488 BACTESIOLOGIC TECSNIC. 



advised that, in making agar (partly also gelatin) plates, the nutrient 

 medium be first allowed to solidify in the dishes, and then the surface 

 be superficially smeared over with the material to be examined by 

 means of a platinum loop, strips of filter-paper, or a platinum brush. 

 Only characteristic surface colonies are obtained in this way. 



( / ) Sugar=agar shake cultures : The contents of a tube are 

 melted in the water-bath and cooled down to about 40°. A loopful of 

 the pure culture is then introduced and thoroughly distributed, and 

 after the medium solidifies the tube is placed in the incubator. 



4. Anaerobic Cultures. 



We have employed almost exclusively the method of H. Buchner: 

 Absorption of oxygen by pyrogallic acid in the presence of potassium 

 hydroxid. ' 



(a) For Stab Cultures. — At the bottom of a glass cylinder, which 

 must be a little longer than the test-tube, is placed a heaping teaspoon- 

 ful of pyrogallic acid and 20 c.c. of 3% potassium hydroxid solution. 

 The inoculated stab culture is then placed in the cylinder, which is 

 closed at one end with a soft rubber stopper or a ground-glass stopper 

 which is sealed with paraffin. According to Kitasato, anaerobes 

 which are less sensitive to oxygen may be cultivated in high stab cul- 

 tures in sugar-agar without pyrogallic acid. A stab 8 to 10 cm. deep 

 is made in sugar-agar with a small loop and the needle turned upon its 

 long axis before being withdrawn. 



(J) For Plate Cultures. — Instead of the glass cylinder, a wide exsic- 

 cator with a ground cover is used. The lower part is filled with sand 

 and the pj'rogallic acid mixture, and then the manipulation is as above 

 (Arens). 



If it is desirable to obtain the most perfect anaerobiosis, the pyro- 

 gallic acid method is combined with either the pumping out of the air 

 with a water-pump or the displacing of the air with hydrogen, so that 

 only a slight trace of oxygen remains to be taken up by the pyrogallic 

 acid. We have employed the latter method many years. The cultures 

 are placed in a roomy exsiccator ^vith sufficient pyrogallic acid and 

 potassium hydroxid, and then, by means of a double perforated rubber 

 cork, hydrogen is allowed to flow through for one-half hour. After 

 closing the opening, we sink the whole apparatus, weighted with lead, 

 in water. 



Kabrhel recommends (C. B. xxv, 555), as a control for the absence 

 of oxygen, that a tube be introduced which contains liquefied nutrient 

 gelatin, to which is added, just before use, O.Zfo to 1.0% grape-sugar, 

 and which is rendered a transparent blue with a strong alcoholic solu- 

 tion of methylene-blue. Such an uninoculated tube is completely 

 decolorized in twenty-four to thirty-six hours only in a chamber 

 entirely free from oxygen. This indicator will also point out how 

 essential it is to remove covers, corks, etc., in the case of anaerobic 

 cultures. 



' Sensitive varieties are said to thrive better in an atmosphere of 

 hydrogen. 



