All stains require filtering, as decomposition occurs with the formation of granules which become deposited on the specimen ; the violet and red stains are particularly liable to do this.

Many, if not most, stains work better and with more rapidity if some mordant be also added to the solution; among these mordants, carbolic acid, aniline oil, and caustic potash are severally employed. Carbolic is the mordant commonly used; a 5 per cent. watery solution is kept made up and only mixed with the alcoholic solution of dye immediately before use.

In some staining processes decolourising agents are employed to differentiate between certain bacteria, some remaining unaffected, others are decolourised (Gram's method).

FIG. 14.-STAND TO HOLD BOTTLES OF STAINS, &c., FOR LABORATORY BENCH.

Formulæ of Stains.

(1) Löffler's Methylene Blue.

Saturated alcoholic solution methylene blue

30 cc.

Potassium hydrate (1 in 10,000 in distilled water).. 100 cc.

Very little liable to overstain, even when left in contact for a long time. Eosin may be used as a counter-stain.

(i.) Saturated alcoholic solution of gentian violet.

(ii.) Saturated watery aniline oil (aniline water).

The aniline water requires filtering, and should be freshly prepared each time by shaking up 5 cc. aniline oil with 200 cc. distilled water. Immediately before use 1 part of the stain is added to 10 parts of the aniline water.

With both aniline gentian-violet and with carbol-fuchsin, methylated spirit should be used to clear for one minute.

These four stains are of general application, and although by no means all that are used by bacteriologists are sufficient for most purposes. Various special methods will now be described, and the composition of the stains given under the various headings.

Gram's Method.-Solutions:

(1) Aniline gentian-violet.

(2) Iodine, 1 gm. ; pot. iod., 2 gm.; distilled water, 300 cc.

(3) Absolute alcohol or rectified spirit.

(1) Stain the section of tissue or coverslip film for five minutes, preferably by floating on the stain in a watch glass.

(2) Place in iodine solution, after washing off excess of stain, till the colour has changed to purple black; time required one-half to two minutes.

(3) Decolourise in absolute alcohol till no more colour can be extracted.

(4) Wash well in xylol, dry and mount in canada balsam dissolved in xylol.

All bacteria do not stain by this method, and it is therefore used as a means of differentiating certain species.

There are various modifications of the process; two only will be mentioned. (a) Weigert, who uses aniline oil as the decolourising agent after the iodine solution, instead of alcohol. (b) Muir and Ritchie, who use carbolic instead of the aniline water (1), and substituting olive-oil for the xylol (4), afterwards washing in xylol. Contrast stains may be employed with this method, and have the advantage of showing any bacteria present that have decolourised by the Gram process.

Safranin and Bismarck brown, saturated alcoholic solution diluted with an equal bulk of distilled water, give good results.

Ziehl-Neelsen method for acid-fast bacteria (tubercle, leprosy, &c.). The tubercle bacillus does not stain well with the ordinary methods

adopted for bacteria generally, but requires an energetic stain plus the application of heat.

Solutions :

(1) Carbol-fuchsin stain.

(2) 25 per cent. of pure sulphuric acid.

(3) Alcohol, absolute or rectified.

(4) Carbol-methylene blue stain.

Method:

(1) Stain the films for five or ten minutes in diluted carbolfuchsin (1 in 3 water) kept hot over a water bath.

steam but not boil.

The stain should

(2) Decolourise with rectified spirit until no more colour is extracted, and wash in water.

(3) Plunge into the sulphuric acid solution and wash in water; repeat the process if there is more than a faint pink tinge on washing.

(4) Stain for half a minute in diluted carbol-methylene blue. Wash, dry, mount. The tubercle bacilli are stained a bright red, the background of epithelial cells and other bacteria, blue.

Spore Staining.—Solutions:

(1) Carbol-fuchsin.

(2) 5 per cent. sulphuric acid.

Method I.

(1) Stain the film as for tubercle bacilli.

(2) Decolourise rapidly in 5 per cent. sulphuric acid and wash. (3) Counterstain with carbol-methylene blue.

(4) Wash, dry, and mount.

The spores are stained a bright red, the bacilli blue.

Möller's method of spore-staining is of considerable advantage in

some cases:

Method II,

(1) Chloroform two minutes.

(2) 5

per cent. chromic acid two minutes.

The remaining steps are similar to Method I.

In all the above preparations the film is prepared as directed on page 41. To simply demonstrate the presence of spores without attempting to stain the organisms the flaming is increased. This makes the spore envelope more permeable to the stain, although the

bacilli themselves are broken up and easily decolourise in the acid solution.

Method III.

(1) Pass coverslip fifteen times through flame. Proceed as in I., but do not counter-stain, as the bacilli are destroyed by the heating.

Capsule Staining.-Some organisms, as the pneumococcus, are possessed of gelatinous capsules, which may be stained by special processes.

Method I.

Solutions: Glacial acetic acid; aniline gentian-violet; sodium chloride cent.

per

(1) Immerse in acetic acid while film is wet for three seconds. (2) Wash off acid with aniline gentian violet.

(3) Wash in 2 per cent. sodium chloride.

(4) Examine in sodium chloride solution.

FIG. 15.-BOSTON'S FORCEPS FOR HOLDING COVERSLIPS DURING STAINING.

FIG. 16.-CORNET'S FORCEPS FOR HOLDING COVERSLIPS DURING STAINING.

MacConkey's: Method II.

Stain Dahlia

Methyl-green (00 crystals)

Sat. alcoholic fuchsin

Distilled water

(1) Prepare film in ordinary way.

1.5 gm.

0.5 gm.

10.0 cc.

200 cc.

(2) Flood coverslip with stain holding it in spring forceps (Cornet's).

(3) Heat till steam is given off, and allow to remain five minutes. (4) Wash, dry, and mount.

The cocci are stained a deep violet, the capsules a faint violet.

Flagella. Flagella staining is one of the most difficult of all bacteriological operations, requiring a good deal of practice and not a little patience. The flagella are invisible under ordinary circumstances, and it is probable that in the process of staining they become swollen and so come into view. Another idea is that the stain is deposited upon and not in the flagella, as is the case in the ordinary staining of bacteria. It is most difficult to get both flagella and bacilli stained and the beginner must be prepared for a good many failures before a successful preparation is obtained.

In staining flagella the greatest care must be exercised that the coverglasses used are perfectly clean, that the films are not too thick, that the organisms are well separated in the emulsion used, that the flagella are not destroyed in flaming, and that the stains used do not precipitate. A young agar culture should be used, and a small quantity of the growth removed with the platinum needle and emulsified with distilled water in a clean watch glass. A drop of this emulsion is carefully spread over the surface of the clean coverslip and allowed to dry, and then passed once or twice through the flame. Three methods are described, although many others have been suggested from time to time. It will be seen that a mordant is used in all cases.

Pittfield's Flagella Stain.

Solutions: A. Saturated alcoholic gentian-violet

10 per cent. solution of potash alum.. 10 parts. B. 10 per cent. solution of tannic acid

Mix equal parts of A and B immediately before use.

Method: Flood the coverslip with the mixture and warm over bunsen flame till steam is given off, but do not boil. Allow the stain to remain on for five minutes; wash off, dry, and mount.