208 APPENDIX 



Ponder's Stain. Kinyoun's modification. 



Toluidine blue 0.1 gram 



Azure 1 0.01 gram 



Methylene blue 0.01 gram 



Glacial acetic acid 1.0 c.cm. 



95 per cent alcohol 5.0 c.cms. 



Distilled water 120 c.cms. 



The films should be stained for two minutes or more. 



Dorset* s Egg Medium. Take 12 fresh eggs, wash the shells 

 with water and then with undiluted formalin; allow to dry. 

 Break the eggs into a graduated cylinder and note the total 

 volume. Add one part of sterile saline solution (0.85 per cent 

 sodium chloride) to three parts of the mixed eggs. Pour into a 

 sterile beaker or basin and whip with an egg whisk; filter 

 through cheese cloth or muslin into a sterile flask and tube 

 10 c.cms. in the usual way. Inspissate at 75 C. for one hour in 

 a sloping position and then add 0.5 c.cm. of sterile glycerine 

 broth (physiological saline containing 6.0 per cent of glycerine) 

 to each tube to prevent drying. Incubate at 37 C. for forty- 

 eight hours and reject all contaminated tubes. Eyre recom- 

 mends adding sufficient alcoholic basic fuchsin to produce a 

 distinct colouration before the medium is tubed. 



Casein agar. To 300 c.cms. of distilled water add 10 grams 

 of casein (C. P. Hammersten) and 7 c.cms. of N. NaOH. Heat 

 to boiling for several hours until thoroughly dissolved. Adjust 

 the weight and bring the reaction to 0.2 per cent acid. The 

 agar solution is prepared by dissolving 10 grams of agar in 500 

 c.cms. of water. Both solutions are filtered, mixed, tubed, 

 and sterilised under pressure. The final reaction should be 

 +0.1 per cent and, if the acidity is higher than this, a portion 

 of the casein will be precipitated during sterilisation. 



