486 APPENDIX 



filter-paper. After either of these methods has been adopted, stain the 

 film for thirty seconds with a concentrated aqueous solution of methylene- 

 blue (or the following solution for the same period of time : Borax, 

 5 parts ; methyl ene-blue, 2 parts ; water, 100 parts). Wash in water, 

 dry with filter-paper, and mount in xylol-balsam under a cover-glass.* 

 Loffler's blue or carbol thionin may be used. For double staining, 

 Jenner's, Romanowsky's, or Leishman's stains may be used. 



To demonstrate flagella, proceed as follows : Take a piece of thick 

 blotting-paper, 3 x 1 J inches, with a round hole in the middle the size 

 of an ordinary cover-glass. Moisten the blotting-paper and place it on 

 a clean slide. Take a drop of the blood on another slide which has 

 been breathed upon, and invert it on the blotting-paper (moist cell). 

 In thirty minutes separate and dry the blood-film on both slides by 

 gentle warming over the lamp. Fix with absolute alcohol, which may 

 be allowed to evaporate or be dried with filter-paper. Wash with 

 acetic acid (15 per cent.) to dissolve out the haemoglobin, wash in water, 

 and dry as before. Stain the dried film with carbol-fuchsin (20 per cent.) 

 for six to eight hours. Wash and mount as before. 



Bacteriological Examination of Oysters 



Particular attention should be paid to the (a) washings of the shell, 

 (b) the liquor in the pallial cavity, and (c) the contents of the alimentary 

 canal of the oyster. The two latter are the chief parts for examination 

 in the ordinary course, and to obtain knowledge of the contained 

 bacteria the method to adopt is as follows : 



Method. Thoroughly cleanse the oyster shells by scrubbing with 

 soap and water, rinse under the tap, and again in sterile water. Also 

 the hands of the bacteriologist should be thoroughly cleansed and 

 rinsed in antiseptic (e.g. 1-1000 corrosive sublimate) and sterile water. 

 Now lay the oysters on the table with the flat shell uppermost, and 

 open with a sterile knife. Pour the pallial liquor into a sterilised flask 

 or capsule, and cut up the body of the oyster, adding the pieces to the 

 liquor or to another flask. Add to the flasks of liquor and of oyster 

 pieces, or to the one flask containing both sufficient sterile water (100 

 c.c. or 1000 c.c. as desired). The emulsion is now to be cultured as 

 follows, adding in each case suitable quantities of the emulsion, e.g. (10 

 c.c. or 5 c.c. or 1 c.c. or '5 c.c.) : Three tubes of broth (for indol forma- 

 tion) ; three tubes of phenolated broth (for B. coli and its allies, and also 

 for secondary plate cultivation) ; three tubes of M'Conkey medium, 

 bile-salt-glucose peptone (for B. coli and its allies, coloration and gas) ; 

 three tubes of freshly sterilised milk, heated after inoculation to 80 C. 

 for 15 minutes, and cultured anaerobically (for B. enteritulis sjwrogenes) ; 

 three tubes of litmus milk (for acid and clotting) ; three gelatine 

 " shake " cultures (for gas production) ; three plates of phenolated 

 gelatine and three of ordinary gelatine ; and three plates of agar for 

 incubation at 37 C. For quantitative estimation of colonies 011 the 



* See also Tropical Diseases (Manson), p. 40. 



