Alcohol, absolute 20 c.c. Picric acid, saturated aqueous solution 10 c.c. Distilled water 10 c.c. During the progress of steps 1-5 the preparation must be repeatedly examined microscopically with the 1/6-inch objective. When properly differentiated the clubs appear brilliant red on greenish ground. 6. Dehydrate in alcohol. 7. Clear in xylol. 8. Mount in xylol balsam. This method serves equally well for films and for sections. VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES. For bacteriological purposes, sections of tissue are most conveniently prepared by either the ~freezing method~ or the ~paraffin method~. The latter is decidedly preferable, but as it is of greater importance to demonstrate the bacteria, if such are present, than to preserve the tissue elements unaltered, the "frozen" sections are often of value. Whichever method is selected, it is necessary to take small pieces of the tissue for sectioning,--2 to 5 mm. cubes when possible, but in any case not exceeding half a centimetre in thickness. Post-mortem material should be secured as soon after the death of the animal as possible. The tissue is prepared for cutting by-- (a) Fixation; that is, by causing the death of the cellular elements in such a manner that they retain their characteristic shape and form. The fixing fluids in general use are: Absolute alcohol; corrosive sublimate, saturated aqueous solution; corrosive sublimate, Lang's solution (_vide_ page 82); formaldehyde, 4 per cent. aqueous solution. (Of these, Lang's corrosive sublimate solution is decidedly the best all-round "fixative.") (b) Hardening; that is, by rendering the tissue of sufficient consistency to admit of thin slices or "sections" being cut from it. This is effected by passing the tissue successively through alcohols of gradually increasing strength: 30 per cent. alcohol, 50 per cent. alcohol, 75 per cent. alcohol, 90 per cent. alcohol, absolute alcohol. In both these processes a large excess of fluid should always be used. FREEZING METHOD. 1. ~Fixation.~ Place the pieces of tissue in a wide-mouthed glass bottle and fill with absolute alcohol. Allow the tissues to remain therein for twenty-four hours. 2. ~Hardening.~ Remove the alcohol (no longer absolute, as it has taken up water from the tissues) from the bottle and replace it with fresh absolute alcohol. Allow the tissues to remain therein for twenty-four hours. [Illustration: FIG. 71.--Washing tissues.] NOTE.--If not needed for cutting immediately, the hardened tissues can be stored in 75 per cent. alcohol. 3. Remove the alcohol from the tissues by soaking in water from one to two hours. Remove the stopper from the bottle; rest a glass funnel in the open mouth and place under a tap of running water. The water of course, overflows, but the tissues remain in the bottle (Fig. 71).