THE TUBERCLE BACILLUS 95 



3. Apply absolute alcohol for 30 seconds. 



4. Wash in water and apply methylene blue for 5 minutes. 



5. Wash in water. 



6. Dry and examine. 



By either of these methods tubercle bacilli will be stained red and every- 

 thing else blue. 



Growth. The tubercle bacillus (human and bovine types) is an aerobic 

 organism which grows at temperatures between 3OC. and 42C., best at 37C. 

 It will not grow on media other than blood serum unless blood, blood serum, 

 tissue or glycerin are added. Development is slow, growth first appears after 

 12 or 14 days' incubation and continues to increase for 3 or 4 weeks. 



Organisms transplanted directly from the human body or sputum to cul- 

 ture media often fail to grow. Isolation of tubercle bacilli from other organ- 

 isms by plating is unsatisfactory. It is best to inoculate a healthy animal with 

 the suspected material or material containing tubercle bacilli or tubercle bacilli 

 and other organisms. After 3 or 4 weeks general tuberculosis develops and if 

 the animal is opened under aseptic precautions and the contents of enlarged 

 tubercular glands removed and planted on appropriate culture media, pure 

 cultures can be obtained. 



Glycerin Bouillon. To obtain growth on this medium several large loops- 

 ful of an actively growing young culture must be obtained and floated on the 

 surface of the glycerin bouillon. The glycerin bouillon may have i per cent, 

 glucose added to it with advantage; it must be slightly alkaline and have a good 

 supply of oxygen, obtained by placing the bouillon in a broad flask with a wide 

 neck. After 2 to 3 weeks' incubation at 37C. growth appears on the surface; 

 at first a small, thin whitish scum, which rapidly spreads over the entire surface 

 and continues to grow thicker for a month or more. The pellicle becomes 

 wrinkled or granular as it ages, portions may fall to the bottom of the flask, and 

 when the culture is old the growth may change from white to yellowish. The 

 medium remains clear. 



Glycerin-agar. Growth appears in about 2 weeks; at first as dry, scaly 

 white spots. These coalesce, forming a thick, dry, corrugated film, described 

 as "bread-crumb" growth. 



Serum-agar. Growth is the same as on glycerin-agar except that this is a 

 more favorable medium for initial cultivation after removal from a guinea-pig. 



Coagulated Serum. Growth is the same as on serum-agar. 



The tubercle bacillus does not form spores. 



Resistance. Tubercle bacilli are most resistant when in tissue, least re- 

 sistant when in culture media. Putrefaction of tissue containing them does not 

 lessen their vitality nor virulence. To destroy tubercle bacilli in tissue, sputum, 

 feces, etc., boiling for ^ hour is required. Their resistance to dry heat is so 

 great that a temperature of i5oC. for several hours is necessary to destroy 

 them. Destruction by chemical germicides is difficult, thorough exposure to 

 strong solutions is necessary. 



The tubercle bacillus resists drying and retains its virulence in the dried 



