METHODS OF INVESTIGATION 19 



frequently used method of overlaying the water with par- 

 affin oil or similar substances is not very satisfactory. A 

 slow diffusion of oxygen does take place through these 

 substances. 



Special precautions are necessary when small aquatic 

 animals, like protozoa, are used, or w^hen processes are 

 studied that go on even at minimal oxygen tension, like 

 the elevation of the fertilization membrane in sea urchin 

 eggs. 



Kitching (1939a) devised suitable methods for proto- 

 zoa ; one of them will be described in some detail. It con- 

 sists in passing oxygen-free gas around a hanging drop 

 containing the organisms. The gas (hydrogen was mostly 

 used), taken from^a cylinder, was purified by being bub- 

 bled through concentrated sulfuric acid, dilute sulfuric 

 acid, dilute potassium hydroxide and distilled water. 

 It was then made oxygen-free by being passed through 

 an electrically heated quartz-tube containing platinized 

 asbestos. ''Finally the gas was carried by pure lead 

 tubing with seals of de Khotinsky cement through a 

 closed glass wash-tube with distilled water in it to the 

 chamber containing the organisms. The organisms were 

 mounted in a hanging drop on a cover glass which was 

 sealed with vaseline or a mixture of vaseline and par- 

 affin wax to the chamber. The hanging drojo was not 

 allowed to touch the vaseline. The chamber itself con- 

 sisted of a glass ring about 2 cm. in diameter and 1 cm. 

 deep, closed underneath by a microscope slide to which 

 it was sealed with de Khotinsky cement, and with glass 

 inlet and outlet tubes. The upper edge of the ring was 

 ground to support a cover glass, and the microscope slide 

 which formed the base of the chamber lEitted onto the 

 mechanical stage of the microscope. ' ' The outflowing gas 

 was bubbled through a suspension of marine luminous 

 bacteria which luminesce at about 0.005 mm. of oxy- 

 gen. They did not luminesce, however, in Kitching 's 



