Unna's alkaline solution of methylene-blue is used. The advantages of this solution are that it stains readily tissues. hardened in Zenker's fluid and brings out nuclei and nuclear figures with great sharpness, while at the same time it stains the protoplasm of certain cells so that they are easily distinguished from other cells. The Heidenhain-Biondi triple stain is useful after fixation in corrosive sublimate, but cannot be employed with celloidin sections, so that its field is limited. The other aniline dyes are used on occasion or for some definite purpose, but not so generally as those mentioned above.

Alum-hematoxylin Stains.-Most alum-hematoxylin solutions will over-stain if the sections are left too long in them. The proper time required depends on the fixing reagent used and on the degree of ripeness of the staining solution. It is therefore advisable to wash a section in water occasionally and decide from the color it has acquired if it be sufficiently stained, or to mount it in water on a slide and examine with the low power of the microscope.

The best results are obtained with alum-hematoxylin solutions by staining sections just deeply enough, washing them thoroughly in several changes of water, and leaving them in a large dish of water over night. This thorough washing is done to rid the tissues of every trace of alum or of acid, so that the color will become a clear blue and will keep indefinitely.

Many microscopists prefer to stain deeply and diffusely in an old, quickly-staining alum-hematoxylin solution, and then to employ a decolorizer. The agents most used for the purpose are alum (1 per cent. aqueous solution for one to two hours), hydrochloric acid (to per cent. aqueous solution, or even the ordinary acid alcohol), and acetic acid (1 to 3 per cent. solution) for a few seconds only. After being sufficiently decolorized the sections must be thoroughly washed in water, preferably for a number of hours, otherwise the stain will fade. The objection to this method is that a pure nuclear stain only is obtained, because the acid removes the color completely from all the rest of the tissue. Under cer

tain circumstances, as when hematoxylin is used as a contrast-stain to fuchsin in staining for tubercle bacilli, such a sharp limitation to the nuclei is desirable.

Alum-hematoxylin stains well and quickly tissues hardened in alcohol, in corrosive sublimate, and in picric acid. It stains much more slowly tissues hardened in solutions. containing chrome salts, such as Zenker's and Müller's fluids.

For counter-staining eosin will usually be found to give the most beautiful contrast, although picric acid, Van Gieson's mixture, and neutral carmine are often of service.

A good alum-hematoxylin solution should have a bluish or purplish color, and should stain celloidin very faintly or not at all.

Aqueous Alum-hematoxylin; Delafield's Hematoxylin; Ehrlich's Acid Hematoxylin (see pages 241 and 242).

1. Stain in one of the above solutions two, five, or thirty minutes, or sometimes even longer.

2. Wash in several changes of water, and then leave sections, if possible, for several hours or over night in a large dish of water.

3. Contrast-stain, usually an aqueous solution of eosin, toper cent., for one to five minutes.

4. Alcohol, 95 per cent., two or three changes to dehydrate and to remove excess of contrast-stain.

5. Clear in oleum origani cretici or in Dunham's oils-ofcloves-and-thyme mixture.

6. Canada balsam.

The staining of the nuclei by Ehlrich's alum-hematoxylin solution is not so sharp as that obtained by the simple aqueous solution.

The more customary method of using Delafield's alumhematoxylin solution is to filter a few drops of it into a dish of water and to stain sections for a long time, even over night, with the very dilute solution thus obtained. It is sometimes advisable to use the aqueous solution in the same way.

Mayer's Hemalum (see page 242).-1. Stain three to five minutes or longer.

2. Wash out in 1 per cent. alum solution until the stain is precise.

3. Wash thoroughly in several changes of water.

4. Alcohol, 95 per cent.

5. Oleum origani cretici.

6. Canada balsam.

The staining is rather diffuse, so that it has to be washed out to some extent with alum-water. Mayer's acid hemalum is more precise, and usually does not need to be decolorized, so that the second step can be omitted.

Hemalum is used for staining tissues in bulk. Twentyfour hours are required for large pieces.

Heidenhain's Hematoxylin Stain.-1. Stain twentyfour to forty-eight hours in a simple per cent. aqueous solution of hematoxylin dissolved by the aid of heat.

2. Transfer the sections directly to a per cent, aqueous solution of simple chromate of potassium for twenty-four to forty-eight hours, changing the solution frequently until no more color is given off by the sections.

3. Wash thoroughly in water.

4. Alcohol.

5. Oil.

6. Canada balsam.

Carmine Stains.-The ordinary carmine solutions give good nuclear stains, but of the finer details in a specimen they bring out much less than a direct alum-hematoxylin stain. They are much less used now than formerly, except as contrast-stains to bacteria and to fibrin in the methods of Gram and Weigert, for which purpose lithium carmine will usually give the best results.

Alum Carmine; Alum Cochineal (see page 244).—1. Water.

2. Stain in either of the above solutions for five to twenty minutes.

3. Wash thoroughly in water. 4. Alcohol, 95 per cent.

5. Oleum origani cretici.

6. Canada balsam.

Over-staining does not occur. The solutions cannot be recommended for tissues which stain with difficulty. When used for staining in bulk, twenty-four to forty-eight hours are required.

Lithium Carmine (see page 244).-I. Water.

2. Stain two to five minutes.

3. Transfer directly to acid alcohol, one or more changes for several minutes or more, until the sections are well differentiated.

4. Wash in water.

5. Alcohol, 95 per cent.

6. Oleum origani cretici.

7. Canada balsam.

This method gives an intense and permanent bright-red nuclear stain. Over-staining is impossible. A trace of picric acid added to the alcohol used for dehydration affords a beautiful contrast-stain.

Aniline Dyes as Nuclear Stains.-Any of the basic aniline dyes may be used as nuclear stains after the following general method:

1. Stain celloidin sections in a strong solution of the dye preferred in water or in dilute alcohol for five to thirty minutes.

2. Wash in water.

3. Dehydrate in 95 per cent. alcohol.

4. Clear in oil of bergamot or origanum.

5. Wash out the oil thoroughly with several changes of xylol.

6. Xylol balsam.

With paraffin sections use absolute alcohol and clear directly in xylol.

As a matter of fact, however, certain dyes and certain solutions are generally used in preference to the others. The use of aniline dyes as stains presents for solution one or two problems. Most of the colors are more or less affected by all clearing reagents except xylol. With paraffin sections

and those from which the celloidin has been removed it is very easy to dehydrate in absolute alcohol and to clear in xylol. With celloidin sections, however, this is impossible, because the absolute alcohol will dissolve out the celloidin, For celloidin sections it

and this is usually not desirable. will therefore generally be found advisable to dehydrate in 95 per cent. alcohol, to clear and further dehydrate in bergamot or origanum oil (the decolorizing effect of these oils. is slight and slow), to wash out the oil with xylol, and then to mount in xylol balsam.

In washing out the excess of color it is sometimes found advantageous to acidulate very slightly either the water or the first alcohol with acetic or hydrochloric acid. This process, if not carried too far, tends to make the nuclear stain sharper.

Safranin is one of the very best nuclear-staining aniline dyes. Tissues may be hardened in alcohol, corrosive sublimate, Flemming's, Hermann's, or Zenker's fluids. Any one of the solutions of safranin given on page 248 may be used.

1. Stain paraffin sections two to five minutes to twentyfour hours according to the staining solution and fixing reagent used.

2. Wash in water.

3. Absolute alcohol, several changes, until the section appears properly differentiated.

4. Xylol.

5. Xylol balsam.

For celloidin sections dehydrate in 95 per cent. alcohol, clear in bergamot or origanum oil, and wash out in xylol. To render the stain more precise a few drops of acid alcohol are sometimes added to the first alcohol.

Eosin and Methylene-blue.-Harden in alcohol, Zenker's fluid, or corrosive sublimate.

1. Stain paraffin sections in a 5 per cent. or saturated aqueous solution of eosin for five to twenty minutes or longer.

2. Wash in water to get rid of excess of eosin.