60 GENERAL BACTERIOLOGY 



objective. The Petri dishes can be inverted, and thus avoid the 

 danger of exposing the culture to contamination from the air except 

 with gelatin where liquefying organisms are present. Observe, a. 

 structure of colony as a whole ; &. character of margin. (See Chap- 

 ter III.) 



SPECIAL DIRECTIONS. Study, write descriptions and make draw- 

 ings of all plate cultures. Use blank pages for description and 

 sketch of cultures. 



EXERCISE 34. USE OF DECOLORIZING AGENTS. 



Make three cover-glass preparations from a 24 hour old culture 

 of B. subtilis, staining them with an aqueous solution of gentian 

 violet. Mount in water and examine. While they are still under 

 the microscope, place at one side of the cover-glass a few drops of 

 one of the following solutions, and by means of a strip of filter paper 

 at the opposite side draw the liquid under the cover glass until all 

 the color is removed. In this way determine the relative value of 

 alcohol (95%), acetic acid (5%), and nitric acid (30%) as decolor- 

 izing agents. 



EXERCISE 35. GRAM'S STAIN. 



EXPLANATORY. This is a differential stain and one of the most 

 useful. Some bacteria when stained by this method exhibit a dark 

 violet color, others remain perfectly colorless, thus rendering pos- 

 sible the differentiation of bacteria which are morphologically nearly 

 or quite identical, and also greatly facilitating the demonstration of 

 certain bacteria in animal tissue. Most of the pathogenic micrococci 

 retain the violet stain, although there are important exceptions. 

 The bacilli and spirilla may or may not remain colored. 



GENERAL DIRECTIONS. 



a. Spread film. 



b. Air dry and fix. 



c. Stain with anilin-oil gentian violet 1% minutes. 



d. Pour off stain and without washing. 



e. Apply Gram's iodine solution (17, 6) 1% minutes. 



/. Apply 96% alcohol 3 minutes, or until drippings do not stain 

 white filter paper. 

 g. Wash in water. 



