Paraffin Method

1. Fixation. Place the pieces of tissue, resting on cotton-wool, in a wide-mouthed glass bottle. Pour on a sufficient quantity of the corrosive sublimate fixing fluid; allow the tissue to remain therein for twelve to twenty-four hours according to size.

2. Pour off the fixing fluid and wash thoroughly in running water for twenty minutes to half an hour to remove the excess of corrosive sublimate.

3. Hardening. Place the tissues in each of the following strengths of alcohol in turn for from twelve to twenty-four hours: 50 per cent., 75 per cent., 90 per cent., absolute.

4. Dehydration is effected by transferring the tissues to fresh absolute alcohol.

5. Clearing. Half fill a wide-mouthed bottle with chloroform. On the surface of the chloroform float a layer of absolute alcohol about five to ten millimetres in depth. Place the pieces of tissue in the layer of alcohol and when they have sunk through this layer, transfer them to pure chloroform for from six to twenty-four hours according to the size of the pieces. When "cleared," the tissue becomes more or less transparent.

6. Infiltration. Place the cleared tissues in fresh chloroform with several pieces of paraffin wax and stand in a warm place, such as on the top of the warm incubator. The warmth gradually melts the paraffin and the tissues should remain in the mixture about twenty-four hours.

7. Transfer the tissues to a vessel containing pure melted paraffin. Place this vessel in a paraffin water-bath regulated for 2° C. above the melting-point of the paraffin used, and allow the tissues to soak for some four to six hours to ensure complete impregnation. The paraffin used should have a melting-point of not more than 58° C. For all ordinary purposes 54°C. will be found quite high enough.

8. Imbed in fresh paraffin in a metal (or paper) mould.

(a) Arrange a pair of L-shaped pieces of metal on a plate of glass to form a rectangular trough (Fig. 72).

(b) Pour fresh melted paraffin into the mould from a special vessel (Fig. 73).

(d) Blow gently on the surface of the paraffin in the mould, and as soon as a film of solid paraffin has formed, carefully lift the glass plate on which the mould is set and lower plate and mould together into a basin of cold water.

(e) When the block is cold, break off the metal L's; trim off the excess of paraffin from around the tissue with a knife, taking care to retain the rectangular shape, and store the block in a pill-box.

When several pieces of tissue have to be imbedded at one time, shapes of stout copper, 10 cm., 5 cm., and 2.5 cm. square respectively, and 0.75 cm. deep (Fig. 74) will be found extremely useful. These placed upon plates of glass replace the pair of L's in the above process. When the paraffin has set firmly the screw a should be loosened to allow the two halves of the flange b to separate slightly—this facilitates removal of the paraffin block.

8. Cement the block on the carrier of a "paraffin" microtome (the Minot, the Jung, or the Cambridge Rocker) with a little melted paraffin. Greater security is obtained if the paraffin around the base of the block is melted by means of a hot metal or glass rod.

9. Cut sections—thin, and if possible in ribbands.

Mounting Paraffin Sections.—

1. Place a large drop of 30 per cent. alcohol on the centre of a slide (or cover-slip) and float the section on to the surface of the drop, from a section lifter.

2. Hold the slide in the fingers of one hand and warm cautiously over the flame of a Bunsen burner, touching the under surface of the glass from time to time on the back of the other hand. As soon as the slide feels distinctly warm to the skin, the paraffin section will flatten out and all wrinkles disappear.

(The slide with the section floating on it may be rested on the top of the paraffin bath for two or three minutes, instead of warming over the flame as here described.)

3. Cautiously tilt up the slide and blot off the excess of spirit with blotting paper, leaving the section attached to the centre of the slide.

4. Place the slide in a wire rack (Fig. 75), section downward, in the "hot" incubator for twelve to twenty-four hours. At the end of this time the section is firmly adherent to the glass, and is treated during the subsequent steps as a "fixed" cover-glass film preparation.

Note.—If large, thick sections have to be manipulated, or if time is of importance or acids are used during the staining process, it is often advisable to add a trace of Mayer's albumin to the alcohol before floating out the section. If this substance is employed, a sojourn of twenty minutes to half an hour in the "hot" incubator will be found ample to ensure firm adhesion of the section to the slide. The albuminous fluid is prepared as follows:

Mayer's Albumin.—

Weigh out

Salicylate of soda 1 gramme

and dissolve in

Glycerine 50 c.c.

Add

White of egg 50 c.c.

Mix thoroughly by means of an egg whisk.

Filter into a clean bottle.

As an alternative method paint a thin layer of Schallibaum's solution on the slide with a camel's hair pencil; lay the section carefully on this film and heat gently to fix the section.

Schallibaum's solution:

Clove oil 30 c.c.

Collodion 10 c.c.

Keep in a dark blue bottle in a cool place.

Staining Paraffin Sections.—

1. Warm paraffin section over the Bunsen flame to soften (but not to melt) the paraffin, then dissolve out the wax with xylol poured on from a drop bottle.

2. Remove xylol by flushing the section with alcohol.

3. If the tissue was originally "fixed" in a corrosive sublimate solution, the section must now be treated with Lugol's iodine solution for two minutes and subsequently immersed in 90 per cent. alcohol to remove all traces of yellow staining.

4. Wash in water.

5. Stain deeply, if using a single stain, as the subsequent processes decolourise.

6. Wash in water, decolourise if necessary.

7. Flood with several changes of absolute alcohol to dehydrate the section.

8. Clear in xylol. (Oil of cloves is not usually employed, as it decolourises the section.)

9. Mount in xylol balsam.