20 General Bacteriology. 
This stain should stand 24 hours and then be filtered. It does not keep well and must 
not be used when more than 14 days old. 
6. Gram’s Iodine solution. 
Todine, - - - - - - - - 1 gm. 
Potassium iodide, - - - - - - 2 gm. 
Distilled water, - - - - - - - 300 ec 
7. Gabbett’s Methylen blue solution. 
Methylen blue (dry), - - - - - 2 gms. 
Sulphuric acid, - - - - - - - 25 ec. 
Distilled water, - - - - - - 75 ee. 
8. Alcohol, 96%. 
REFERENCES. A. 156; H.75; M. & W. 245; M.&R. 
103; McF. 90; P. 200. 
SpEecIaL DrrEcTIONS. Prepare the solutions of dyes 
from the saturated alcoholic solutions (furnished) and 
place them in 2 oz. bottles arranged with pipettes and neatly 
labeled. ‘The bottles are conveniently kept in a block. 
Fig. 8. 
EXERCISE XVII. SIMPLE COVER-GLASS PREPARATION. 
Fic. 8. Block for stain bottles. 
GENERAL Directions. Bacteria may be studied under the microscope in a living 
condition in a hanging drop preparation (XIX); buton account of their hyaline charac- 
ter, which makes the examination difficult, the student should first learn to stain them 
and later make the hanging drop preparation. With a few exceptions all bacteria can 
be stained by the following process: <A small drop of distilled water is placed on a clean 
cover-glass by means of the platinumloop. With a sterile needle a portion of the material 
to be examined is secured and while the cover-glass is held in the fingers of the left hand 
the bacteria on the needle are introduced into the water, thoroughly mixed and then 
spread in a thin film over as much of the surface of the cover-glass as possible. When 
the bacteria are taken from fluid media a drop of water will not be necessary. In this 
case use a loop. The film is now allowed to dry. Ifthe drop is sufficiently small this 
will be ashort process. It may be hastened by holding the cover-glass high over the 
flame, but it should always be held in the hand to prevent over-heating, which spoils the 
preparation. 
When the film is thoroughly dry place the cover-glass in a pair of Cornet or Stewart 
forceps and ‘‘fix’’ the bacteria in the flame. This is done by passing the preparation 
through the upper portion of a gas flame, film side up. Three passages should be made, 
each consuming about one second of time. The forceps are now placed on the table and 
the film flooded with one of the anilin dyes. After the stain has acted for five or ten 
minutes it is washed off into a waste dish with a stream of distilled water, and while 
the cover-glass is still wet it is placed, bacteria side down, on a clean glass slide, being 
careful to avoid air bubbles. The surplus water is then taken up by means of a small 
piece of blotting or filter paper. 
a preparation is now ready for microscopical examination. (For directions see 
XVIII). 
