FIG. 65. (a) pinchcock ; 

 (6) mercury. 



SOURCES OF ENERGY IN ANAEROBES 535 



researches of Traube (1874), Hiifner (1876), Nageli (1879), Nencki (1880), 

 Liborius (1886), and others 1 . 



Methods. The following arrangement may be employed to demonstrate the 

 existence of anaerobic organisms. A flask of about 300 cc. capacity is two-thirds 

 filled with a slightly alkaline nutrient solution containing 

 5 per cent, sugar, i per cent, meat-extract and i per 

 cent, peptone, and coloured blue by indigo-carmine. 

 After introducing a trace of a suitable putrefying fluid, 

 hydrogen is passed through, a and b are closed, and the 

 whole apparatus is placed in a warm room. The indigo 

 is soon reduced as the developing bacteria absorb the 

 last traces of oxygen, and the increasing turbidity is 

 obviously due to the development of anaerobes. At the 

 end of the experiment oxygen may be readmitted, when 

 the reduced indigo-carmine at once turns blue 2 . 



The air may also be removed from an inoculated flask by means of an air- 

 pump. The enclosed fluid is then caused to boil for some time at about 30 C. 

 until about one-third of the contents have evaporated, when the tapering neck of the 

 flask may be drawn out and sealed in a Bunsen flame. When gelatine cultures are 

 desired, hydrogen may either be passed through the gelatine while still fluid, or 

 through special chambers into which the gelatine has been previously poured. 

 The most sensitive anaerobes are able to develop upon Petri plates or in test-tubes 

 placed in a chamber containing no oxygen, as in Fig. 66. The bell-jar is repeatedly 

 exhausted and then re-filled with hydrogen, and if the taps are air-tight and the 

 hydrogen pure this method suffices for 

 all requirements. To attain the latter 

 end the acid in the Kipp's generating 

 apparatus should be covered with a layer 

 of liquid parafin and the hydrogen passed 

 through pyrogallol and potash in addi- 

 tion to the usual purifying tubes. (Figure 

 in Unters. a. d. Bot. Inst. z. Tiibingen, 

 1885, Bd. i, p. 637.) 



The usual methods of isolation and 

 cultivation are applicable here also (cf. 

 Sect. 66). The development may be. 

 followed microscopically by the use of gasr 

 chambers s , in which any given pressure 

 of oxygen may be maintained by partial evacuation or by the introduction of 



FIG. 66. (e) vessel containing water. 



1 For literature, see Huppe, Methoden d. Bakterienforschung, 1891, 5. Aufl., p. 354; Fliigge, I.e., 



p. 125. 



* For an account of the different methods employed in the culture of anaerobes, cf. Huppe, 

 Methoden d. Bakterienforschung, 1891, 5. Aufl., p. 354; Novy, Centralbl. f. Bact., 1893, Bd. XIV, 

 'pp. 566, 581 ; Beyerinck, Butylferment, 1893, pp. 17, &c. 



' 3 Clark, Ber. d. Bot. Ges., 1888, p. 273. Cf. Bot. Zeitung, 1887, p. 31. 



