APPENDIX 477 



and 950 e.c. of distilled water. To this is added 50 c.c. of glacial acetic 

 acid. Stain II. consists of 2 grammes of vesuvin dissolved in 1000 c.c. 

 of boiling distilled water. Both stains are filtered before use. Prepare 

 films in usual way, and stain with No. I. for thirty seconds. Wash in 

 water and then stain with No. II. for thirty seconds. Wash, dry, 

 and mount. The bacilli are stained brown by vesuvin and the meta- 

 chromatin granules blue-black. Some bacteriologists place great 

 reliance upon the diagnostic value of Neisser's stain for the diphtheria 

 bacillus from blood serum cultures and from swabs. In the latter case 

 the stain is sometimes used as a "rapid method of diagnosis." It is not, 

 however, absolutely reliable. 



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 1 to 2 c.c. of sterile salt solution. Inoculate agar plates 

 with a drop of this dUution, and incubate at 37° C. When the colonies 

 appear, sub-culture those resembling streptococcus colonies in bouillon, 

 and on blood serum. Sub-culture from the bouillon in milk, gelatine, 

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

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

 groin or intra-peritoneally. An acute purulent inflammation will be 

 set up in the exudation of which streptococcus will occur in large 

 numbers. 



Method of Staining. — Gram's method is the most satisfactory. Next to 

 Gram's stain the most useful is Loffler's blue. It may be noted that 

 most of the putrefactive organisms do not hold Gram's stain. 



Bacillus COli communis. — (a) Dilute the milk to be examined 500 

 or 1000 times. Take a sterilised brush, dip it in the dilution, and brush 

 over the surface of six agar plates without recharging the brush. 

 Incubate at 42° C, and sub-culture the coliform colonies (bouillon, milk, 

 litmus milk, gelatine " shake " cultures, bile-salt-glucose-peptone, etc.). 



(V) Take six tubes of phenol bouillon (0'05 per cent, of carbolic acid), 

 and inoculate them with crude or diluted milk. Those which show 

 abundant turbidity after twenty-four to forty-eight hours at 37° C. may 

 be plated out on phenol gelatine, incubated at 20° C, and the coli 

 colonies sub-cultured ; or diluted milk may be at once plated out on 

 phenol gelatine, and colonies sub-cultured on such media as will show the 

 characteristics of the organisms. 



The main characters of the B. co/i group of organisms may be briefly 

 restated here, though particulars will be found elsewhere in the present 

 volume: — (1) They are non-sporing and non-liquefying; (2) they rarely 

 stain by Gram's method ; (3) they are motile ; (4) they produce acid and 

 gas in glucose and lactose media ; (5) they produce acid in milk, and 

 usually coagulate it ; (6) they grow well at a temperature of 42° C. 

 Referring to the isolation of B. coli, Houston writes : " No test based on 

 observation of a change or changes produced in the nutrient medium, 

 and supposed to be characteristic of B. co/i, can compare with isolation 

 from plate cultivations of the microbes suspected to be B. coli, and the 



