OXYGEN SUPPLY OF CERTAIN ANIMALS 319 



The tank of commercial nitrogen used contained a small amount of 

 oxygen, as was determined by means of the Hemple gas pipette. The 

 oxygen was removed by passing the gas through two bottles of alkaline 

 pyrogallol (Pi — Po). 



Flasks of 150-cc. capacity were used in order to observe the reactions 

 of the organisms studied in the deoxj^genated water. Each observa- 

 tion flask (F) was provided with two tubes which were inserted through 

 the stopper. The intake tube was longer, reaching nearly to the 

 bottom of the flask, while the exit tube was level with the stopper. 

 Both tubes were provided with stopcocks, which were closed while 

 the experiment was in progress. 



To fill the observation flask (F) with deoxygenated water without 

 contact with air, the flask was first filled with tap water, its intake 

 tube connected with the exit tube of the large flask (.4), and the obser- 

 vation flask inverted. Nitrogen was then led from the tank (A^), 

 purified in the p}- rogallol bottles (Pi — Po) , through the tube (C) passing 

 around the boiling flask (A), and thence into the observation flask (F), 

 where it drove out the water. Following this the flask (F) was righted, 

 the stopcock on tube C was closed, and the stopcocks on tubes / and 

 E opened. Nitrogen was then passed through tube I to the bottom of 

 the boiling flask (A) thus stirring up any mud which might have been 

 boiled with the water. The pressure forced the oxygen-free mud and 

 water out of the flask through the tube E and into the observation 

 flask (F) where it displaced the contained nitrogen. Organisms to be 

 observed in the oxygen-free water were placed in the observation 

 flask before the filling process began, care being taken that they were 

 not washed out during the filling. 



As soon as one observation flask was filled, it was disconnected from 

 tube E and another could be put in its place. Series of flasks contain- 

 ing deoxygenated water and organisms were sealed with paraffin or 

 asphaltum and kept in the cellar where light and temperature factors 

 were as close as possible to those under normal conditions at the bot- 

 tom of Lake Mendota. 



6. Methods used in experimental work on animals 



The guaiacum used was freshly prepared 1 per cent solution of gum 

 guaiacum in 95 per cent alcohol. The benzidine solution was made by 

 dissolving 3 to 4 mgr. of benzidine crystals in 2 cc. of glacial acetic acid. 

 Commercial hydrogen peroxide was used. 



Aqueous extracts of animals were made by grinding them in a watch 

 crystal with a pestle. 



To determine whether or not the enzyme complex which was found 

 in the chironomid larvae would oxidize guaiacum in the absence of 

 molecular oxygen, the writer constructed the apparatus shown in 

 figure 2. This apparatus provided for the removal of oxygen from an 

 experimental chamber, in which reactions were then allowed to take 

 place. 



