340 THE TUBERCLE BACILLUS 



drop of normal soda solution, add '25-0 '50 gram of pancreatin and incubate at 

 37 C. for 2 or 3 hours. Then centrifuge or pour into a conical glass vessel with a 

 crystal of thymol added to hinder putrefaction and leave to stand for 12 or 14 hours : 

 decant the supernatant liquid and prepare films with the deposit. 



6. Jousset's method. Digest the sputum for 2 or 3 hours in the warm incubator 

 (38 C.) with 10-30 c.c. of artificial gastric juice made as follows : 



Pepsin, - 2 grams. 



Pure glycerin. 1 __ in 



Hydrochloric acid,}' ' aa ' 10 C ' C ' 



Sodium fluoride, - - 3 grams. 



Distilled water, Q.S. to 1000 c.c. 



When completely peptonized, centrifuge and examine the deposit for tubercle 

 bacilli (p. 342). 



Films made from sputum or from the deposit after solution of the sputum 

 are stained by one or other of the methods already described, Ziehl-Neelsen's 

 method being the most satisfactory. 



2. Inoculations. In a doubtful case of pulmonary phthisis when no bacilli 

 can be found in the sputum on microscopical examination, animal inoculation 

 must be resorted to for purposes of diagnosis. The sputum must be collected 

 as free from other organisms as possible (p. 192) and an emulsion of it, pre- 

 pared by rubbing up in sterile water, inoculated beneath the skin of a guinea- 

 pig. Should the sputum contain tubercle bacilli the animal soon shows 

 signs of infection (p. 297). On account of the risk of setting up a fatal septic 

 peritonitis sputum should never be inoculated into the peritoneal cavity [but 

 see p. 338, 2. Inoculations]. 



3. Cultures. It is very rarely that cultures are sown with sputum for 

 purposes of diagnosis ; indeed for a long time it was thought impossible to 

 obtain a pure culture of the bacillus from sputum. Kitasato, Pastor and 

 others have, however, described methods by which it may be effected. 



(a) Kitasato' s method. After the mouth has been washed out with sterile water, 

 induce the patient to cough, and collect the sputum in a sterile glass vessel. Wash 

 the sputum in a number (10) of glass vessels containing sterile water (p. 192) then, 

 adopting the ordinary precautions to prevent contamination, lift a small fragment 

 from the centre of the purulent mass and spread it on glycerin-serum. Sow a large 

 number of tubes. Growth appears after the tubes have been incubated at 38 C. 

 for about 10 or 12 days. 



(6) Pastor's method. Collect the sputum with the same precautions as in Kita- 

 sato's method. Emulsify in a little sterile water and filter through a piece of sterile 

 gauze. Sow a tube of liquefied gelatin with a few drops of the filtrate, pour into a 

 Petri dish and allow it to set. After incubating for 3 or 4 days at 20 C. .any con- 

 taminating organisms that may have been present will have grown giving rise to 

 numerous colonies. With a sterile scalpel cut out those portions of the gelatin on 

 which there is no growth and transfer them to tubes of glycerin-serum ; if a number of 

 tubes be sown a pure culture of the tubercle bacillus will be obtained on some of them. 



(c) Hesse's method. Hesse's medium (p. 317) is most useful for isolating the 

 tubercle bacillus from sputum. The sputum is washed by Kitasato's method 

 and sown on the agar in Petri dishes. After incubating at 37 C. for about 

 10 hours the number of tubercle bacilli in the fragments of sputum will be 

 found to have considerably increased and colonies will be visible to the 

 naked eye at about the sixth day. 



(d) Jockmann's method. Jockmann uses the following medium which is similar 

 to Hesse's : 



Heyden's albumose (Nahrstoff Heyden), .... 5 grams. 



Salt, - 5 



Neutral glycerin, - 30 ,, 



Normal soda solution, - - - - - - - 5 c.c. 



Water, 1000 



