APPENDIX 247 



dry, and fix by means of methyl alcohol for five minutes ; then 

 pour off the spirit and allow the films to dry again. Next spread 

 a little fresh blood-serum (obtained from a Wright's pipette in the 

 usual way) in a thin film over the specimen, using the end of 

 a slide as a spreader. When dry, flood the slide (or cover-glass) 

 with Leishman's stain diluted with an equal volume of distilled 

 water. Allow it to act for one hour, wash, blot, and dry. 



IV 



Demonstration of Tubercle Bacilli in Clots from 

 Pleuritic Fluid, etc — Improved Method. 



This method yields better results than the simple digestion of 

 the clot previously referred to. Remove the clot from the fluid 

 and place it on a large filter-paper on a funnel, so that the fluid 

 can drain away. When it has shrunk to a small size remove it 

 and place it in a large volume of water (not necessarily distilled), 

 and keep in gentle movement for a few minutes. Repeat the 

 process two or three times, so as to wash away all haemoglobin, 

 soluble proteid, etc., from the clot. Place the latter in a clean 

 (preferably new) test-tube, add 4 to 5 c.c. of water, a drop of 

 dilute (i to 10) HCl, and a pinch of pepsin, shaking until the 

 latter is dissolved. Keep it in the incubator until the clot is com- 

 pletely dissolved, adding a crystal of thymol to prevent excessive 

 bacterial growth. Then shake the fluid, put it in a clean centri- 

 fugalizing tube, and centrifugalize thoroughly. Prepare films 

 from the exudate and stain in the usual way, taking great care to 

 avoid over-decolorization ; one or two dips in 2I per cent, sul- 

 phuric acid will usually suffice. 



The clot from a pint or more of fluid can be treated in this 

 way ; the more the material taken, the greater the chance of 

 success. 



