The Elements of Bacteriological Technique Part 25

8. Dry and mount.

NOTE.--The cultivation from which the films are prepared must be upon blood-serum which has been incubated at 37C.

for from nine to eighteen hours.

The bacilli are stained a light red by the neutral red, which contrasts well with the two or three black spots, situated at the poles and occasionally one in the centre representing protoplasmic aggregations (?

metachromatic granules) stained by the acid methylene-blue.

~Wheal and Chown (Oxford) Method.~--(To demonstrate actinomyces.)

1. Stain briefly with Ehrlich's haematoxylin (until nuclei are faint blue after washing with tap water).

2. Wash in tap water.

3. Stain in hot carbol-fuchsin (as for tubercle bacilli) for five to ten minutes.

4. Wash in tap water.

5. Decolourise with Spengler's picric acid alcohol. This is prepared by mixing:

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 pa.s.sing 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 gla.s.s 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.

[Ill.u.s.tration: 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 gla.s.s 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 gla.s.s 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 gla.s.s 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.