306 BACTERIOLOGY. 
before death, a more important rdle than the associated 
bacteria. . 
The great majority of stained smears from specimens 
of sputa show not only the tubercle bacilli stained in 
red, but many other bacteria stained blue. Some of 
these associated bacteria have come from the diseased 
areas of the lungs, while others were merely added 
to the sputa as it passed through the mouth, or have 
developed after gathering. To separate the one from 
the other we wash the sputa. 
Sputum Washing. The first essential is that the 
material be washed within a few minutes, and certainly 
within an hour, of being expectorated. If a longer 
time is allowed to intervene, the bacteria from the 
mouth will penetrate into the interior of the mucus, and 
thus appear as if they came from the lungs. Sputum 
treated twenty-four hours after its expectoration is use- 
less for examining for anything except the tubercle 
‘ bacillus. A rough method is to pour some of the speci- 
men of sputum to be examined into a convenient re- 
ceptacle containing sterile water, and withdraw, by 
means of a sterilized platinum wire, one of the cheesy 
masses or thick “balls’’ of mucus. Pass this loop five 
times through sterile water in a dish; repeat the oper- 
ation in fresh water in a second and third dish. Spread 
what remains of the mass on cover-glasses and make 
smear preparations; stain and examine. With another 
mass inoculate ascitic bouillon in tubes and agar in 
plates. 
If it is desired to examine the specimens for capsule 
bacteria, pneumococci, etc., they may be stained by 
Welch’s acetic-acid method (page 203) or by Gram’s 
method (page 203). 
