, for 7–17 days. Smears, taken daily, were stained by Von Kossa's method for the demonstration of calcification. 18 species showed evidence of calcification. In addition to the previously reported Bacterionema matruchotii, Streptococcus salivarius, Streptococcus sanguis (Sherman type 1), Veillonela alcalescens and a Neisseria species, calcification was also demonstrated in Actinomyces naeslundii, 1 strain of Actinomyces viscosus, an unidentified catalase positive facultative Gram‐positive rod, Staphylococcus epidermidis, Micrococcus varians, Eikenella corrodens, a Campylobacter sp., Eubacterium saburreum, an unidentified Gram‐negative rod, Haemophilus aphrophilus, Haemophilus segnis, Bacteroides melaninogenicus and Propionibacterium acnes. The Gram‐negative flore of calculus thus shares with the more frequently studied Gram‐positive species the property of in vitro calcification. Since the surface of calculus is covered with plaque containing a high proportion of Gram‐negative microorganisms, it is suggested that they may play a larger part in the production of calculus than had been indicated previously.
Deposits obtained from the in vitro calcificaqtion of vialble adn formalin killed microorganisms have been examined by Von Kossa staining and X‐ray diffraction analysis. 34 of the 35 species examined were isolated from, and represcented, the flora of human calculus. Twenty‐one of the viable and 23 of the formalin‐killed cultures gave positive Von Kossa smears but idebntical results for live and dead preparations were obtained in only 25 of the 35 organisms examined. In viable cultres only, teh proportion of calcified organisms increased with time suggesting that degenrative change is probably a prerequistie for cellular calcification. The X‐ray diffraction analysis shwed that in clacified viable cultures hydroxyapatitie was always found (22) and in the majority of cases (18) was the sole from of crystalline with octocalcium phosphate and/or brushite (10), gave a weaker pattern than that given by similar viable cultures. In 9 deposits octocalcium phosphate and brushite were the only form s of calcium phosphate detected adn 6 of these gave positive Von Koss smears. The formation in vitro of these two forms suggests that, as previously recorded for hydroxyapatiti, the plaque microbiota may play a part in theri in vivo formation.
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