48 SPOROZOA. 



100 c.c. of methyl alcohol. Next ripen the stain by placing 

 the bottle of stain overnight (but not longer) in the 37° C. 

 incubator. 



In order to stain, lay the blood-film, film side upwards, on 

 a staining rack. Drop the stain on to the sHde until the 

 whole surface is covered with the stain. The methyl alcohol 

 in the stain fixes the film. This takes half a minute only ; 

 after that interval drop on to the shde double the number of 

 drops of pure distilled water. By tilting the end of the shde 

 allow the water and stain to mix thoroughly, and allow the stain 

 to act for 5 minutes or more. Wash the stain ofi" by immersing 

 the slide in distilled water. Put the shde to soak in a clean 

 Petri dish containing fresh distilled water and rock gently. 

 The film turns at first greenish-blue, then pink. This takes 

 about half a minute or less. When the film is just turning 

 pink remove the slide and lean it against a vertical surface 

 to dry. 



Giemsa's Stain. — To prepare the stain grind in a glass mortar 

 with a glass pestle 3 grm. of azur-II-eosin and 0-8 grm, of 

 azur-II (Griibler's or Merck's) into thorough solution in 250 c.c. 

 of the purest anhydrous glycerine. Add 250 c.c. of the purest 

 acetone-free methyl alcohol and mix thoroughly. Allow to 

 stand overnight and next day filter the stain through filter 

 paper. Prepared Giemsa's stain is stocked by many firms, 

 and is also supphed by the Central Research Institute, Kasauli. 



Fix the film by covering it for 3 to 5 minutes Avith pure 

 methyl alcohol or by dipping it for 10 minutes into absolute 

 alcohol, and wash thoroughly in distilled water. Dilute 

 1 part of Giemsa's stain with 14 parts of distilled water 

 (10 or 15 drops in as many c.c. of distilled water), and pour 

 it over the slide placed in a Petri dish. Stain for half an hour 

 or longer. Remove the slide, flush with distilled water, and 

 put it in a bath of fresh distilled water until it commences to 

 turn pink. Remove the slide and let it dry by leaning it 

 against a vertical surface. 



The Panoptic Method of Staining. — Lay the shde on a stain- 

 ing rack and cover it for half a minute with undiluted Leish- 

 man's stain ; dilute the stain with double the number of drops 

 of distilled water, and allow to stain for 5 or 10 minutes. 

 Wash the film with distilled water. Then lay the shde in 

 a Petri dish and flood with diluted Giemsa's stain, one drop to 

 each c.c. of water. Stain for 1 to 24 hours, covering the 

 Petri dish to prevent evaporation. Wash the fihn with distilled 

 water and transfer it to a bath of 1 in 1000 acetic acid. When 

 the film begins to turn pink, remove, wash rapidly with dis- 

 tilled water, and slant the slide against a vertical surface to dry. 



A more rapid alternative method is as follows : — Cover the 

 film with undiluted Leishman's stain for half a minute ; 



