474 THE SPIROCHETES 



The agar is melted, cooled to about 45° C, and added quickly to the saline, hemoglobin, and 

 serum mixture, which has been warmed to about the same temperature. Thorough mixing 

 is important ; a medium containing flocculent agar particles does not give as good growth as 

 one of uniformly gelatinous consistency. The hemoglobin is made by laking one part of de- 

 fibrinated rabbit blood with three parts of distilled water. The addition of the hemoglobin 

 is not essential for the growth of leptospiras, however, although it is probably very important 

 in the cultivation of certain other organisms for which this same medium is used (e.g., 

 Bartonella bacilliformis) . 



If cultures of spirochetes are to be kept some time, it is desirable to add a layer of parafhn 

 oil, to prevent evaporation of the medium, which is noticeable after six to eight weeks. If 

 transfers are made every two months, however, the paraffin oil is not necessary. The infer- 

 ence drawn by some workers that L. iclerohaemorrhagiac is a facultative anaerobe because 

 it can grow under a layer of paraffin oil is based on the erroneous assumption that liquid 

 paraffin can hinder the entrance of oxygen. 



The cultural conditions for L. icteroides and L. hebdomadis are the same as for L. ic- 

 ier ohaemorrhagiae, and the saprophytic leptospiras of water grow readily on this medium 

 also. 



Bauer's technique' for obtaining initial cultures of the water leptospiras is as follows: The 

 water containing the organisms is filtered once through paper, once through a Berkefeld V 

 and twice through Berkefeld N candles. To 180 cc. of the filtrate is added 20 cc. of 2 per 

 cent hormone agar and i per cent fresh defibrinated rat's blood, and the mixture is distrib- 

 uted into tall tubes, about 8-10 cc. to a tube, which are kept at room temperature. Bauer 

 obtained thirteen strains of leptospiras and a strain of another water spirochete from samples 

 of water collected in Lagos, on the west coast of Africa. The technique was applied to lepto- 

 spira containing water from icebox drains in the writer's laboratory, with excellent results. 

 Angerer^ and Dimitroff •J have also cultivated water leptospiras. Dimitroff first obtained im- 

 pure cultures on a medium consisting of 2 per cent human feces in tap water, purified these 

 by filtration, and obtained pure growth on a similar medium and also on 4 per cent rabbit 

 serum in sodium chloride. Dimitroff, who obtained thirty-seven strains from drinking and 

 pond waters, puddles, etc., emphasizes the fact that it is well to test the filters used for their 

 ability to pass the leptospiras, only two of a lot of twenty tested by him having been found 

 suitable. The filtration method might well be tried for the isolation of Hoffmann's Lepto- 

 spira dentium,^ which has not yet been cultivated. 



INFLUENCE OF CARBOHYDRATES ON THE CULTIVATION OF SPIROCHETES 



Akatsus tested the effect of a number of carbohydrates on the growth of T. palli- 

 dum, T. calligyrum, T. microdentium, T. mucosum, and T. refringens. There were no 

 visible alterations in the appearance of media with the growth of the organisms, and 

 no unusual morphological changes of the spirochetes. In the case of the aerobic trepo- 

 nemas, the addition of sugars to the culture medium renders it less suitable for the 

 growth of the organisms. 



The leptospiras so far tested are non-fermenters and are indifferent to the pres- 

 ence of carbohydrates in the medium.'' 



' Bauer, J. H.: loc. cit. 



2 Angerer, K, V,: loc. cit. i Dimitroff, V. T.: loc. cit. 



4 Hoffmann, E.: Deutsche med. Wchnschr., No. 23. 1920; Centnilbl.f. BaktcrioL, 86, 134. 1921. 



sAkatsu, S.: J. Expcr. Med., 25, 375. 191 7. 



^Noguchi, H.: ibid., 27, 604. 1918; 30, iS. 1919. 



