28 STAINING METHODS. 
one hundred of water) is used most bacteria are stained within a 
few seconds toa minute. At the end of this time the staining solu- 
tion is to be washed away by means of a gentle stream of water, or 
by moving the cover glass about in a vessel containing distilled 
water. 
Decolorization.—It often happens that the albuminous material 
associated with the bacteria which we propose to examine is stained 
so deeply as to obscure the view of these; and, generally, we will 
obtain more satisfactory preparations by the use of a decolorizing 
agent, by which the background is cleared up and the outlines of the 
cells more clearly defined. The agents chiefly used for this purpose 
are alcohol, diluted acids, and solution of iodine with potassium 
iodide (Gram’s solution). 
Koch recommends a solution containing sixty parts of alcohol to 
forty parts of water. The cover glass is to be quickly passed 
through this solution two or three times. Some bacteriologists pre- 
fer to use absolute alcohol. 
Or we may use dilute acetic acid (one-half to one per cent) or 
very dilute hydrochloric acid (ten drops to half a litre of water). 
For decolorizing preparations containing the tubercle bacillus 
strong solutions of the mineral acids are employed (one part of ni- 
tric or of sulphuric acid to three parts of water). 
_ Gram’s solution contains one part of iodine and two parts of 
potassic iodide in three hundred parts of water. Special directions 
will be given for the use of these agents when we give an account 
of the staining methods most useful for the various pathogenic 
organisms. : 
Double Staining.—After decolorizing the background of albu- 
minous material we may again stain this with a contrast stain, 
such as eosin or vesuvin. In mounts made from pure cultures, 
either liquid or solid, a single stain, for the bacteria only, is all that 
we require, and our aim is to have the background as free as possi- 
ble from any material which would obscure the view. 
After staining, decolorizing, and washing the preparation the 
cover glass or slide is again dried by exposure to the air or gentle 
heat, and is then ready for the permanent mounting in Canada bal- 
sam. If the bacteria have been stained upon the slide, a small drop 
of balsam dissolved in xylol is placed in the middle of the prepara- 
tion and a clean, thin glass cover applied. 
If it is the intention to make the microscopical examination with 
an immersion objective of high power, or to make photomicro- 
graphs from it, only the thinnest glass covers should be used—one- 
two-hundredths of an inch or less. 
If the preparation is not intended for permanent preservation, 
