88 ANAEROBIC ORGANISMS: ISOLATION AND CULTURE 



Petri dish isolation on agar {Klein's method). — Melt a tube of 

 glucose formate agar, pour out into a Petri dish and set aside to 

 cool. After the medium has thoroughly set, a trace of the material 

 under examination, or a drop of the whey of a typical milk culture, 

 or a drop of fluid from a serum culture, is diluted in a sterile 

 watch glass with from \ to i c.c. of sterile salt solution. A 

 platinum spatula of the form shown on Fig. 21 is sterilised in the 

 flame, dipped into the dilution, and is then drawn over the surface 

 of the set agar, so as to rub the fluid adhering to the spatula 

 uniformly over the whole surface of the agar. The Petri dish is 

 closed with its cover and placed on a small wire triangle above a 

 smaller and deeper glass dish, set upon a ground-glass plate con- 



FlG. 21. — Platinum spatula for Klein's method. 



taining a sufficient quantity of pyrogallic acid. A sufficient 

 quantity of liquor potassae is added to the pyrogallic acid in the 

 lower glass dish, and the whole apparatus is then covered with a 

 small bell glass, the free rim of which has been previously well 

 smeared with resin ointment so as to ensure the bell glass, on 

 being well pressed down on the ground-glass plate, an air-tight 

 seal. 



4. Petri dish cultivation in vacuo or in hydrogen ^— 

 When a supply of hydrogen and the vacuum pump are avail- 

 able, a modification of the ordinary method of isolation by means 

 of Petri dish cultivation can be employed. Either gelatine or 

 agar media can be used, but in each case glucose should be added 

 in the proportion of 2 to 3 per cent. 



Cultures in glucose gelatine — 



The medium should be boiled for ten minutes in a water bath 

 in order to expel the free oxygen, and then quickly cooled down 

 under the tap to below 40" (a temperature approaching that of the 

 solidifying point). The tube is then rapidly inoculated in the 

 usual manner, and poured out without delay into the Petri dish, 



' If the most stringent anaerobic conditions are desired, solidification should 

 take place either in vacuo or in a current of hydrogen, but under ordinary 

 circumstances the above will be found sufficient for the purpose, the glucose 

 acting as a reducer of any free oxygen present in the medium. 



