EXAMINATION OF BACTERIA IN TISSUES. 91 



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 down- 

 wards 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 spirit. After this treatment the films are stained and treated as 

 if they were sections. 



() Formol-alcohol formalin i 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 grm. 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 satu- 

 rating 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 (<z) 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, but far finer results can be obtained by 



