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. Grain's Iodine solution. 



Iodine, 



Potassium iodide, 

 Distilled water, 



7. Gabbett's Methylen blue solution. 



Methylen blue (dry), 

 Sulphuric acid, 

 Distilled water, 



8. Alcohol, 96%. 



REFERENCES. A. 156; H. 75; M. & W. 245; M. & R. 

 103; McF. 90; P. 200. 



SPECIAL DIRECTIONS. 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. 



1 gm. 



2 gm. 

 300 cc. 



2 gms. 

 25 cc. 

 75 cc. 



Block for stain bottles . 



GENERAL DIRECTIONS. Bacteria may be studied under the microscope in a living 

 condition in a hanging drop preparation (XIX) ; but on 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 platinum loop. 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. If the drop is sufficiently small this 

 will be a short 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. 



The preparation is now ready for microscopical examination. (For directions see 

 XVIII). 



