476 APPENDIX 



formed crystals of the salt. Wash again in water, blot off excess of 

 moisture, and apply stain. This fixing agent should be used on all 

 occasions when dealing with morbid material or cultures of a specially 

 virulent nature. 



METHODS OF EXAMINATION FOR SPECIAL MICRO-ORGANISMS IN MILK 



Bacillus pseudo-tuberculosis Of Pfeiffer (found in London milk 

 by K/eiri). By the centrifuge or by sedimentation in an ice-chest for 

 twenty-tour hours, obtain the particulate matter of the milk to be 

 examined. Inoculate 2 c.c. into a guinea-pig subcutaneously or intra- 

 peritoneally. In the course of three to four weeks caseo-puruleiit 

 nodules will occur in the inguinal glands (if subcutaneously inoculated), 

 or in the omentum and pancreas and other organs (if intra-peritoiieally). 

 Cultures may be obtained best from glands, spleen, pancreas, or liver. 

 Examine the nodules by staining and culture. They will have the 

 following characters if the disease be pseudo-tuberculosis : (a) Absence 

 of giant cells ; (6) absence of the true tubercle bacillus ; (c) presence of 

 large numbers of B. pseudo-tuberculosis ; and (d) signs of a rapid and not 

 a slow development. 



Method 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 counter-staining in a mixture 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 diphtherias. By centrifuge or sedimentation obtain the 

 particulate matter of the milk under examination and inoculate it into a 

 guinea-pig. Sub-culturing 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 1 part the broth to contain 

 glucose 1 per cent., peptone 1 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. 459) will be found the most satisfactory, but the methylene-blue 

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

 alcoholic solution of methylene-blue added to 100 c.c. of a '01 per cent, 

 solution of caustic potash. By this stain the striped appearance of the 

 bacilli of older cultures on blood serum is obtained more readily than by 

 other methods. 



Neissers Method for Differentiation of the Diphtheria Bacillus. This 

 method consists in applying two stains as follows. Stain I. is made of 

 1 gramme of methylene-blue dissolved in 20 c.c. of a 95 per cent, alcohol, 



