164 * TUBERCULOSIS 
impossible. Sections of the tissues containing young lesions properly 
stained are often helpful. In case the tubercle bacteria are not found 
in smears or sections, guinea pigs should be imoculated.* If the 
inoculated guinea pigs are chloroformed in about 25 days young 
tubercles can usually be detected in the adjacent lymph glands from 
which cultures may be made. The tubercle bacteria are usually 
readily demonstrated by making film preparations from the lesions. 
fuchsin. Place a few drops of the stain on the film side of the cover-glass preparation 
and hold it over a flame with forceps until steam is given off. Allow the hot stain to 
act for from 3 to 5 minutes, or the preparation may be floated on the carbol fuchsin in 
a watch glass without heat. In this case it is allowed to act for from 10 to 15 minutes. 
The preparation is then rinsed in water and decolorized by treating it with a 10% 
solution of nitric or sulphuric acid for from 14 to 1 minute. It is again rinsed in water, 
when it is ready for examination. It can be dried and mounted permanently in bal- 
sam. The tubercle bacteria should be stained a deep reddish color. All other bacteria 
or animal] tissue in the preparation should be nearly or quite decolorized. If desired, a 
counter-stain, such as alkaline methylene blue, may be used after decolorizing; that 
is, the preparation should again be stained for about 1 minute in alkaline methylene 
blue, rinsed in water, and examined as before. In these preparations the tubercle 
bacteria are red and the other organisms and cells are blue. A counter-stain is of little 
value in preparations made for simple diagnostic purposes. When a counter-stain is 
desired Gabbett’s decolorizing and counter-staining solution is very convenient. 
GABBETT’S SOLUTION 
Methylene blue (powder) ............00 000 ccc ccc eee eee eee 2 grams 
TOO Sua PUTO ACL: oy So 2 ccc ce a seacesecniden 4 epergud Sob eotlpctoc ses <P etonusers- aidase tare 100 ce. 
After staining with the carbol fuchsin treat the preparations with this mixture until 
the film has a faintly bluish tint. This solution decolorizes and counter-stains at the 
same time. Care must be taken not to confuse the other acid fast bacteria with those 
of tuberculosis. The acid fast bacteria other than tubercle are decolorized with 
acidulated alcoho] (3 per cent hydrochloric acid in 95 per cent. alcohol). 
*In animal inoculation for the purpose of diagnosis guinea pigs are preferable. 
Rabbits rarely develop the disease when inoculated with the human variety but 
usually do when infected with the bovine variety. With tuberculous tissue either of 
the two methods described below may be employed. 
A small piece (about the size of a pea or bean) of the tissue may be inserted under 
the skin by first making an incision with a sharp scalpel through the skin and superficial 
fascia, and then with a pair of fine forceps insert the bit of tissue well under the skin 
and close the opening with one or more sutures. 
The tissue may be crushed in a mortar and thoroughly mixed with a few cubic 
centimeters of sterile water or bouillon and then injected with a hypodermic syringe. 
The needle should be of large calibre. If it is suspected milk, it may be injected into 
the abdominal cavity. If the material is tuberculous and contains living tubercle 
bacteria, the death of the animal follows in from three weeks to four months. Usually 
the lymphatic glands in the groin and axilla are enlarged and often caseous. If a guinea 
pig is used, the liver, spleen, lungs and kidneys are liable to be affected, in the order 
named; if a rabbit, the lungs are often the first of the viscera to be attacked. 
In avian tuberculosis it is necessary to use chickens instead of guinea pigs. They 
may be inoculated subcutaneously or into the abdominal cavity. Several weeks may 
id necessary for the lesions to develop sufficiently to enable one to distinguish the 
isease. 
