36 General Bacteriology. 



eessfully with those species which liquefy this medium. In the case of agar the tubes 

 should be placed in a horizontal position a few hours (over night) until the medium has 

 become attached to the tube ; afterwards they can be stored in the usual receptacles for 

 tube cultures. 



REFERENCES. A. 131; M. & R. 65; McF. 143. 



SPECIAL DIRECTIONS. . Melt a tube of gelatin and without inoculating it practice 

 making a roll-culture as described above. Avoid tipping the tube enough to get medium 

 on cotton plug. Remelt and roll again until the knack is acquired. 



b. Make two roll-cultures in gelatin of B. coli (XIII), using a water-blank instead 

 of gelatin tube No. 1. 



c. Make two agar cultures of B. subtilis in same way. 



d. Incubate b. in cool chamber, andc. at 28 C. 



EXERCISE XXIX. STUDY OF PLATE CULTURES. 



MACROSCOPIC. As the colonies appear, note: . form, b. size, c. surface elevation, 

 d. consistency, t. color. Both the surface and deep colonies should be described as they 

 are frequently very different. Drawings should always be made wherever they will be of 

 value; study should be continued as long as changes are noticed. (See Chapter III, 

 I. A. a.-f.) 



MICROSCOPIC. The colonies appearing on the plates are to be studied under a low 

 power of the microscope. Use a f in. (16 mm.) 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, . structure of 

 colony as a whole ; 6. character of margin. (See Chapter III. I. A./ifcgr.) 



REFERENCES. P. B. C. (Cheesman's Charts.) 



SPECIAL DIRECTIONS. Study, write descriptions and make drawings of all plate 

 cultures. Use blank pages for description and sketch of cultures. 



EXERCISE XXX. USE OF DECOLORIZING AGENTS. 



Make three cover-glass preparations from a 24 hour old culture of B. subtilis, stain- 

 ing 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 cov er-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 decoloring agents. 



EXERCISE XXXI. GRAM'S STAIN. 



EXPLANATORY. This is a differential stain and one of the most useful. Some bac- 

 teria when stained by this method exhibit a dark violet color, others remain perfectly 

 colorless, thus rendering possible the differentiation of bacteria which are morphologically 

 nearly or quite identical, and also greatly facilitating the demonstration of certain bac- 

 teria 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. 



6. Air dry and fix. 



