and dissolve in

Distilled water 350 c.c.

Filter.

This solution will keep active for several days, but fresh solution must be used for each preparation.

METHOD.--

1. Prepare emulsion, make and fix films as above in the preceding method, steps 1 to 4.

2. Pour on the film as much of the fixing solution as the cover-gla.s.s will hold, heat carefully over the flame till steam rises, and allow the steaming fixing fluid to act for five minutes.

3. Wash well in water.

4. Wash in absolute alcohol.

5. Wash in distilled water.

6. Pour some of the sensitising solution on the film and allow it to act for from thirty seconds to one minute; blot off the excess of fluid with filter paper.

7. Without washing, transfer the film to a watch-gla.s.s containing the reducing solution and allow it to remain therein for from thirty seconds to one minute; blot off the excess of fluid with filter paper.

8. Without washing, again treat the film with the sensitising solution, this time until the film commences to turn black.

9. Wash in distilled water.

10. Dry and mount.

~To Stain Nuclei of Yeast Cells.~

1. Prepare and fix film in the usual manner.

2. Soak in ferric ammonia sulphate 3 per cent. aqueous solution for two hours.

3. Wash thoroughly in water.

4. Stain in haematoxylin solution (see page 95) for thirty minutes.

5. Wash in water.

6. Differentiate in ferric ammonia sulphate solution for 1-1/2-2 minutes, examining wet under microscope during the process.

~To Stain Spores.~

~1. Single Stain.~--

1. Prepare cover-slip film in the usual way.

2. In fixing, pa.s.s the cover-slip film fifteen or thirty times through the flame instead of only three. This destroys the resisting power of the spore membrane and allows the stain to reach the interior.

3. Stain in the usual way with methylene-blue or fuchsin.

4. Wash in water.

5. Dry and mount.

~2. Double Stain.~--

1. Prepare and fix film in the usual way--i. e., pa.s.s three times through flame to fix.

2. Cover the film with hot carbol-fuchsin and hold in the forceps above a small flame until the fluid begins to steam. Set the cover-slip down and allow it to cool. Repeat the process when the stain ceases to steam and continue to repeat until the stain has been in contact with the film for twenty minutes. (This stains both spores and bacteria.)

3. Wash in water.

4. Decolourise in alcohol, 2 parts; acetic acid, 1 per cent., 1 part.

(This removes the stain from everything but the spores.)

5. Wash in water.

6. Mount the cover-slip in water and examine microscopically with the 1/6-inch objective. (Spores should be red, and the rest of the film colourless or a very light pink.) If satisfactory, pa.s.s on to section 7; if unsatisfactory, repeat steps 2 to 5.

7. Counterstain in weak methylene-blue. (Now spores red, bacilli blue.)

8. Wash in water.

9. Dry and mount.

The spores of different bacilli differ greatly in their resistance to decolourising reagents; even the spores of the same species of organisms vary according to their age. Young spores are more easily decolourised than those more mature.

Sulphuric acid, 1 per cent. aqueous solution, and hydrochloric acid, 0.5 per cent. alcoholic (90 per cent.) solution, are useful decolourising reagents.

~3. Moeller's Method.~--

1. Prepare and fix films in the usual manner.

2. Immerse in absolute alcohol for two minutes, then in chloroform for two minutes; wash in water. This dissolves out any fat or crystals that might otherwise retain the "spore" stain.

3. Immerse in chromic acid, 5 per cent. aqueous solution, for one minute; wash in water.

4. Pour Ziehl's carbolic fuchsin on the film, warm as in previous methods, and allow it to act for ten minutes.

5. Wash in water.

6. Decolourise in sulphuric acid, 5 per cent. aqueous solution, for five seconds.

7. Wash in water.