for want of time, and there is no pursuit in which patience and time for experimenting are more required than in double staining.

The student must not be discouraged by many failures, as there is always some new fact to be learnt and noted, and in the application of these to future experiments some brilliant results are sure to be obtained.

PICRO-CARMINE AND LOGWOOD.

This combination has been used for a long time, and gives very satisfactory results. The student should begin with sections of scalp, skin, or tongue, and the result, if the process be carefully carried out, will be found very satisfactory.

The sections must be first stained in picro-carmine and then in logwood. Make a dilute solution of picro.carmine in distilled water, about 10 drops to the watch-glass, and let the sections remain in it for from twenty minutes to half an hour, then wash in water and place in distilled water acidulated with 1 or 2 drops of acetic or picric acid. Let them remain in this for about an hour. Remove the sections from the acidulated water and place them in dilute logwood stain; this should not be too strong, from 5 to 7 drops to the watch-glass of distilled water; do not let them stain too deeply. When sufficiently coloured, which will be shown by their becoming a faint lilac colour, they must be washed to remove

the excess of logwood, and mounted in the usual

manner.

This double stain is very effective when used with fresh tissues, such as serous membranes; it brings out the connective tissue corpuscles in the mesentery of the newt, and at the same time shows the nonstriped muscle tissue very well. It is also useful in bringing out the delicate tissue in the tubuli seminiferi of the testis and showing the developing spermatozoa there.

In any tissue where there are elements of different kinds, such as scalp or developing bone, it will be found to give very good results. The logwood stain must not be too deep, as it is a very opaque colour.

CARMINE AND INDIGO-CARMINE.

This is a useful double stain and it is especially applicable to sections made from material hardened in chromic acid, as they do not require to be passed through a solution of carbonate of soda, but can be placed in the stain as soon as the mucilage has been washed out of them. In this staining process three solutions are necessary.

1. Borax-carmine solution, page 41.

2. Hydrochloric acid, I part; absolute alcohol, 201 parts.

3. Indigo-carmine solution, page 42.

STAINING PROCESS.

Take a few drops of No. 1 in a watch-glass and immerse the sections, let them remain for two or three minutes, and then remove them to a watchglass containing a small quantity of No. 2.

Let them remain in this until they take on a bright rose colour, which will be in a few seconds, then wash them in methylated spirit to get rid of the acid. They must be washed in several changes of spirit.

When the acid has been thoroughly removed, place the sections in a watch-glass of No. 3 undiluted, and let them remain in it until they show a distinct blue tinge, the proper depth of this staining will be learnt by practice.

When carefully used this process is an admirable one, but there are one or two points that have to be attended to, or the two colours will not be sufficiently differentiated.

If the sections are left too long in the acid mixture, the carmine will be taken out of the edges and these parts will afterwards take on the blue stain too deeply, and so give a result the very opposite of that intended, as the whole value of double staining depends on one colour picking out the whole of one particular tissue throughout the section, and if this is not done the specimen is of no use.

If the carmine stain is only just sufficiently acted on by the acid, so as to change the original dull purple colour to a bright rose, and the edges of the

specimen are not bleached, it will, when put into the indigo-carmine solution, stain evenly throughout.

If the acid solution be too strong, it will have the same effect as a too long immersion in a weaker solution, and a few seconds will bleach the edges. This process will be found very useful in pathological investigation, as the carmine picks out very distinctly all the new growths.

INDIGO-CARMIME AND VESUVIN.

This is a useful double stain for gland tissue, especially for the pancreas.

Make a five per cent. solution of vesuvin in distilled water.

Then

Place the sections in this for ten minutes. wash well in distilled water to get rid of the superfluous colour.

Then place them in a five per cent. of indigo-carmine until they become a deep blue colour. Wash well in distilled water, then in alcohol, and mount in Canada Balsam.

This double stain is very effectual with tissue hardened in chromic acid, and requires no neutralising with carbonate of soda.

* See Quarterly Journal of Microscopical Science, Vol. xxiv., 1884, p. 183.

PICRO-CARMINE AND ANILINE Colours.

Some very good results may be obtained by staining sections first in picro carmine, then letting them remain in acidulated water for an hour, and afterwards staining them with various solutions of aniline colours.

Safranine, after picro-carmine, gives a good double stain, as the picro-carmine colours all the connective tissue and nuclei, while the safranine stains muscle, epithelium, &c.; but the two colours are not sufficiently different to give as good a result as logwood and picro-carmine, although they will be found useful where great transparency is desired.

Picro-carmine and iodine green give a very beautiful effect when it is wanted to isolate gland tissue; such as Peyer's patches, or the glands in the tongue, œsophagus, or solitary glands in the large intestine. The picro-carmine staining everything but the glands, which remain a bright green. When this result is not obtained, methyl- not iodine-green has been used.

Eosin and aniline blue give good results, but require to be used cautiously, as if the staining is too deep the section becomes opaque. To get the best effect, the section should be very thin, and must be well washed after staining with eosin, and then just immersed for a few seconds in the aniline blue.

A great many other combinations will suggest themselves to the student, and he will be amply rewarded by experimenting further with the various staining agents mentioned.