182 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



4. Flagella. Preparation of Film. 1 (a) Add enough of an eighteen 

 to twenty-four-hour agar culture to a test-tube containing 5 c.c. of 

 sterile salt solution to produce a faint turbidity in the upper half of 

 the solution. 



(6) Incubate at 37 C. for thirty to sixty minutes. 



(c) Place two or three 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. 



Staining Flagella. Pittsfield's Flagella Stain. 2 



Preparation of Stain. 



(a) Mordant: 



Tannic acid, 10 per cent, aqueous solution 10 c.c. 



Mercuric chloride, saturated aqueous solution 5 c.c. 



Alum, saturated aqueous solution 5 c.c. 



Carbol fuchsin 5 c.c. 



(6) The Stain: 



Alum, saturated aqueous solution 10 c.c. 



Carbol fuchsin 5 c.c. or, 



Gentian violet 2 c.c. 



Flood the dried and fixed film with the mordant and steam gently 

 for one minute. Wash in running water, air-dry and flood with the 

 stain. Heat gently two minutes, wash thoroughly in water, air-dry 

 and mount. 



F. Differential Stains for Bacteria. 1. Gram Stain* A most impor- 

 tant differential method of staining bacteria is that of Gram. Bacteria 

 may be divided into two groups: those which retain the initial stain 

 Gram-positive organisms and those which fail to retain the initial 

 stain but color with the counter stain Gram-negative bacteria. 



It was believed formerly that the organisms which retained the 

 initial stain the Gram-positive bacteria contained within their 

 protoplasm, a substance of unknown composition which united 

 chemically with gentian violet (or other pararoseanilin dye) and 

 iodin to form a compound relatively insoluble in alcohol. Gram- 

 negative bacteria did not contain the hypothetical substance, which, 

 in association with the dye and iodin, was insoluble in alcohol. Treat- 

 ment of the latter group with alcohol, therefore, would remove the 



1 Kendall, Jour. Applied Microscopy, 1901, v, 1836. 



2 Medical News, September 7, 1895. 



3 Gram, Fortschr. d. Med., 1894, ii. 



