

DETECTION OF THE BACILLUS 341 



Sterilize. To 20 c.c. of this broth add 10 c.c. of sputum, mix thoroughly and 

 incubate at 37 C. After incubating for 24 hours the bacilli will have increased in 

 number and films can be prepared with the deposit and stained with carbol-fuchsin. 

 This method facilitates the detection of tubercle bacilli when they are present in 

 the sputum in small numbers only. 



(e) Spengler's method. Tubercle bacilli are more resistant to the action 

 of formalin vapour than most other bacteria and it is upon this fact that the 

 following method is based. Dworetzky states that he has not had good 

 results. 



Cover the bottom of a Petri dish with a filter paper, spread 3 c.c. of sputum 

 in a layer not more than 2 '5 mm. thick and sprinkle with pancreatin to facilitate 

 digestion of the mucus. Line the lid of the dish with filter paper soaked in a few 

 drops of commercial formalin. Incubate the dish and its contents at 20-25 C. 

 After incubating for 2 hours it is said that all micro-organisms other than the tubercle 

 bacillus are killed so that if tubes of glycerin-agar be now sown with the sputum 

 a pure culture of the tubercle bacillus will be obtained. 



[(/) Griffith's method. A. S. Griffith (for the English Commission) obtained 

 cultures direct from sputum by using antiformin. 1 The method he employed 

 at first was that recommended by Uhlenhuth ; diluted sputum and antiformin 

 were mixed together to form a 15 per cent, antiformin-sputum mixture and 

 allowed to stand for 3 hours ; the solution was then centrifuged, the deposit 

 washed and recentrifuged, and the second deposit sown on to culture 

 media. In subsequent experiments however he found that a 10 per cent, 

 dilution of antiformin allowed to act for 20-30 minutes gave better results 

 (see fig. 208 (a), p. 317).] 



(g) Spengler recommends another method based on the resistance of the 

 tubercle bacillus to heat and only applicable to nummular sputum. 



Take up a large fragment of the nummular sputum in a platinum loop and hold it 

 near a flame so that the sputum is roasted but not detached from the loop. Repeat 

 the process three or four times then sow the flamed sputum on 2 per cent, glycerin- 

 serum or on glycerin-agar. Growth appears in a week to 10 days. Spengler acknow- 

 ledges that this requires a certain amount of skill. 



In a case of advanced pulmonary tuberculosis recorded by Bertarelli the 

 sputum [appeared to] consist solely of tubercle bacilli which when sown 

 direct on to glycerin-serum and blood-agar readily gave a pure culture of 

 the bacillus. Such cases are very exceptional ; and undoubtedly the most 

 certain method of obtaining a pure culture from sputum is to inoculate a 

 guinea-pig and sow cultures from the lesions which develop (p. 314). 



B. Blood. 



The tubercle bacillus rarely passes into the blood of persons affected with 

 tuberculosis. Lustig and others have, however, succeeded in staining the 

 bacillus in films prepared with blood obtained by pricking the finger or the 

 spleen (in miliary tuberculosis). The bacillus is more easily demonstrated 

 in the clots formed post mortem in the heart and blood vessels. The carbol- 

 fuchsin method should be employed for staining the preparations. 



Bezangon and Griffon, and Jousset have described methods designed to facili- 

 tate the detection of the organism in the blood. The results of the methods are, 

 however, vitiated by the occurrence in the surrounding air of acid-fast bacilli 

 and of saprophytic bacilli which acquire acid-fast properties in organic 

 products. To secure the best results from these methods they ought to be 

 carried out under strictly aseptic conditions, and this in practice is difficult 



[ l Antiformin is a mixture of sodium hydroxide and Eau de Javelle. It has the power 

 of dissolving albuminous substances and of killing and dissolving all bacteria except 

 those which like the tubercle bacillus possess a waxy envelope.] 



