208 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
(h) Remove excess of stain with acid alcohol. 
(c) Wash in water. 
(d) Dehydrate in 95 per cent alcohol. 
(e) Expose to ether vapor to fix section to slide. 
(/) Stain with anilin oil methyl violet for five to twenty minutes. 
(g) Remove excess stain with normal salt solution. 
(h) Gram-iodine solution for one minute. 
(i) Remove excess iodine with water. 
(j) Remove moisture as thoroughly as possible with filter paper. 
(k) Dehydrate in several changes of anilin oil. 
(I) Clear with several changes of xylol. 
(m) Mount in neutral xylol balsam. 
Staining Tubercle Bacilli in Tissues. — (a) Paraffin sections are 
covered with carbol-fuchsin and steamed gently for five minutes. 
(b) The excess stain is removed with water. 
(c) Decolorize and counterstain with Gabbet methylene blue sul- 
phuric acid stain about one minute. 
(d) Remove excess of stain and acid with water. 
(e) Dehydrate with absolute alcohol. 
(/) Clear section in xylol. 
(g) Mount in xylol balsam. 
Staining Actinomyces in Tissues.— Mallory Method.'^— {a) Stain 
paraffin sections with saturated aqueous eosin for ten minutes. 
(6) Remove excess stain with water. 
(c) Stain with anilin oil methyl violet for two to five minutes. 
{d) Remove excess stain with normal salt solution. 
{e) Remove excess water with filter paper. 
(/) Clear in anilin oil, 
{g) Remove anilin oil with several changes of xylol. 
(/;) Mount in neutral xylol balsam. 
The clubs stain pink, the filaments blue. 
IV. Methods and Media for the Cultivation of Bacteria.— One of 
the most important procedures in bacteriology is the preparation of 
nutritive media in which the morphology, chemistry and cultural 
characteristics of the organism may be studied; furthermore, it is 
possible by cultural methods to separate one type of bacterium in 
pure culture from associated organisms, and to study its reactions 
apart from all contaminating microorganisms. The technique of 
isolating and cultivating bacteria is exacting at every step of the pro- 
cess, from the preparation of glassware to the selection of suitable 
nutritive media. This technique, furthermore, requires not only 
scrupulous cleanliness; it necessitates a most rigorous maintenance of 
sterility. 
Bacterial cultivation is usually carried out in glass vessels— test- 
tubes, flasks, fermentation tubes and Petri dishes— because glass is 
1 Loc. cit., p. 433. 
