68 THE BACTERIOLOGICAL EXAMINATION OF MILK 



giant cells ; {b) absence of the true tubercle bacillus ; {c) presence 

 of large numbers oi B. pseudo-tuberculosis ; and {d) signs of a rapid 

 rather than a slow development. 



MetJiod of staining. — Make films in the ordinary way and stain 

 with Loffler's methylene-blue, heating the stain till it steams 

 (Klein). Wash in distilled water. Nodules may be hardened in 

 Miiller's fluid and spirit and sections cut, and stained by placing in 

 Loffler's blue for twenty-four hours and counterstaining in a mix- 

 ture of eosin and methylene-blue. Loffler's blue may also be used 

 for staining the bacillus in milk-films made from sediment. Gram's 

 method is also applicable, but the bacillus is not acid-fast and will 

 not hold the Ziehl-Neelsen stain. 



Bacillus diphtheriae — 



By centrifuge or sedimentation obtain the particulate matter of 

 the milk under examination and inoculate it into a guinea-pig. 

 Subculturing from the tissues of the guinea-pig, or, having obtained 

 sediment as above, inoculate six tubes or plates of Loffler's medium 

 (ox serum 3 parts, veal broth i part — the broth to contain glucose 

 I per cent, peptone i per cent, and sodium chloride 0-5 per cent). 

 Upon this medium the Klebs-Loffler bacillus grows rapidly in 

 twelve to twenty hours, producing scattered, nucleated, round, white 

 colonies which later become yellow. 



Method of staining. — Gram's method as modified by Nicolle {see 

 p. 59) will be found the most satisfactory, but the methylene-blue 

 solution of Loffler is often used. This consists of 30 c.c. of a satu- 

 rated alcoholic solution of methylene-blue added to ICXD c.c. of a -oi 

 per cent solution of caustic potash. By this stain the striped appear- 

 ance of the bacilli in older cultures on blood serum is obtained. 

 For differential purposes it is best to use Neisser's method. 



Streptococcus in milk — 



By centrifuge or sedimentation obtain the particulate matter 

 of the milk. Take a sterilised platinum loop, dip in the sediment, 

 and remove a drop of it Distribute this in a test tube containing 

 I to 2 C.C. of sterile salt solution. Inoculate agar plates with a drop 

 of this dilution, and incubate at 37° C. When the colonies appear, 

 subculture those resembling streptococcus colonies in bouillon, and 

 on blood serum. Subculture from the bouillon in milk, gelatine, 

 and agar, carefully noting the characters of growth, etc. Or 

 guinea-pigs may be inoculated in the subcutaneous tissue of the 

 groin or intraperitoneally. An acute purulent inflammation will 



