EXAMINATION OF BACTERIA IN TISSUES 89 



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 spirit. After this treatment the 

 films are stained and treated as if they were sections. 



(b) 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 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 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- 

 ments in which the freezing is accomplished by compressed 

 carbonic acid gas), but far finer results can be obtained by 

 embedding the tissue in solid paraffin and cutting with some of 

 the more delicate microtomes of which, for pathological purposes, 

 the small Cambridge rocker is by far the best. For bacterio- 

 logical purposes embedding in celloidin is not advisable, as the 

 celloidin takes on the aniline dyes which are used for staining 

 bacteria, and is apt thus to spoil the preparation, and besides 

 thinner sections can be obtained by the paraffin method. 



The Fixation and Hardening of Tissues. The following 

 are amongst the best methods for bacteriological purposes : 



(a) Absolute alcohol may be used for the double purpose of fixing and 

 hardening. If the piece of tissue is not more than ^ inch in thickness it 



