AND CHARLES D. JEFFRIES. Isolation and properties of an exocellular nuclease of Serratia marcescens. J. Bacteriol. 85:273-278. 1963.-The exocellular nuclease of Serratia marcescens, isolated by anion-exchange chromatography on diethylaminoethyl-Sephadex, depolymerized deoxyribonucleic acid, ribonucleic acid, and the polynucleotide which is refractory to pancreatic ribonuclease activity. The enzyme was tentatively classified as a nonspecific phosphodiesterase. Magnesium was essential for activity, which was optimal at pH 8.8. The purified enzyme was completely inactivated by heating at 50 C for 15 nmn. Exocellular nucleases produced by bacteria are usually reported as deoxyribonucleases and ribonucleases, depending upon the substrate used for their detection. For example, Masui et al. (1956) reported that Staphylococcus aureus and Bacillus subtilis, among others, secreted both types of nucleodepolymerases into the culture medium. Nishimura (1960) later crystallized an exocellular ribonuclease from B. subtilis, which implies that ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are degraded by distinct species of nucleolytic enzymes in this bacterium. In contrast, Cunningham (1959) reported that S. aureus secretes a phosphodiesterase which depolymerizes both DNA and RNA.

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IT has been known for some time that leptospirae consist of protoplasmic spirals and an axial filament. Timmermann (1927, quoted by van Thiel, 1948) and Herreweghe (1943) showed that axial flaments were demonstrable in stained preparations of leptospirae in which the spirals had been decolourised, or in unstained preparations treated with spec& antisera or sodium taurocholate. Other authors, however, have found no evidence of such an elastic axial filament m a differentiated structure in their electron micrograph (Morton and Anderson, 1943) and doubt its existence in leptospirae (van Thiel and van Iterson, 1947).Recently Babudieri (1948, 1949) concluded from examination of electron micrographs that leptospirae consist of a cylinder of homogeneous protoplasm enveloped by a very thin anistic membrane and wrapped round a rigid central filament-the axistyle. This structure is said to be particularly evident in partially autolyeed leptospirae, and in old cultures in which the destruction of the cellular membrane and disintegration of the cytoplasm have set it free.In the experiments here reported, a gradual step-by-step disintegration of leptospirae by sodium desoxycholate has been induced and followed up by electron microscopy.* MATERIALS AND METHODS Organisme. These were pure cultures of the Wijnberg and Lisboa strains of LeptoF'ra ~cterohaemorrhagice and of Leptoqira caniwla, strain Utrecht, all kindly supplied by Dr J. C. Broom of the Wellcome Laboratories of Tropical Medicine, London ; &o of a strain of leptoepira (L. k d a t) isolated from 8 cam of " abacterial " cystitis (Czekalowski and Horne, 1951 ; Czekalowski and McLeod, 1954).Medium and cultivation. The organisms were grown in 8 medium composed of 12.5 per cent. meat extract, 0.1 per cent. Witte peptone and 10 per cent. of 8 suitable rabbit serum inactivated at 56" C. for 30 minutes. The completed Certain aspects of this work (the extra-cytopla6c position of the axistyle. its mode of attachment to the protoplasmic cylinder and its separation by means of sodium desoxycholate) were summarised in the discussion on the paper presented by Bradfield and Cater to the Pathological Society of Great Britain and Ireland at the Cambridge meeting in January 1952 (this J o u d , 1952, Iraiv, 243).t This strain was referred to in Czekalowski and Horne (1951) and Czekalomeki and Eaveg (1954) 88 L. czekalowski. J. PAW. BAOF.-VOL. I X I X (1856) 128 I Ren. Ist. Super. Sanita, Rome, xi, J . Hyg., Csmb., xlvii, 390. Quart. J . Micros. Sci., xciv, 351. Nature, Lond., clxix, 944. J . Bact., 1 x 6 , 619. 1046. Brit. Med. J., ii, 879. This Journal, lxvii, 43. Actu Biol. Belg., iii-iv, 245. Biochim. et Biophys. Acta, i, 527. Elements of bacterial cytology, Ithacu, N . Y . J . Bact., xlv, 143. J . Exp. Med., lxxvi, 103. Arch. Path., xxxiv, 199. The leptospiroses, Leiden, pp. 2 Proceed. Kon. Ned. Akad. v. and 4.Wetensclt., i, 976.

There is general agreement that the cytoplasmic eosinophilic inclusions found in cells infected with fowl-pox virus consist of aggregations of virus particles, possibly embedded in a matrix. It seemed likely that these inclusions might also be sites of virus multiplication, and therefore that electron microscopy of sections through them might afford some evidence of the way in which virus multiplication takes place. MATERIALS AND METHODSThe strain of fowl pox was kindly sent to us from the Ministry of Agriculture and Fisheries Laboratory at Weybridge as frozen-dried infected chorioallantoic membranes. This strain was isolated in Great Britain 6 or 7 years ago, and since then has been passaged at irregular intervals through fowls and embryonated eggs. An emulsion of the frozen-dried membranes we received was inoculated on to chorioallantoic membranes of 12-day hens' eggs. After 3 days' incubation at 350 C. the membranes were harvested and ground up in a mixture of 0 85 % saline (3 parts) and 'Lemco' broth (1 part). The suspension was centrifuged at 1500 g for 10 min., and the supernatant was inoculated in serial ten-fold dilutions on to fresh chorioallantoic membranes of 12-day chick embryos. Eggs receiving a dose which produced three to six pocks per membrane were incubated for 5 days. The membranes of these eggs then showed large pocks, 5-6 mm. diameter, almost completely transparent, with ill-defined edges. There was no naked-eye evidence of necrosis.2 % (w/v) osmium tetroxide solution in M/30 sodium-veronal-acetic acid buffer, pH 7 35, was dropped on to the membranes and left for 15 min. The membranes were then removed from the egg; pieces (about 1-2 mm. square) were cut from the centres and edges of the lesions, and fixed for a further 31 hr. in the same fixative.They were then embedded in methacrylate, and thin sections (about 200 A. thick) were cut by the method described by Eaves & Flewett (1954). The sections were photographed in a RCA EMU 2C electron microscope equipped with a 200,u objective aperture. RESULTSSections 1 It thick were examined by phase-contrast microscopy. Thin sections were cut from the same block for electron microscopy. It was observed by the first method, and confirmed by the second, that a great proliferation of chorionic epithelium had taken place, and that it was many cell layers thick. No sign of cell necrosis was seen, though the connective tissue of the membrane showed some fibroblastic proliferation and infiltration with macrophages.

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