910 THE SPIROCHETES 



that it contains nucleic acid (see Zuelzer 1925). The spirochaetes that stain with 

 methylene blue are generally coloured blue by Giemsa, whereas those that do 

 not are generally coloured red. But the exact tint that results from Giemsa' s 

 stain depends to some extent on the medium in which the organism is grown ; 

 thus in the blood, Trep. recurrentis stains blue with occasional reddish granules, 

 whereas in culture it stains red. In the larger spirochsetes it is possible by such 

 stains as Giemsa's and iron hsematoxylin to bring out the finer details of structure, 

 but in the smaller ones this is practically impossible. The reaction to Gram's stain 

 is negative, though this stain is rarely used in practice. 



For the demonstration of spirochaetes in tissue-sections Levaditi's method is 

 one of the most successful. It depends on the ability of the organisms, when 

 treated with silver nitrate followed by reduction with a formol-pyrogallic acid 

 mixture, to become impregnated with metallic silver, and therefore to appear 

 black. Fo • the demonstration of spirochaetes in films the Fontana method of silver 

 impregnation is most useful. 



Many species, particularly the leptospirae, are able to pass through the usual 

 bacterial filter candles. This property they owe to their extreme tenuity. Exact 

 measurements by Hindle and Elford (1933), made with graded collodion membranes, 

 show that the width of Trep. pallidum is about 0-2 /j, and of Lepto. hiflexa 0-1 jjl. 

 Use is often made of their filtrability to separate them from contaminating bacteria. 



Cultivation.- — The cultivation of spirochaetes in vitro is not so simple as that 

 of most bacteria. Nearly all the methods that have proved successful involve 

 the use of a medium containing native animal protein, such as blood, serum, or 

 ascitic fluid. Whether this acts chemically as a nutrient material, or physically 

 as a protective colloid preventing the organisms from being poisoned by the 

 products of their own metabolism, is not clear. A further requirement for many 

 spirochaetes is a low oxygen pressure ; this is obtained by culturing them in 

 narrow tubes containing a high column of medium ; or by adding a piece of sterile 

 kidney, which produces a zone of anaerobiosis in its neighbourhood (Theobald 

 Smith's method). It is usual to cover the medium with a layer of parafiin oil ; 

 this was at first believed to prevent the ingress of oxygen, but it appears now 

 that this explanation is incorrect. The beneficial action of the oil probably 

 depends partly on the prevention of evaporation of water from the medium, and 

 partly on the prevention of the loss of COg from the medium, which would other- 

 wise become progressively more alkaline (Gates and Olitsky 1921, Kligler and 

 Robertson 1922). Spirochaetes have been cultivated mainly in liquid or in semi- 

 solid media. Colony production on the surface of solid media under aerobic or 

 anaerobic conditions has been reported by a few workers (Twort 1921, Gates 1923, 

 Aksjanzew-Malkiu 1933, Seguin and Vinzent 1938), but in general little success 

 has been obtained with this method. 



Multiplication occurs rather slowly, and may not be evident for a week or more. 

 When a clear medium is used, growth may be evident from the appearance of a 

 faint turbidity ; but generally microscopical examination is necessary, particu- 

 larly for motility and signs of transverse division. Most spirochaetes seem to prefer 

 a slightly alkaline medium, about pH 7-2-7-6. Subculture has to be performed 

 every few days as a rule, the exact time depending on the particular organism. In 

 young cultures the organisms are actively motile and under dark-ground illumin- 

 ation appear uniformly refractile ; but in older cultures when degeneration sets 

 in, they lose their motility, tend to agglutinate into clumps, and become granular ; 



