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The fine structure of Azotobacter vinelandii has been studied by means of electron microscopy of ultrathin sections made of the encysting and germinating cells. The organisms were fixed with KMnO4 and embedded in epoxy resin. On an encystment medium the rod-shaped bacteria begin to assume an almost spherical form and then bark-like exine appears in 11/~ to 2 days. The exine thickens and an electron permeable intine forms between it and the shrinking cell body. In 5 days the intine makes up more than half of the cyst volume and begins to show a definite two-layered structure. Meanwhile the peripheral bodies, which may be extensions of the cell membrane of the vegetative cell, disappear as the encystment progresses. The cell wall and membrane of the vegetative cell remain demonstrable as the confining structure of the shrinking central body of the mature cyst. In this central body lipoidal globules appear together with aggregations of nuclear material. Cyst germination begins with an increase in the size of the central body at the expense of the intine. The nuclear aggregations become more diffuse and the lipoidal globules disappear. The exine may be pushed outward and the bark-like fragments separate as the emerging vegetative cell develops. Invagination of the cell wall and membrane may occur at this stage leading to cell division. Empty exines remain as horseshoe-shaped structures.

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 :

A saline pond in a region in Antarctia where other lakes and ponds are frozen remains unfrozen at the prevailing low temperatures. The ecology of the pond is unique. A distinctive aerobic microbial population, though restricted to this natural habitat, adapts to growth in artificial culture. The growth habit of these organisms, as seen in nature and in laboratory culture, indicates a possible relationship between growth at high salt concentration, at low temperatures, and in media of low organic content.

The Azotobacter cysts were found to be more resistant than the vegetative cells to various harmful agents. Studies involving ultraviolet irradiation indicated that cysts required twice as great a dosage, as correspondingly treated vegetative cells, to be 90% inactivated. The acquisition of ultraviolet resistance during the encystment process was gradual and appeared to be related to the formation of exine and intine. A slow loss of ultraviolet resistance during germination was also noted. The cysts exhibited no marked resistance to heat, although they were extremely resistant to gamma radiation, sonic treatment, and desiccation. Evidence was presented indicating that the cyst is not a bacterial endospore. The eneystment process may confer a survival advantage upon the organism by coupling the low endogenous respiration rate with the ability to withstand desiccation. Bacteria possess the capability of forming several structural devices which enable them to resist the detrimental effects of the physical environment. The most apparent of these devices is the endospore, a discrete body which exhibits extreme resistance to deleterious agents. Another