Isolation 663 



but as the smegma bacillus (q.v.) is apt to be present in the urine, 

 the precaution should be taken to use Pappenheim's solution 

 for differentiation or to wash the stained film with absolute alcohol, 

 that it may be decolorized and confusion avoided. 



Staining the Bacillus in Feces. — It is difficult to find tubercle 

 bacilli in the feces because of the relatively small number of bacilli 

 and large bulk of feces. 



Staining the Bacillus in Sections of Tissue. — Ehrlich's Method 

 for Sections. — Ehrlich's method must be recommended as the most 

 certain and best. The sections of tissue, embedded in paraffin, 

 should be cemented to the slide and then freed from the embedding 

 material. 



They are then placed in the stain for from twelve to twenty-four hours and kept 

 at a temperature of 37°C. Upon removal they are allowed to lie in water for 

 about ten minutes. The washing in nitric acid (20 percent.) which follows may 

 have to be continued for as long as two minutes. Thorough washing in 60 per 

 cent, alcohol follows, after which the sections can be counterstained, washed, 

 dehydrated in 96 per cent, and absolute alcohol, cleared in zylol, and mounted in 

 Canada balsam. 



Unna's Method for Sections. — Unna's method is as follows : The sections are 

 placed in a dish of twenty-four-hour-old, newly filtered Ehrlich's solution, and 

 allowed to remain twelve to twenty-four hours at the room temperature or one to 

 two hours in the incubator. From the stain they are placed in water, where they 

 remain for about ten minutes to wash. They are then immersed in acid (20 per 

 cent, nitric acid) for about two minutes, and become greenish black. From the 

 acid they are placed in absolute alcohol and gently moved to and fro until the 

 pale-blue color returns. They are then washed in three or four changes of clean 

 water until they become almost colorless, and then removed to the slide by means 

 of a section-lifter. The water is absorbed with filter-paper, and then the slide is 

 heated over a Bunsen burner until the section becomes shining, when it receives a 

 drop of xylol balsam and a cover-glass. 



It is said that sections stained in this manner do not fade so quickly as those 

 stained by Ehrlich's method. 



Gram's Method. — The tubercle bacillus stains well by Gram's method and by 

 Weigert's modification of it, but these methods are not adapted for differentiation. 

 They should not be neglected when no tubercle bacilli are demonstrable by the 

 other methods, as they are particularly well adapted to the demonstration of such 

 of the organisms as may not be acid-proof. 



Isolation. — Piatkowski* has suggested that the cultivation of the 

 tubercle bacillus and other "acid-proof" organisms may be achieved 

 by taking advantage of their ability to resist the action of formal- 

 dehyd. The material containing the acid-proof organism is mixed 

 thoroughly with 10 cc. of water or bouillon, which receives an ad- 

 dition of 2 or 3 drops of 40 per cent, formaldehyd or "formalin." 

 After standing from fifteen to thirty minutes transfers are made to 

 appropriate culture-media, when the acid-proof organisms may 

 develop, the others having been destroyed by the formaldehyd. 



Still further improvement in the means by which the tubercle 

 bacilli can be secured free from contamination with other organisms 

 and from surrounding unnecessary and undesirable materials, has 

 accrued from the use of antiformin. This commercial product, 

 patented in 1909 by Axel Sjoo and Tornell, consists of Javelle water 

 * "Deutsche med. Wochenschrift," June 9, 1904, No. 23, p. 878. 



