HIDEYO NOGUCHI 473 



same medium as that which I had employed for the blood treponemas. They found, how- 

 ever, that the optimum temperature was 2 2°-25° in the case of the leptospira, the organisms 

 remaining alive much longer than at 37° C. Ito and Matsuzaki' later reported favorable 

 growth of the same organism on solid medium containing agar or gelatin and certain types of 

 whole or defibrinated blood. Reiter and Ramme^ found that the German strains grew luxuri- 

 antly on a rabbit serum saline, which is composed of one part of serum and five parts of 

 0.85 per cent saHne solution. Beef serum, i cc, to Ringer or Locke solution, 9 cc, may also 

 be used. Uhlenhuth's medium^ consists of a mixture of i cc. of rabbit serum and 30 cc. of 

 tap water, or i cc. of the infected blood of the guinea pig to 30 cc. of tap water. Martin 

 and Pettit'' regard the rabbit serum diluted with isotonic salt solution as the best medium 

 for the ictcrohacmorrhagidc, and the writer's experience is that no other animal serum, ex- 

 cept perhaps sheep serum, gives as rich a growth of organisms of the leptospira group as does 

 rabbit serum. Wenyons adds a drop or two of rabbit blood to each tube. Verwoort^ uses 

 horse serum. 



An interesting and important phenomenon in the cultivation of leptospiras is their pecu- 

 liar tendency to multiply more abundantly in a semisolid particle, such as a piece of agar or 

 fibrin, than in clear liquid media. The writer noticed this fact early in his work, and it has been 

 repeatedly confirmed that the addition of melted nutrient agar to the rabbit-serum saline 

 to a final concentration of 0.1-0.3 P^r cent furnishes the optimum semisolid condition for 

 abundant growth. Too much agar may not be used, for the organisms are obligatory aerobes, 

 and the depth and rapidity of penetration of oxygen into the medium are less the more agar 

 there is added. With a concentration of 0.3 per cent or less, the surface layer of 2-3 cm. is 

 easily penetrated by oxygen, as can be demonstrated by using methylene blue as indicator. 

 The leptospira itself may be used as an indicator of the penetration of oxygen into the me- 

 dium, for it grows only in the aerated zone, the same zone to which the color of methylene 

 blue is found to return after removal of a vasehne seal. A rich culture is easily recognized by 

 the opalescent layer near the surface, sharply demarcated from the clear region below. 



When cultures in large quantities are desired to furnish material for the immunization 

 of horses or for preparing material for the vaccination of human beings, the medium may 

 be placed in a flask like that shown in the illustration (Fig. 87) to a depth of 1-2 cm. Growth 

 then occurs uniformly throughout the medium, and within two to three weeks the leptospiras 

 are present to a concentration of about 16,000,000,000 per cubic centimeter. Rich cultures 

 may be diluted with three parts of saline, killed by heating for one half hour at 55° C. and 

 used as vaccine. The standardization of the concentration of the vaccine is less easy than in 

 the case of bacteria from fluid media, but darkfield examination of the final product should 

 show large numbers of dead organisms per field. 



The medium now used for routine cultivation of all leptospiras in the writer's laboratory 

 is as follows: 



Parts 



0.9 per cent sodium chloride 800 



Rabbit serum 100 



Hemoglobin solution o. i-o. 2 



2 per cent nutrient agar 100 



' Ito, T., and Matsuzaki, H.: /. Expcr. Med., 23, 557. 1916. 



^Reiter, H.: Deutsche med. Wchnschr., 42, 1282. 1916. 



3Uhlenhuth, P.: ihid., 63, 152, 511. 1917. ''Martin, L., and Pettit, A.: loc. cit. 



sWenyon, C. M.: Protozoology, 2, 1299. 1926. 



^ Verwoort, H.: Geneesk. Tijdschr. v. Nederl.-Indie, 62, 697. 1922; 63, 800. 1923; Far Eastern 

 Assoc. Trop. Med., 5th Bienn,. Cong., Singapore, p. 683. 1923 (Abstract, Trop. Dis. Bull., 20, 583. 

 1923; 21, 262. 1924; 22, 175. 1925). 



