METHODS OF STAINING 79 
objects must be of low refractive index. This method of observing 
bacteria has been especially valuable for the study of Treponema 
pallidum and flagella of ordinary bacteria. 
THEORY AND Mrtuops or STAINING 
The staining of bacteria should be regarded as a delicate procedure 
and one requiring much care. It should not be considered—although 
it probably is in many cases—~as a saturation of the cell with the dye. 
Rather should it be regarded as a chemical reaction involving the 
same laws which govern chemical changes. It doubtless follows the 
law of mass action. 
Koch (1877) was one of the first to recognize the value of aniline 
dyes for staining. For convenience the aniline dyes have been divided 
into two classes—the basic and acid dyes. The basic aniline dyes 
contain amido groups while the acid dyes contain the hydroxyl groups. 
The basic dyes are usually used as the salts of hydrochloric acid while 
the acid dyes are secured as sodium or potassium salts. Unna (1888) 
has mentioned that these dyes are not always basic but may be salts 
neutral in character. They are called basic simply because the colored 
component is basic. The aniline dye does not separate into two parts, 
the basic or colored part attacking the cell protoplasm, but rather the 
entire molecule plays its part in the staining process. That the stain- 
ing ability is determined by the solvent condition of the dye is shown 
by the following observations. 
1. Pure alcoholic water-free solutions of the dyes do not stain. 
2. Pure absolute alcohol will not decolorize while alcohol diluted 
with water decolorizes quickly. 
3. The more perfect the solution of the dye in the solvent the 
lesser does its staining ability become. This helps to explain why 
absolute alcohol does not decolorize or why pure alcoholic solutions 
do not stain. 
One of the most important essentials of the staining procedure 
is clean apparatus. All grease and foreign matter must be removed 
from the slides and cover glasses according to the procedure outlined 
under cleaning apparatus in the first chapter. After the slides have 
been thoroughly cleaned they may be kept in a salt-mouth bottle in 
alcohol. The cover slips may be kept in a short wide-mouth bottle 
containing alcohol or ether. In this way both the slides and cover 
glasses will be free from grease provided they are thoroughly cleaned 
and rinsed in distilled water. 
