216 LABORATORY EXERCISES IN BACTERIOLOGY. 



of gentian violet and warm until it steams. Wash well in water; moisten 

 with two per cent, acetic acid solution for ten seconds; wash in water; 

 cover, and examine in water with one-seventh inch dry lens. If not suc- 

 cessful, remove cover and repeat. If pneumonic sputum be not on hand, 

 use tubercular sputum containing Micrococcus tetragenus, which also pre- 

 sents a well-marked capsule. 



Exercise 50. Staining Flagella (Lowit). Have five slides perfectly 

 free from fat (first washing with soap and water, rinsing in water, then in 

 alcohol and ether, drying with paper; fluid should spread evenly and re- 

 main in a thin film, not in drops, if slides be clean). Use young cultures, 

 twenty to twenty-four hours old, on agar, of the following organisms: 

 Planococcus agilis, Pseudomonas pyocyanea, Bacillus typhosus, Bacillus coli 

 and Microspira comma, for the preparation of films. On each slide place 

 three small drops of distilled water. With usual precautions take from the 

 first culture a bit of the surface of the growth, and with a single, circular, 

 sweeping movement diffuse some of the bacteria in the first drop of the 

 water on one of the prepared slides. Sterilize needle in flame. Take a 

 little of this first drop and in a similar manner diffuse in the second. Flame 

 needle. Repeat diffusion from second to third drop. Dry all three drops 

 in the air and fix films by passing slide through the flame only once or twice, 

 taking care not to overheat. In the same manner prepare and fix films 

 from each of the remaining cultures on the other slides, and in turn stain 

 each preparation as follows : Apply the following freshly prepared mordant, 

 allowing it to act for two or three minutes, cold. 



Tan.nic acid, 5 



Distilled water 20 



Dissolve and filter twice. To ten cubic centimeters of the solution add five cubic centi- 

 meters of a saturated aqueous solution of copper sulphate, well filtered, and one cubic centi- 

 meter of a saturated alcoholic solution of fuchsin. Filter the mixture twice. 



After action of this mordant, wash preparation well in water; there- 

 after stain with aniline- water-gentian-violet for five minutes, cold. Wash 

 well in water and for one or two seconds in fifty per cent, alcohol. Dry and 

 examine as usual. If unsuccessful, repeat. Note the different types of 

 flagellation presented by these organisms. Does Bacillus typhosus or Ba- 

 cillus coli exhibit the greater number of peritrichous flagella ? 



4. Size of Bacteria. Individual bacteria of the same kind are fairly uniform in 

 size, a feature of some importance in identification. One must recognize, however, a 

 certain variation as occurring in case of the same microorganism, particularly when 

 grown under different conditions and at the beginning of growth as well as in old 

 colonies. The statement of size must, therefore, refer to average individuals, or must 



