1. Prepare and fix films in the usual way.

2. Pour stain I on the film and allow it to act for thirty seconds.

3. Wash thoroughly in water.

4. Pour stain II on to the film and allow it to act for thirty seconds.

5. Wash thoroughly in water.

6. Dry and mount.

NOTE. The cultivation from which the films are prepared must be upon blood-serum which has been incubated at 37° C. for from nine to eighteen hours.

The bacilli are stained a light brown by the vesuvin, which contrasts well with the two or three black spots, situated at the poles and occasionally one in the centre representing protoplasmic aggregations (? meta-chromatic granules) stained by the acid methylene-blue.

To Demonstrate the (?) Syphilis Bacillus (Lustgarten's Method).

1. Prepare and fix the film in the usual manner. 2. Stain in aniline gentian violet or aniline fuchsin, twentyfour hours.

3. Wash in water.

4. Place in 1.5 per cent. solution of permanganate of

potash (a brown precipitate of oxide of manganese forms), three to four seconds.

5. Wash in watery solution of pure SO,. If not decolourised, repeat sections 4 and 5 until satisfactory.

6. Wash in water, dry, and mount.

Or

1. Prepare and fix the film in the usual manner.

2. Stain in hot carbol-fuchsin.

3. Wash in water to which has been added 2 or 3 drops of chloride of lime.

4. Decolourise in concentrated solution of chloride of iron. 5. Wash thoroughly in water.

6. Dry and mount.

Syphilis bacilli remain red.

NOTE. These methods are equally applicable to tissue sections.

VI. METHODS OF DEMONSTRATING

BACTERIA IN TISSUES.

FOR bacteriological purposes, sections of tissues 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,―0.5 c.c. cubes when possible,-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 76); formaldehyde, 1 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, rectified 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 widemouthed 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 50 per cent. alco

hol.

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. 53. Washing tissues.

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.

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, 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 50 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).

Alternative Method.

Sa. 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.

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

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

8d. Drop on xylol to clear the section.

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

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.