STAINING OF FILM PREPARATIONS 727 



() Staining of films. 



Giemsa's methods. A. Slow method. Recommended. 1. Dry the films 

 in the air ; fix in absolute alcohol for half an hour. 



2. Stain for 20 hours in Petri dishes in the following solution : 



Giemsa's solution 1 (Griibler), '- - - xxxv drops. 2 



Distilled water, 20 c.c. 



3. Wash in distilled water. Dry with filter paper, or in the warm 

 incubator. Examine with an oil-immersion lens 



without a cover-glass, or mount in balsam. 

 The Spirochaetes are stained reddish-violet. 



B. Quick method. Recommended. 1. Fix for 

 30 minutes in absolute alcohol ; or better, for 

 a few seconds in the vapour of osmic acid 

 (Schaudinn). 



2. Stain for 1 hour in the following solution 

 which must be freshly prepared : 



Giemsa's solution, ... 10 drops 

 1 per cent, aqueous solution of 



carbonate of sodium, - - 10 



Distilled water, - - - 10 c.c. 



3. Wash in distilled water. If the preparation FiQ.Stf.Treponcmapattidum. 

 be over-stained leave it in water for from 1-5 x 1 io(K) repar 



minutes. Dry. Examine. 



C. Rapid method with heat. Recommended. 1. Make very thin films 

 on a slide. Dry. Fix by passing three times through a small Bunsen 

 flame. 



2. Drop a large drop of the following mixture, recently made up in a per- 

 fectly clean vessel, on to the slide. 



Distilled water, ..... 10 c.c. 



Giemsa's solution, ..... 10 drops. 



1 per cent, solution sodium carbonate, - - - 5 to 10 



Heat the slide with the stain on it for a few seconds over a small flame 

 until steam rises but avoid boiling the solution. 



Pour off the stain and flood the slide with a fresh quantity. Heat again. 

 Change the stain in this way three times but let the third lot of stain act for 

 1-2 minutes. 



3. Wash in water. Dry with filter paper. Mount in balsam. Examine. 



The methods given above are better than Giemsa's original method which was 

 used by Schaudinn in his early work on the spirochaste. Schaudinn fixed his prepara- 

 tions as above and then stained for 16-24 hours in the following solution : 



Aqueous solution of Giemsa's eosin (0*05 per 1000), 12 parts. 



Aqueous solution of Azur I. (1 per 1000), - 3 



Aqueous solution of Azur II. (0'8 per 1000), - 2 



Marino's method. Recommended. This is a rapid method and does not 

 entail fixing the films. 



1 Giemsa's solution may be obtained ready made from Griibler of Leipzic. It is pre- 

 pared by mixing : 



Azur II. Eosin, - - 3 grams. 



Azur II., - - 0-80 gram. 



Glycerin, - - 250 c.c. 



60 per cent, methyl alcohol, - 350 ,, 



Dissolve the stains in the alcohol then add the glycerin. Leave for 24 hours. Filter. 



2 1 c.c. xxx drops. 



