EXAMINATION OF BACTERIA IN TISSUES 93 



urine it is an advantage to place a drop of distilled water on the 

 film and heat gently to dissolve the deposit of salts ; then gently 

 wash in water and dry. In this way a much clearer picture is 

 obtained when the preparation is stained. 



Films dried and fixed by the above methods are now ready to 

 be stained by the methods to be described below. 



(b) Wet Method. — If it is desired to examine the fine 

 histological structure of the cells of a discharge as well as to 

 investigate the bacteria present, it is advisable to substitute 

 " wet " films for the " dried " films, the preparation of which has 

 been described. The nuclear structure, mitotic figures, etc., are 

 by this method well preserved, whereas these are considerably 

 distorted in dried films. The initial stages in the preparation 

 of wet films are the same as above, but instead of being dried 

 in air they are placed, while still wet, film downwards in 

 the fixative. The following are some of the best fixing 

 methods : — 



(a) A saturated solution of perchloride of mercury in '75 per cent, 

 sodium chloride ; fix for five minutes. Then place the films for half an 

 hour, with occasional gentle shaking, in 75 per cent, sodium chloride 

 solution to wash out the corrosive sublimate ; they are thereafter washed 

 in successive strengths of methylated »pirit. After this treatment the 

 films are stained and treated as if they were sections. 



(ft) Formol-alcohol — formalin 1 part, absolute alcohol 9. Fix films 

 for three minutes ; then wash well in methylated spirit. This is an 

 excellent and very rapid method. 



(c) Another excellent method of fixing has been devised by Gulland. 

 The fixing solution has the composition — absolute alcohol 25 c.c, pure 

 ether 25 c.c, alcoholic solution of corrosive sublimate (2 grms. in 10 c.c. 

 of alcohol) about 5 drops. The films are placed in this solution for five 

 minutes or longer. They are then washed well in water, and are ready 

 for staining. A contrast stain can be applied at the same time as the 

 fixing solution, by saturating the 25 c.c. of alcohol with eosin before 

 mixing. Thereafter the bacteria, etc., may be stained with methylene- 

 blue or other stain, as described below. This method has the advantage 

 over (a) that, as a small amount of corrosive sublimate is used, less 

 washing is necessary to remove it from the preparation, and deposits are 

 less liable to occur. 



3. Examination of Bacteria in Tissues. — For the examina- 

 tion of bacteria in the tissues, the latter must be fixed and 

 hardened, in preparation for being cut with a microtome. 

 Fixation consists in so treating a tissue that it shall permanently 

 maintain, as far as possible, the condition it was in when re- 

 moved from the body. Hardening consists in giving such a 

 fixed tissue sufficient consistence to enable a thin section of it 

 to be cut. A tissue, after being hardened, may be cut in a 

 freezing microtome {e.g., Cathcart's or one of the newer instru- 



