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Actinomycetes, involved in oral and periodontal diseases, cause serious infections in immunocompromised hosts. Severely scorbutic guinea pig leukocytes killed only 12% of phagocytosed actinomycetes, had distorted nuclear morphology, had 16 times less ascorbate, and had no chemotactic responses in vitro. Ascorbate reversed these indices and also prevented nitrosamine formation by oral organisms. Degranulating leukocytes release lactoferrin and ascorbate that chelate iron, essential for microorganisms. Ascorbic acid, 2,2'-bipyridine and 1,10-phenanthroline were bactericidal to several bacterial pathogens at millimolar concentrations. Iron alone reversed this effect. In in vivo experiments an Actinomyces viscosus monoflora was implanted in rhesus monkeys. Plaque and serum samples showed decreased (by six orders of magnitude) bacterial counts and decreased actinomycete antibody titers in animals given 1 g ascorbate/d. Removing ascorbate returned counts and titers to preascorbate concentrations. Fifteen marmosets, receiving twice daily topical applications of ascorbate or water, had comparatively lower gingival, calculus, and plaque indices and only slightly lowered actinomycete counts.

S U M M A R YTris and EDTA combined to form a homologue which has far greater chelating ability than EDTA alone. The weaker serine and NH,Cl homologues of EDTA replaced EDTA+tris in causing toxicity and permitting lysozyme lysis in Azotobacter vinelandii. Serine could effectively substitute for tris in these systems for Pseudomonas aeruginosa. These compounds had relatively little effect on Escherichia coli. The toxicity of EDTA+tris for these organisms could be alleviated by pre-incubation in physiological saline (0.15 M-NaCl). Subsequent lysozyme lysis was prevented by this treatment. The removal of metals from A . vinelandii cysts by EDTA+tris was also inhibited. The EDTA + tris + metallo chelate complexes were inactivated (dissociated) by saline. I N T R O D U C T I O NMany Gram-negative organisms which are resistant to lysozyme will lyse when ethylenediaminetetra-acetic acid (EDTA) and tris buffer (2-amino-2-hydroxymethylpropane-lY3-diol) are also present in addition to lysozyme (Repaske, 1956(Repaske, , 1958 McQuillen, 1960 a). Repaske first established a requirement for these compounds in this lytic system but did not determine the toxicity of the individual components. Socolofsky & Wyss (1961) were able to rupture cysts of Azotobacter vinelandii by using EDTA and tris. The released ' central bodies ' were not viable and were lysed by adding lysozyme. Goldschmidt & Wyss (1 966 : to be published), while studying this toxic effect in greater detail, observed that the EDTA+ tris system was toxic to vegetative organisms as well as to cysts even under conditions where cyst rupture did not occur (i.e. in 0.15 M-NaCl). EDTA suspended in water or 0.025 M-NaCl also ruptured the cyst exine, but the central bodies remained viable under these conditions. They also found that various other substances such as amines, amino acids and NH4C1 could replace tris in producing toxicity in the presence of EDTA; these compounds also substituted for tris in the tris+EDTA+lysozyme system. These results are presented in the present paper. During this work, other bacteria were tested for their sensitivity to the EDTA+ tris system (Goldschmidt, Goldschmidt & Wyss, 1967). Repaske (1958) noted that Escherichia coli was sensitive to his lytic system. Goldschmidt et al. (1967) found that male strains of E. coli (Hfr, male) were sensitive to the EDTA+ tris system (65-95 % kill within 4 min.) while female strains and E. coli B/r (which is ' F" ' and does not mate) were not killed. There are many reports in the literature discussing differ-* Present address :

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