PLATE XLVIII)THE observations which prompted this investigation were that most strains of coagulase-positive staphylococci (Staph. pyogenes) produced opacity when grown in media containing egg yolk and that this reaction was strongly inhibited by staphylococcus antitoxin. Although the opacity developed more slowly than the lecitho-vitellin reactions of Clostridium welchii (Nagler, 1939 ; Macfarlane, Oakley and Anderson, 1941) and Bacillus cereus (McGaughey and Chu, 1948 ; Chu, 1949), and was not due t o a lecithinase of the type produced by these organisms, its superficial appearance was similar. The objects of this paper are to describe the reaction, to indicate its relationship t o some other properties of staphylococci, and to report the results of preliminary attempts to determine its nature. MATERIALS AND METHODSYolk both was prepared by the method of McGaughey and Chu : 5 parts by weight of fresh hen-egg yolk were added to 100 parts of digest or infusion broth. Kieselguhr (2 g. per 100 ml.) was added after thorough stirring, and the mixture stirred again and allowed to stand for about 30 minutes. It waa then filtered through filter-paper pulp. As opacity production was enhanced in the presence of glucose (probably due to the effect of lowered pH, as described below), one per cent. of this sugar was added to the medium, which was sterilised by filtration through a Seitz pad. Yolk saline waa made by substituting 0.85 per cent. NaCl for broth and omitting the glucose. These reagents were stored a t 4-6" C., and could be used up to a t least 4 weeks after preparation.The antiserum used in the experiments was Burroughs Wellcome therapeutic r e h e d staphylococcus antitoxin, which was stated to contain 2500 units in 1.9 ml.The staphylococci, with the exception of a few obtained from the National Collection of Type Cultures, were isolated from routine specimens of pus, etc., and from nasal and throat swabs of patients and members of hospital staff; they were usually tested soon after isolation. Cultures were maintained on nutrient agar (infusion base) slopes in screw-capped bottles at room temperature.Coagulase tests were done both by the tube method (Gillespie, 1943), and by Berger's modi6cation (1943) of the slide method of Cadness-Graves et al. (1943).It was an almost clear yellow fluid, of pH 7.2.

GILLESPIE and Alder (1952) described an egg-yolk (E.Y.) reaction of Staphylocoecw aurew (coagulase-positive staphylococcus). About 80 per cent. of strains isolated from human Bources outside hospital produced opacity when grown in egg-yolk broth and were called egg-yolk-positive (E.Y. +) strains. The remaining 20 per cent. were egg-yolk-negative (E.Y. -).The distribution of the reaction among strains isolated from in-patients of this hospital was very different. Most of t'he hospital cultures were E.Y.-.The difference was found to be due to the widespread dissemination among in-patients of a few strains that were penicillin-resistant and E.Y.-at the same time. , 411 of the 60 coagulase-negative staphylococci examined were found t,o be E.Y.-.Egg-yolk reactivity was found to be a stable property of staphylococci. A strong but not absolute correlation was demonstrated between it and the lipolytic activities of the organisms against triglycerides of higher fatty acids, but the mechanism of the reaction was not elucidated.In the present paper we report the reault,s of a further survey of' the distribut,ion of the E.Y. reaction among staphylococci from human and animal sources and describe the relationship found between the react,ion and the clinical evidence of pathogenicity of human coagulasepositive strains. MATERIALS AND METHODSCultures of staphylococci were maintained on infusion-base nutrient-agar stabs, which, after incubation overnight at 37' C., were stored a t c. 4' C.Coagulase tests were done on freshly-isolated cultures by the slide method (Cadness-Graves et al., 1943). Most of the coagulase-positive cultures, including all that were found to be E.Y.-, were re-tested by the tube method (Gillespie, 1943). This extra precaution was taken to ensure that no coagulase-negative strain was listed in error as coagulase-positive. This sort of mistake would have been serious, since all coagulase-negative strains were E.Y.and most coagulasepositive strains E.Y. +. All the coagulase-negative staphylococci examined were tested by the tube method. The E.P. reactions of the organisms were tested by growing them at 37" cin glucose-yolk broth, with and without antitoxin, as previody described. Known E.Y.+ and E.Y.strains were included as controls with each batch of tests. Experience has shown that storage of the yolk broth at ca. 4' C. for 1-2 months before use speeds up the reaction and increases the tendency of the separated material to form a curd. Medium which had been thus allowed t o mature was used ip the later part of the work. A few Staph. aurew strains from animals produced relatively faint turbidity J. PATH. BACT.-VOL. LXVI (1953) 205

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