110 Animal Microloyy 



rinsed again iu water, and examined in the ordinary way. The bacteria 

 of virulent diphtheria should appear as pale-brown rods, some of which 

 show at one or both ends bluish-black oval bodies of greater diameter 

 than the rod. Such dark bodies will not be seen in the pseudo-diph- 

 theria bacilli. 



The bacilli must have been grown for from 12 to 18 hours on Loff- 

 ler's blood-serum which is a mixture of glucose bouillon 1 part and beef- 

 blood serum 3 parts. The mixture is run into test-tubes and coagulated 

 at 100 C.; the tube should be tilted to one side to give a slanting surface 

 for culture purposes. The formula for glucose bouillon is as follows: 

 dry glucose, 10 grams; Liebig's extract of beef, 3 grams; peptone, 10 

 grams; sodium chloride, 5 grams; water 1,000 c.c. 



e) Gabbefs solution for demonstrating tubercle bacilli. 



Methylen blue 1 to 2 grams 



Distilled water 75 c.c. 



Concentrated sulphuric acid 25 c.c. 



The acid decolorizes, while the methylen blue serves as a contrast stain. 

 The solution acts rapidly. A modification of the method to be com- 

 mended is first to stain the preparation with carbol fuchsin (see c) by 

 warming the stain on the slide until it steams, rinsing in water and 

 then proceeding with the methylen-blue solution. Smegrna, leprosy, 

 and syphilis bacilli are also stained by this method. Tubercle bacilli 

 are also stained by Gram's method (see /). To examine sputum for 

 tubercle bacilli, the sputum is carefully inspected for small yellowish- 

 white cheesy masses varying in size from the diameter of a pin-head to 

 that of a small pea. Very thin smear preparations (see A) are made 

 from such masses. 



/) Gram's method. 



Gram's solution. 



Iodine crystals 1 gram 



Iodide of potassium 2 grams 



Distilled water 300 c.c. 



The preparations are first stained in anilin gentian violet (memorandum 

 3d), and then immersed in Gram's solution for from 1 to 2 minutes. 

 They are then rinsed in alcohol until the violet color is no longer visible 

 to the naked eye. To decolorize them sufficiently, it may be necessary 

 to treat them again with the iodine solution. Finally rinse in water and 

 examine, or if a permanent preparation is desired, rinse in absolute alco- 

 hol, transfer to xylol, and mount in balsam. If the preparations are 

 from cultures, it should be borne in mind that the method works well 

 only when applied to bacteria from actively growing cultures; old cul- 

 tures seldom yield satisfactory results. 



