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, pass 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., pass 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, pass 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