Mat 24, 1907] 



SCIENCE 



817 



they persist for a day or two longer than 

 is the ease with the active virus. 



When the sac is allowed to remain in 

 the rat for seven days the spirilla de- 

 crease greatly in numbers and may even 

 disappear. In the culture sacs after re- 

 moval from the rat, and kept at room 

 temperature, the spirilla die out in a day 

 or two. 



Throughout this series the spirilla have 

 preserved their form unchanged. They 

 appear either as single cells (8 microns) 

 or of double length (16 microns) but at 

 times even longer spirals are found. The 

 latter are the result of end to end union 

 by means of flagella as we have hereto- 

 fore shown. As in the case of blood pre- 

 parations no evidence is observed of di- 

 vision other than transverse. One obser- 

 vation in this connection is deserving of 

 special emphasis owing to its bearing upon 

 the question as to whether spirochetes 

 multiply by transverse or longitudinal 

 division. In these cultures it is not un- 

 usual to find short spirals of two or three 

 turns, and from 4 to 8 microns in length. 

 These may occur singly or in pairs (8 to 

 12 microns long) showing the pale division 

 zone. The width of the short form is the 

 same as that of the longer cells. The oc- 

 currence of these short spirals is readily 

 explainable as the result of transverse divi- 

 sion. It may further be stated that the 

 cultural spirals usually stain solid by the 

 Romanowsky method but at times they may 

 show granulations which to some extent 

 may be due to granules deposited from the 

 medium. 



Sac Cultures in Rat Serum. — In view of 

 the fact that Prowazek and others are in- 

 clined to consider spirochetes as protozoa 

 and cell parasites it was desirable to ascer- 

 tain whether or not the spirilla could be 

 maintained in active multiplication in a 

 clear serum. Accordingly, the spirilla 

 were inoculated into rat serum, completely 



freed from corpuscles by centrifugation. 

 Up to the present time we have effected 7 

 consecutive passages in such serum in the 

 space of 24 days. At each passage a con- 

 trol sac containing defibrinated rat blood 

 was placed in the rat. The serum cultures 

 although totally devoid of corpuscles were 

 in every respect as rich in spirilla as the 

 blood cultures. The conclusion to be de- 

 duced from these experiments is that multi- 

 plication of spirilla may take place without 

 _any intracellular stage. The occasional 

 presence of spirilla in a cell is to be re- 

 garded as an accident rather than as an 

 expression of an unrecognized cycle. 



A New Flagella Stain for Ps. radicola: F, 



C. Harrison. 



Take a loop of the mucilaginous or viscid 

 growth from an agar culture of Ps. radici- 

 cola two days to several months old and 

 spread it on a clean slide, lashing it out in 

 slender tongues, let the film dry in air 

 without killing or fixing, flood the film a 

 moment with a saturated alcoholic solution 

 of gentian violet, wash under the tap, dry 

 between folds of filter paper and examine 

 with the oil immersion lens. The mucilage 

 in which the cells lie will be found deeply 

 and evenly stained, and the bacteria 

 scarcely stained at all, so that the prepara- 

 tion presents the appearance of a photo- 

 graphic negative. The unequal density of 

 the protoplasm of the cells is clearly seen, 

 as indeed it is in the living cells when 

 examined from a hanging drop. (See 

 photograph. ) 



The single polar flagellum is also clearly 

 demonstrated by this stain since it, like the 

 protoplasm of the cells, refuses the stain, 

 and so it appears as a clear or uncolored 

 streak in the surrounding deeply stained 

 mucilage. The flagella are best stained at 

 the margins of the films and in thin places. 

 In parts of the film where the culture is 

 thickly spread, the mucilage is intensely 



