HUMAN SPIROCH^TOSIS 715 



The spirochsetes never form spores : reproduction takes place according to 

 some observers by transverse division (Metchnikoff, Bardach) and according 

 to others by longitudinal division (Schaudinn, [Nuttall]). 



Spirochsetes can be kept alive for several days in hanging drop preparations 

 but the addition of normal saline solution destroys their motility and causes 

 them to agglutinate in a few minutes (Karlinski). 



If the blood be collected as soon as they begin to appear in it the spirochsetes can 

 be kept alive for 40 days but if collected just before the crisis when they are about 

 to vanish from the circulation they die in a day or two (Novy and Knapp). 



Staining reactions. The S. recurrentis is somewhat difficult to stain and 

 special methods have to be adopted. It is gram-negative. 



1. Blood-films. Blood-films should be dried in the air or in an oven at 

 60-70 C. but should on no account be passed through the flame. To render 

 the parasites more conspicuous the haemoglobin may be dissolved out of the 

 red cells before staining the films. The best method for staining is Giinther's 

 but any of the methods described as suitable for staining the Treponema 

 pallidum may be used. 



Giinther's method. Follow the instructions given on p. 206 using Ehrlich's 

 aniline violet as the dye and leaving it to act for 8-10 minutes. 



The red cells are unstained, the white cells and the parasites are stained 

 violet. 



By staining the blood-films not only are the Spirochsetes more easily seen but they 

 appear much more numerous than in fresh, unstained preparations. 



2. Sections. Pieces of the spleen should be fixed in absolute alcohol and 

 embedded in paraffin. The sections may be stained by NikiforofTs method. 



Nikiforoff's method. 1. Stain the sections for 2448 hours at the ordinary 

 temperature of the laboratory in the following solution : 



Saturated aqueous solution of methylene blue, - 10 c.c. 



Distilled water, - 10 



1 per cent, alcoholic solution of tropeolein, - 1 ,, 



10 per cent, solution of potash, - - 4 drops. 



2. Wash in distilled water, then in alcohol-ether. 



3. Clear in clove oil and xylol and mount in balsam. 



2. Cultivation of the parasites. 



Most attempts to grow Spirochsetes in artificial culture have failed. 

 Norris, however, and Pappenheimer and Flourney by sowing a few drops 

 of the blood of an infected rat in citrated human or rat blood noticed a con- 

 siderable multiplication of the Spirochsetes after 24 hours but the cultures 

 could not be maintained further than the second generation. 



Levaditi sowed S. duttoni in macacus serum previously heated to 70 C. 

 and placed the culture in collodion sacs in the peritoneal cavities of rabbits ; 

 in this way he obtained luxuriant cultures of the organism. Adopting 

 Levaditi's technique but using non-coagulated rat blood in the sacs in the 

 early cultures Novy was able to grow an Indian Spirochsete for twenty 

 generations : the cultures which were always scanty were virulent for rats 

 and retained their vitality for 7 days. 



Duval and Todd seem to have been able to obtain a certain amount of 

 growth through two generations by sowing an American Spirochsete in a 

 complicated culture medium. A broth was made with skinned mice and 

 sterilized : egg yolk and defibrinated mouse blood was then added and 

 allowed to macerate in sealed vessels in the incubator at 37 C. for 6 or 8 

 weeks. The medium was then sown with the blood of an infected mouse. 



