METHOD.

I.

Prepare the sections for staining, secundum artem.

2. Stain sections in the warm (e. g., in the hot incubator) for twenty-four hours.

3. Wash with water.

4. Decolourise lightly with acetic acid, 1 per cent. 5. Dehydrate rapidly with absolute alcohol.

6. Clear with xylol.

7. Mount in xylol balsam.

To Demonstrate Acid-fast Bacilli.

1. Prepare the sections for staining in the usual

way.

2. Stain with hæmatin solution ten to twenty seconds, to obtain a pure nuclear stain; then wash in water. 3. Stain with carbolic fuchsin twenty to thirty minutes at 47° C.; then wash in water.

4. Treat with aniline hydrochlorate, 2 per cent. aqueous solution, for two to five seconds.

5. Decolourise in 75 per cent. alcohol till section appears free from stain-fifteen to thirty minutes. 6. Dehydrate with absolute alcohol.

7. Clear very rapidly with xylol.

8. Mount in xylol balsam.

VII. CLASSIFICATION OF FUNGI.

FUNGI are divided into:

1. Hymenomycetes (including the mushrooms, etc.). 2. Hyphomycetes (moulds).

3. Blastomycetes (yeasts and torule).

4. Schizomycetes (bacteria).

NOTE.-Formerly myxomycetes were included in the fungi; they are now recognised as belonging to the animal kingdom, and are termed "mycetozoa."

MORPHOLOGY OF THE HYPHOMYCETES.

At the commencement of his studies, the attention of the student is directed to the various non-pathogenic moulds and yeasts, not only that he may gain the necessary technique whilst handling cultivations of harmless organisms, but also because these very species are amongst the commonest of those that may accidentally contaminate his future preparations.

The hyphomycetes are composed of a mycelium of short jointed rods or "hyphæ" springing from an axis or germinal tube which develops from the spore. Hypha are—

(a) Nutritive or submerged.

(b) Reproductive or aerial.

The protoplasm of these cells contains granules, pigment, oil globules, and sometimes crystals of calcium oxalate.

Reproduction. Apical spore formation-asexual; zoospores-sexual. Mucorinæ.-Mucor (Fig. 59).-Note the branching filaments-"mycelium" (a), “hyphæ” (b). Note the asexual reproduction.

1. A filament grows upwards. At its apex a septum forms, then a globular swelling appears-"sporagium" (d). This possesses a definite membrane.

2. From the septum grows a club-shaped mass of protoplasm-" columella" (c).

3. The rest of the contained protoplasm breaks up into "swarm spores" (e).

Finally the membrane ruptures and spores escape. Perisporaceæ.-Aspergillus (Fig. 60).-Note the branching filaments-"mycelium" (a).

Fig. 61.-Penicillium.

Note the asexual

reproduction.

1. A filament (b) grows upwards, its termination becomes clubbed; on the clubbed extremity flask-shaped cells appear"sterigmata"

(c).

2. At free end of each sterigma is formed an oval body-a spore or "gonidium" (d), which, when ripe, is thrown off from the sterigma.

Penicillium (Fig. 61).—Note the branching filaments -"mycelium" (a) (frequently containing globules). Note the asexual reproduction.

1. A filament grows upwards-" goniodophore” (b)— and its apex divides up into several branches"basidia "(c).

2. At the apex of each branch a flask-shaped cell, "sterigma" (d), appears.

3. At the apex of each sterigma appears a row of oval cells-" spores" or "conidia" (e). These, when ripe, are cast off from the sterigmata.

Ascomycetæ.-Oïdium (Fig. 62).-(This family is perhaps as nearly re

segments, "oïdia," and behave as spores.

Note the asexual reproduction. From the pseudomycelium arise true hyphæ (b), each of which in turn ends in a chain of spores (c).

MORPHOLOGY OF THE BLASTOMYCETES.

The blastomycetes are composed of spherical or oval cells (8 to 9.5 μ in diameter), which, when rapidly multiplying by budding, may form a spurious mycelium. A thin cell-wall encloses the granular protoplasm, in which vacuoles and sometimes a nucleus may be noted. This latter is best seen when stained with osmic acid or hæmatoxylin.

During their growth and multiplication the blasto

mycetes split up solutions containing sugar into alcohol

and CO2.

Reproduction.-Budding, ascospores-asexual.

(Torulæ, whilst resembling yeasts in almost every other respect, never form spores.)

Saccharomyces (Fig. 63).-Note the round or oval cells of granular protoplasm (a) containing solid particles and vacuoles (c), and surrounded by a definite envelope.

Note the asexual reproduction.

1. "Gemmation"-that is, the budding out of daughter cells (b) from various parts of the gradually enlarging mother cell. These are eventually cast off

and in turn become mother cells and form fresh groups of buds.

2. Spore formation-"ascospores" (e). These are formed at definite temperatures and within well-defined periods; e. g., Saccharomyces cerevisiæ, thirty hours at 25° to 37° C., or ten days at 12° C.

Torulæ (Fig. 64).-Note the points in morphology, etc., as above.

Note the absence of ascospore formation.