574 PROCEEDINGS OF SECTION G2. 



slight liberation of gas and a faint opalescence of the fluid. In 12 

 to 15 hours definite cloudiness will be apparent, and much gas 

 formation will be evident. Although at first sight this growth 

 seems to be serobic in nature, I am convinced this is not strictly 

 so. Before any cloudiness is evidenced the gas bubbles can be seen 

 to definitely escape from the swollen muscle tissue, and, further, the 

 cloudiness can be seen to spread upward therefrom, being always 

 most definite at the bottom of the tube. The multipHcation of the 

 organisms evidently therefore occurs within the tissue, the newly- 

 formed bacilli passing outwards into the surrounding fluid. Such 

 a piortion of muscle may be transferred from tube to tube, still 

 giving rise to fresh growths after the medium has been cleared by all 

 the bacilli becoming deposited at the bottom of the fluid, but this 

 transference cannot be continued indefinitely, six times being the 

 limit in our experience. On the whole, I am not inclined to consider 

 the second and later growths resulting from the transference as 

 subcultures, though, perhaps, strictly they have a right to be 

 considered such. Without the portion of muscle tissue no sub- 

 cultures can be made in serobic broth of the same or any other 

 nature. It is my custom to test for accidental aerobes by pressing 

 a few drops of the liquid culture in a fresh broth tube. 



Anaerobic sub-cultures can most conveniently be made in the 

 liquid media already specified when covered with a thin layer, about 

 ^ to 1 cm., of pure olive oil, after the manner recommended by 

 Ham,ilton. The routine now employed by me is as follows : — To 

 glucose broth or ordinary broth (+ 1 to phenolphthalein) one-third of 

 dilute alkaline serum is added, and covered by a layer of oil. The 

 whole is then gently raised to boiling point in order to free the 

 liquid of aU trace of air, and especially care must be taken to see 

 that the oil also is boiled, otherwise the traces of ox3'gen retained 

 therein may be sufficient to inhibit the growth of the bacillus. 

 The inoculations from tube to tube are best made by means of a 

 sterile Pasteur pipette. 



Cultures in Solid Media. — In Stab glucose agar the growth is 

 seen on the second day as a faint streak, wliich develops a beaded 

 appearance not unlike a culture of Blackleg, but without any radial 

 penetration whatever of the medium ; in such a growth there is 

 little gas formation. In shake cultures on glucose agar, where the 

 bacilli are separated so as to produce separate colonies, the shape 

 of the colonies, which appear in 24 hours at 37° C, is very character- 

 istic. No colonies are to be found for a centimetre below the surface, 

 no matter how rapidly cooled. The size of these colonies varies 

 with the number present, and they show no tendency to coalesce. 

 Whatever the size of these colonies, they are very uniform in the 

 tube, and show no tendency to increase beyond a certain point. 

 The size varies between 0.2 mm. and 1 mm. in diameter, depending 

 on the number present. On examining these colonies with a lens 

 they are seen to be biconvex discs, with no tendency what- 

 ever to form radiations. They occupy different planes to each 



