36 General Bacteriology. 
cessfully 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. & RB. 65; McF. 148. 
SpeciaL DirEcTIoNS. a. Melta 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, and c. at 28° C. 
EXERCISE XXIX. STUDY OF PLATE CULTURES. 
Macroscopic. As the colonies appear, note: a. form, b. size, c. surface elevation, 
d. consistency, e. 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.-e.) 
Microscopic. The colonies appearing on the plates are to be studied under a low 
power of the microscope. Use a¥%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, a. structure of 
colony as a whole; b. character of margin. (See Chapter III. I. A. fdég.) 
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 
(80%) as decoloring agents. 
EXERCISE XXXI. GRAM’S STAIN. 
Expuanatory. 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. 
b. Air dry and fix. 
