SIMPLE STAINING 



141 



place a drop of a broth culture about its centre and spread the drop with 

 the end of a pipette ; or 



Place a small drop of filtered water on the cover-glass, mix a trace 

 of the growth from a solid medium with it and spread the mixture with a 

 platinum wire. 



2. Dry the film gently either by holding it above the pilot flame of a 

 Bunsen or by placing it on a Koch's drying stage (fig. 127) heated to 45 

 or 50 C. 



FIG. 127. Koch's drying stage. 



While the film is drying keep the liquid evenly spread over the cover-glass 

 to prevent the formation of concentric circles. 



3. NoWjfce the film to prevent the organisms being washed off by the stain, 

 etc. This may be done either (a) by passing the cover-glass, film upwards, 

 two or three times through the ordinary Bunsen flame ; the organisms are 

 liable to be distorted and shrivelled by this procedure so that it is better 

 (b) to pour two or three drops of alcohol-ether on the film side of the cover- 

 glass and let it evaporate. This method produces no distortion of the 

 organisms. 



Alcohol-ether. 



Absolute alcohol, - - 50 c.c. 



Ether pur., - 50 



In special cases it is better to fix the films by immersing them in absolute alcohol 

 for 15 or 20 minutes or by exposing them to the vapour of osmic acid (vide Tre- 

 ponema pallidum). 



4. Filter two or three drops of stain straight on to the film (diluted carbol- 

 fuchsin, carbol-thionin, alkaline blue, etc., may any of them be used). Be 

 careful not to let the stain run on to the under surface of the cover-glass. 

 Stain for J to 1 minute. 



5. Wash off the stain by running a gentle stream of water from the wash- 

 bottle on to a corner of the cover-glass. The water ought not to be poured 

 on to the centre of the film for fear of washing it off. 



6. The film may now be examined (with the ^ in. immersion lens and a 

 No. II. eyepiece, for preference) : 



(a) Provisionally, in water, by inverting the wet cover-glass on to a slide, 

 blotting the upper surface of the cover-glass with a fine cloth [or filter paper] 

 and then placing a drop of immersion oil on the blotted surface. 



(b) After drying and mounting in Canada balsam. If the film is to be 

 mounted, dry the cover-glass either in the air or by heating it gently, and 

 place a small drop of balsam with the end of a fine glass rod on the film side, 

 invert it on to a slide and press gently to spread the balsam. 



To sum up : Spread a drop of culture on a cover-glass, dry, fix, stain, wash 

 in water, dry, mount in balsam and examine. 



Notes. (a) It is important in staining films to remember which is the film side 

 of the cover-glass. If this should be forgotten, gently scratch the surfaces of the 



