MMTIIODS or CULTIVATING ANAKROBIC BACTKIUA 141 



well. Thus 11. Koch, in 1884, proposed to cover the gelatin plates with a film 

 of mica, which, however, according to the ex- 

 perience of P. LIBORIUS (I.), is not always 

 sufficient in the case of strictly anaerobic 

 bacteria. On the other hand, a second 

 method (also emanating from the German 

 State Board of Health) has proved highly 

 suitable, viz., that of cultures in deep layers, 

 as prescribed by W. Hesse in 1885. For 

 these a puncture culture is made in test- 

 tubes containing nutrient gelatin or agar- 

 agar, the medium being covered, after 

 successful inoculation, with a sterile stratum 

 of the same liquefied substance. 



Another method, which has also been 

 variously modified, is that first employed by 

 Pasteur in connection with his studies of 

 the " vibrion septigue," which consists in ex- 

 hausting the air (oxygen) from the vessel 

 containing the nutrient medium inoculated 

 with an anaerobic organism. The modifica- 

 tion made by MAX GRUBER (II.) is con- 

 venient and reliable, and is in general use, 

 especially in the laboratories of Fermenta- 

 tion Physiologists. Strong test-tubes (Fig. 

 45), about 7 inches long, with a much con- 

 stricted portion in the upper third of their 

 length, are used. These are filled with 

 about 10 c.c. of nutrient medium, then 

 closed by a cotton plug, and after inoculation 

 are immersed in water at about 30 35 C., 

 and then connected with an air-pump. The 

 air is all driven out by the water vapour given 

 off under the diminished pressure, where- 

 upon the narrow part of the tube is closed 

 by fusion and the upper portion removed. 

 By the use of nutrient gelatin this method 

 also facilitates the cultivation of colonies, so 

 that the individual anaerobic species in a 

 bacterial mixture can be isolated. For this 

 purpose the still warm liquid contents of 

 the tubes are converted into Esmarch roll- 

 cultures, as shown in Fig. 46. 



In place of removing the air from the 

 culture vessel by mechanical means pump- 

 ing or driving it out by vapour recourse 

 may be had to oxygen-absorbing chemicals. 

 For this purpose a solution of pyrogallic 

 acid [v-C 6 H 3 (OH) 3 ] in caustic potash, a 

 mixture that takes up oxygen with avidity, 

 and which, as is well known, has long been 

 in use in gas analysis, is employed. It was 

 introduced into physiological work by Nencki in 1880, as a test for the presence 

 of anaerobic organisms, but it was not until the publication of H, BUCHNER'S (VII.) 



FIG. 46. 



Gruber's anaerobic 

 tube exhausted of air. 



Upper portion removed 

 by fusing-. Contents 

 arranged as an Es- 

 march culture where- 

 in the germs have 

 developedto colonies. 

 Somewhat reduced in 

 size. (After Gruber.) 



FIG. 47- 



Buchner's anaerobic 

 tube. 



The pyrogallol solution 

 is at />, the wire sup- 

 port resting therein 

 and carrying- the 

 test-tube with the 

 inocnla ed nutrient 

 medium (n). Some- 

 what reduced. (After 

 Bttchner.) 



21 



