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.

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).

4. Impregnate the tissues with mucilage for twelve to twenty-four hours, according to size. Transfer the pieces of tissue to a bottle containing sterilised gum mixture.

Formula.—

Gum arabic 5 grammes

Saccharose 1 gramme

Boric acid 1 gramme

Water 100 c.c.

5. Place the tissue on the plate of a freezing microtome (Cathcart's is perhaps the best form), cover and surround with fresh gum mixture; freeze with ether, or for preference, carbon dioxide, and cut sections.

6. Float the sections off the knife into a glass dish containing tepid water and allow them to remain therein for about an hour to dissolve out the gum.

(If not required at once, store in 90 per cent. alcohol.)

7. Transfer to a glass capsule containing the selected staining fluid, by means of a section lifter.

8. Transfer the sections in turn to a capsule containing absolute alcohol (to dehydrate) and to one containing xylol or oil of cloves (to clear).

9. Mount in xylol balsam.

Alternative Rapid Method.—

1. Cut very small blocks of the tissue.

2. Fix in formalin 10 per cent. aqueous solution (fixation fluid No. 7, page 82) for 24 hours.

3. Transfer block to plate of freezing microtome and freeze with carbon dioxide vapour.

4. Float the sections off the knife into a glass dish of tepid water.

5. Stain the sections in glass capsules containing selected stains.

6. Place the stained section in a dish of clean water and introduce a glass slide obliquely beneath the section; with a mounted needle draw the section on to the slide and hold it there; gently remove the slide from the water, taking care that any folds in the section are floated out before the slide is finally removed from the water.

7. Drain away as much water as possible from the section. Drop absolute alcohol on to the section from a drop bottle, to dehydrate it.

8. Double a piece of blotting paper and gently press it on the section to dry it.

9. Drop on xylol to clear the section.

10. Place a large drop of xylol balsam on the section and carefully lower a cover-glass on to the balsam.