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tained with little decrease for as long as 10 and 15 days.The Eastern strain of equine encephalomyelitis has been grown with a dilution activity of lO-' in whole minced chick embryo in serum-ultrafiltrate at room temperature and 37" C.A vaccine, containing very small amounts of protein, prepared from the clear supernatant fluid of serum-ultrafiltrate cultures protected guinea pigs against 1,000 to 10, OOO m.1.d. doses of virus (Western strain).In a preceding paper1 we reported the cultivation of rabies virus in developing chick embryos in 2 series through 9 and 6 successive passages respectively. Since then, one series has been carried through 16 subcultures; the second series has been passaged through 47 subcultures during a period of 18 months and is still being maintained. The purpose of this note is to summarize our observations on the behaviour of this virus to different hosts after prolonged cultivation in the developing chick embryo.A fixed virus, presumably derived from the original Pasteur strain, was used for initiating the chick embryo cultures. The first inoculation was made by dropping 0.1 cc of a 10% saline emulsion of infected mouse brain on the allantois immediately over the embryo using the Burnet2 technique. The same technique was used for subcultures : pieces of the inoculated allantois, or preferably the brains of the embryos of 3 to 6 infected eggs, were ground in a sterile glass mortar and enough saline added to make a 10% emulsion. Each passage material was titrated intracerebrally in mice in tenfold dilutions. In the first 12 passages, 5-day-old embryos were infected ; subsequently 6-day-old embryos were used because they were more resistant to the manipulations involved. Passages were usually made 6-1 1, sometimes up to 14, days after the inoculation.The infection in the chick embryo. The embryos of earlier as well

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After the cultivation of strains of the trachoma agent in fertilized eggs had been accomplished,'s2 it seemed an obvious next step to use cell cultures for the same purpose. The first successful cultivation in vitro was reported by Gordon et aL.,3 who grew a strain isolated in Taiwan in explants of yolk sac cells. In these cultures a study of certain morphological stages in the development of the agent could be carried out. Infection was, however, confined to a minority of cells, and it was difficult to obtain more than a single cycle of multiplication. Pollard et aZ. 4 reported observations made with the fluorescence microscope on the growth of a trachoma strain, isolated in Arabia, in the Fernandes line of human amnion and the McCoy line of human synovial cells. Five successive passages could be carried out in the latter cells. The present work has been carried out with one of the strains isolated in China by T'ang et aL. ' This strain, obtained through the kindness of Dr. Collier, was grown in the FL line of human amnion cells.6 The same strain has been grown in HeLa cells by Furness et aL6In FL cell cultures the T'ang strain has so far undergone more than 60 passages. The medium used contained Earle's salt solution, lactalbumin hydrolyzate, yeast extract, and 20 per cent human serum, to which streptomycin and mycostatin were added. The same cell line and medium proved to be unsuitable for the growth of another trachoma strain which was isolated more recently in IsraeL7 In a subline of the FL strain, on the other hand, which is cultivated in bovine instead of human serum, the T'ang strain did not reach full maturation, even when the bovine was replaced by human serum at the time of infection.Multiplication of the T'ang strain in FL cells cultivated continuously in human serum is accompanied by a characteristic cytopathic effect. Large vacuoles full of elementary bodies develop in the infected cells. Their enumeration in infected monolayers gives a good quantitative estimate of the infectivity of a preparation. The method has been described in detail by Furness et al? and has also been used in the present work. Growth Cwve of the AgentAll experiments were carried out after the agent had undergone 22 or more continuous passages in cell cultures.From 30 to 40 tubes containing monolayer cultures 4 to 5 days old were infected with a multiplicity of 5 to 10. After an adsorption period of 6 and in some experiments 2 and 3 hours a t 37" C., the cultures were washed with sev-