DETECTION OF THE BACILLUS 391 



In no case can an absolute diagnosis be made on microscopical evidence 

 alone. 



(a) Films. Films should be stained first with methylene blue or carbol- 

 thionin and then by Gram's method. 



Films from the spleen often contain only a few organisms, but if the tissue be first 

 incubated films made from it will be found to be very rich in bacilli. Wash the 

 surface of the organ in a 1 in 1,000 solution of perchloride of mercury, wrap it up in 

 a cloth wrung out of the same solution and incubate at 37 C. for 24 hours (Cornil) ; 

 or if preferred a little of the pulp may be drawn up into a number of Pasteur pipettes 

 and incubated (Gasser). 



Gasser prefers to stain the films obtained in this way by Gram's method, using 

 dilute carbol-fuchsin as a counterstain : the typhoid bacilli and the groundwork 

 are then stained red while if any gram-positive organisms are present they are, of 

 course, stained violet. 



(b) Sections. Fix the tissues to be cut in alcohol or acid perchloride solu- 

 tion (p. 189) and embed in paraffin. Stain the section by any of the methods 

 applicable to the staining of gram-negative organisms preferably with 

 thionin or by Nicolle's tannin method (p. 217). 



2. Cultures. 



Culture media broth, agar (and, for isolating the bacillus when con- 

 taminations are present, gelatin plates) should be sown with scrapings 

 from the spleen, with fluid exudates, products from puncture of the tonsil, 

 urine collected under aseptic precautions, etc. 



Attention has already been drawn (p. 198) to the dangers attending puncture of the 

 spleen in the living subject. This practice should never be resorted to as a matter 

 of routine and since the serum reaction is now available for the purposes of diagnosis 

 (see p. 384) and the bacilli can be isolated from the blood there is no justification for 

 running the risk attending the operation. 



Examination of the blood, (a) Courmont's method. Collect the blood 

 aseptically by puncture of a vein at the bend of the elbow (p. 193) and sow 

 2-4 c.c. immediately in a large volume (200-300 c.c.) of ordinary broth. 

 Incubate at 37 C. If no turbidity appears after 24 hours shake the flask 

 to promote the growth of the organism and then incubate again, and examine 

 the culture daily. In some cases when the typhoid blood has powerful 

 agglutinating properties growth may be delayed, being invisible until the 

 third or fourth day, and may then occur solely as clumps in the deposit which 

 forms at the bottom of the liquid. 



(/?) Busquet's method. To minimize the inconvenience caused by the agglutinating 

 and bactericidal properties of the blood, Busquet sows a number of flasks each con- 

 taining 250 c.c. of peptone broth with a few drops only of blood. 



Sacquepee and Perquis adopt the additional precaution of defibrinating the 

 blood as it leaves the vein. 



(y) Laff orgue s method. Lafforgue eliminates the serum, which contains the 

 bactericidal substances. The blood is rendered non-coagulable by the addition of 

 sodium citrate (2 drops of a 1 in 5 solution of sodium citrate to 2 c.c. of blood) and 

 then centrifuged. The deposit only is sown in broth in the proportion of 20 c.c. of 

 broth to the deposit from 2 c.c. of blood. Under these conditions the typhoid 

 bacillus grows rapidly. 



(S) Conradi's method. Conradi has shown that bile is an excellent medium, 

 since it renders the blood non-coagulable and inhibits its bactericidal action. 

 Cultures in bile-containing media are recommended for the detection of the 

 typhoid bacillus in blood. 



The principle may be applied in different ways. The simplest methods 

 are those of Zeidler and Kayser. 



1. Methods of Zeidler and Kayser. Zeidler adds 1 c.c. of blood (30 drops 



