METHODS FOR MICROSCOPIC STUDY OF BACTERIA 201 
3. Polar Bodies. — Albert Stain. ^ 
Solution A Albert Stain^ Solution B {Gram Iodine solution). 
Toluidin blue .... O.lSgm. Iodine 2 gm. 
Malachite green 0.20 gm. Potassium iodide 3 gm. 
Glacial acetic acid . 1.00 cc. Distilled water . 300 cc. 
Alcohol, 95 per cent . 2.00 cc. 
Distilled water . . . 100.00 cc. 
(a) Make a thin smear, fix with gentle heat. 
(b) Flood with solution A. Stain five minutes. 
(c) Drain ofi" stain; wash rapidly with solution B. to prevent precipi- 
tate forming. 
(d) Flood with solution B, allow to act for one minute. 
(e) Wash with water. 
(/) Blot dry, examine. 
Granules are black, bodies of bacteria are light green, the barred 
forms of diphtheria bacilli are colored dark green with light green 
interspaces. 
4. Flagella. — Preparation of Film. ^— (a) Add enough bacteria from 
an eighteen to twenty-four-hour agar culture to a test-tube containing 
5 cc. of sterile salt solution to produce a faint turbidity in the upper half 
of the solution.^ 
(b) Incubate at 37° C. for thirty to sixty minutes. 
(c) Place 2 or 3 loopfuls of the suspension upon a perfectly clean 
cover-glass and allow to dry spontaneously in the air or in the incubator. 
Do not attempt to spread the films with the platinum loop; agita- 
tion breaks off flagella. 
Flagella Stain. — Plimmer and Paine.'^—The Mordant.— 
Tannic acid 10.0 gm. 
Aluminum chloride (hydrated) 18.0 gm. 
Zinc chloride 10.0 gm. 
Roseanilin hydrochloride 1.5 gm. 
Alcohol, 60 per cent 40 . cc. 
The several ingredients, immediately after weighing, are triturated 
with the alcohol: at first with 10 to 15 cc. until a fine suspension is 
obtained, then with the remainder. 
To use the stain : 
1. Absolutely clean slide should be used. 
2. The culture should be eighteen-to twenty-four hours old. The 
growth is carefully removed with a large loop, and stirred into water to 
give a faintly turbid suspension. 
3. This is allowed to settle. 
1 Jour. Am. Med. Assn., 1920, 74, 28; 1921, 76, 240; also Laybourn: Ibid., 1924, 
83, 121. 
■ Kendall: Jour. Applied Microscopy, 1901, 5, 1836. 
3 An excellent discussion of dark-field illumination as a means of demonstrating 
flagella is by K. Reichert: Centralbl. f. Bakteriol., orig., 1909, 51, 14. 
* Jour. Pathol, and Bacteriol., 1921, 24, 286. 
