484 PATHOGENIC MICROORGANISMS 



produced by the contaminating organisms and succumb later (four to 

 six weeks) to tuberculosis. The bacilli may then be obtained by culti- 

 vations from lymph nodes or other foci which contain only tubercle 

 bacilli. When isolation from sputum is attempted, whether directly 

 or by means of animal inoculation, the sputum may be rendered com- 

 paratively free from contaminating bacteria by washing. The sputum 

 is rinsed in running water to free it from pharyngeal mucus. It is then 

 washed in eight or ten changes of sterile water. The material selected 

 is taken from the center of the washed mass, if possible from the flakes 

 of caseous material visible in such sputum. 



For the isolation of tubercle bacilli from sputum and other materials in 

 which contaminating bacteria of other species are present, Petroff 1 has devised 

 an excellent method which has been tried, out and used with success in our 

 laboratory by Dr. H. R. Miller. The principles on which Petroff's method 

 rests are, first of all, the bactericidal power of 3% sodium hydroxid on non- 

 acid-fast bacteria, and the selective action of dyes like gentian violet on bac- 

 terial growth, as first practically utilized by Churchman (See page 140). 



The medium used by Petroff is made as follows: 



I. Meat Juice. 500 grams of beef or veal are infused in 500 c.c. of a 15% 

 solution of glycerin in water, in a cool place. After 24 hours the meat is squeezed 

 in a sterile press and the infusion collected in a sterile beaker. 



II. Eggs. The shells of the eggs are sterilized by 10 minute immersion in 

 70% alcohol. They are broken into a sterile beaker, well mixed and filtered 

 through "sterile gauze. One part of meat juice is added to two parts of egg by 

 volume. 



III. Gentian Violet. 1% alcoholic solution of gentian violet is added to 

 make a final proportion of 1 : 10,000. 



The three ingredients are well mixed. The medium is tubed and inspissated 

 as usual. 



Petroff recommends for sputum the following technique: Equal parts of 

 sputum and 3% sodium hydroxid are shaken and incubated at 38 C. for 15 to 30 

 minutes, the time depending on the consistency of the sputum. The mixture 

 is neutralized to litmus with hydrochloric acid and centrifugalized. The sedi- 

 ment is inoculated into the medium described above. Pure cultures are ob- 

 tained in a large proportion of cases. 



Petroff's method has been applied by him to feces, in which the problem 

 is made more difficult by the presence of many spore-formers which resist 

 sodium hydroxid. Feces is collected and diluted with three volumes of water, 

 and then filtered through several thicknesses of gauze. The filtrate is satu- 

 rated with sodium chlorid and left for half an hour. The floating film of bac- 



1 Petroff, Johns Hopkins Hosp. Bull., vol. xxvi, No. 294, August, 1915, p. 276. 



