TEXT-BOOK OF BACTERIOLOGY. 113 



pecially if, after some time, we add a little pure pyrogallic acid. 

 Then the test-tube culture — Esmarch's rolled system — which con- 

 tains the anaerobia to be developed is fixed in this larger tube with 

 a small wire support, and the whole immediately closed again with 

 the gutta-percha stopper. 



Yet this method does not, by any means, remove all the oxygen, 

 and strictly anaerobic bacteria often fail to thrive with it ; besides 

 which, it is very unpleasant to work with pyrogallic acid, and even 

 the most enthusiastic investigators dislike it. 



Gruber obtains a complete exclusion of air, and therefore of 

 oxygen, by the aid of the air-pump. About 15 cm. from the bot- 

 tom of a test-tube of easily-fusible glass he makes a narrow neck. 

 The tube is then provided with a cotton plug, sterilized in the hot- 

 air oven, and about 10 cm. of nutrient gelatin are poured into it and 

 rendered germ-free in the accustomed manner. Then the gelatin 

 is liquefied, and the inoculation takes place with the platinum 

 needle, the cotton plug being removed only as long as is necessary. 

 An air-tight India-rubber stopper is then put on, through which 

 passes a glass tube bent to a right angle and open at both ends. 

 This is placed in connection with the pump. By placing the test- 

 tube in water of 30° C. or 35° C, the gelatin is made to boil in the 

 rarefied air, and bj' pumping and boiling at the same time, the air 

 is removed in about fifteen minutes. The neck of the glass is now 

 melted and sealed hermetically. The gelatin slowly cools and, at 

 the proper moment, is rolled over the inside of the glass by Es- 

 march's method. 



This process has its decided advantages, so that it can be recom- 

 mended for many purposes. A microscopic examination of the 

 growing colonies offers no difficulty. The removal of portions for 

 inoculation can proceed as in the ordinary Esmarch tubes, after 

 the neck has been reopened and the removal of the oxygen is 

 thorough and absolute. If an air-pump be not at hand, or if for 

 any other reason we wish to employ gas instead of a vacuum, we 

 can only take hj^drogen. It has been found that this is the only 

 gas which is harmless for bacteria and does not work prejudicially 

 on their development; while carbonic-acid gas, for example, which 

 was formerly often employed, kills quite a number of different 

 micro-organisms. 



Prepare the hj^drogen in Kipp's apparatus with zinc and sul- 

 phuric acid, and conduct the gas, before emplo^-ing it, through two 

 purifying-bottles, one of which contains an alkaline solution of lead, 

 the other being fllled with alkaline pyrogallic solution, to remove 

 anv sulphuretted hvdrogen or small remains of oxygen. 

 8 



