Sodium chloride 0.75 gramme Hydrarg. perchloride 12.00 grammes Acetic acid 5.00 grammes In distilled water 100.00 c.c.

Filter.

6. Gulland's solution, for five minutes. This solution is prepared by mixing:

Absolute alcohol 25.0 c.c.

Ether 25.0 c.c.

Corrosive sublimate, 20 per cent. alcoholic solution 0.4 c.c.

7. Formalin 10 per cent. aqueous solution (= 4 per cent. aqueous solution of formaldehyde since formalin is a 40 per cent. solution of the gas in water).

Either of these methods of fixation coagulates the alb.u.minous material and ensures perfect adhesion of the film to the cover-slip.

_Clearing._--Wash the cover-slip thoroughly in running water and proceed with the staining.

If the film has been prepared from broth, liquefied gelatine, or pus or other morbid exudations, saturate the film after fixation with acetic acid 2 per cent. and allow it to act for two minutes.

Wash with alcohol, then let the alcohol remain on the cover-slip for two minutes. (This will "clear" the groundwork and give a much sharper and cleaner film than would otherwise be obtained.)

If the film has been prepared from blood or bloodstained fluid, treat with acetic acid 2 per cent. for two minutes after fixation. Wash with water, dry, and proceed with the staining. (This will remove the haemoglobin and facilitate examination.)

_Staining._--

1. Rest the cover-slip, film side uppermost, on the rubber mat.

2. By means of a drop-bottle, cover the film side of the cover-slip with the selected stain, allow it to act for a few minutes, then wash off the excess in running water.

The penetrating power of stains is increased by (a) physical means--e. g., heating the stain; (b) chemical means--e. g., by the addition of carbolic acid, 5 per cent. aqueous solution; caustic alkalies, 2 per cent. aqueous solutions; water saturated with aniline oil; borax, 0.5 per cent. aqueous solution.

The most commonly used dyes for cover-slip film preparations are the aniline dyes.

(A) Basic: (a) Methylene-blue.

(b) Gentian violet.

(c) Fuchsin.

These dyes are kept in saturated alcoholic (90 per cent.) solutions so that decomposition may be r.e.t.a.r.ded.

Two or three drops of alcoholic solution of these dyes to, say, 4 c.c.

water, usually makes a sufficiently strong staining fluid for cover-slip film preparations.

Carbolic methylene-blue (C.M.B.) and carbol fuchsin (C.F.) are prepared by covering the cover-slip with 5 per cent. solution of carbolic acid and adding a few drops of the saturated alcoholic solution of methylene-blue or fuchsin respectively to it. For aniline gentian violet (A.G.V.) the stain is added to a saturated solution of aniline oil in water.

(d) Thionine blue.

(e) Bismarck brown.

(f) Neutral red.

(B) Acid: (a) Eosin, aqueous yellowish.

(b) Safranine.

These dyes are kept in 1 per cent. aqueous solution to which is added 5 per cent. of alcohol, as a preservative. They are generally used in this form.

A few nuclear stains (carmine, haematoxylin) are occasionally used more especially in "section" work.

_Decolourisation._--After overstaining, films may be decolourised by washing for a longer or shorter time in one of the following reagents arranged in ascending order of power

1. Water.

2. Chloroform.

3. Acetic acid, 1 per cent.

4. Alcohol.

5. Alcohol absolute, } equal parts.

Acetic acid, 1 per cent., }

{Hydrochloric, 1 per cent. aqueous solution.

{Hydrochloric, 1 per cent. Alcoholic { (90 per cent.) solution.

6. Mineral acids: {Sulphuric, 25 per cent. aqueous solution.

{Nitric, 33 per cent. aqueous solution.

_Counterstaining._--Use colours which will contrast with the first stain; e. g.,

Vesuvin, } Neutral red, }for films stained by methylene-blue or Eosin, }Gram's method.

Fuchsin, }

Methylene-blue, }for films stained by fuchsin.

Gentian violet, }

8. _Mounting._--

1. Wash the film carefully in running water.

2. Blot off the superfluous water with the filter paper, or dry more completely between two folds of blotting paper.

3. Complete the drying in the air, or by holding the cover-slip in the fingers at a safe distance above the flame of the Bunsen burner.

4. Place a drop of xylol balsam on the centre of a clean 3 by 1 gla.s.s slide and invert the cover-slip over the balsam, and lower it carefully to avoid the inclusion of air bubbles.

NOTE.--Xylol is used in preference to chloroform to dissolve Canada balsam, as it does not decolourise the specimen.

~Impression films~ (_Klatschpraeparat_) are prepared from isolated colonies of bacteria in order that their characteristic formation may be examined by higher powers than can be brought to bear on the living cultivation. They are prepared from plate cultivations (_vide_ page 230) in the following manner.

1. Remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the spirit.

2. Open the plate and rest one edge of the cover-slip on the surface of the medium a little to one side of the selected colony. Lower it cautiously over the colony until horizontal. Avoid any lateral movement or the inclusion of bubbles of air.

3. Make gentle vertical pressure on the centre of the cover-slip with the points of the forceps to ensure perfect contact with the colony.

4. Steady one edge of the cover-slip with the forceps and pa.s.s the point of a mounted needle just under the opposite edge and raise the cover-slip carefully; the colony will be adherent to it. When nearly vertical, grasp the cover-slip with the forceps and remove it from the plate. Re-cover the plate.

5. Place the cover-slip, film uppermost, on the rubber mat, and cover it with an inverted watch-gla.s.s until dry.

6. Fix by immersing in one of the fixing fluids previously mentioned (_vide_ page 82).