9O E. NEWTON* HARVEY. 



There are four convenient methods of removing the last trace 

 of oxygen from biological fluids. I say methods for biological 

 fluids because none of these methods involves very great changes 

 in H-ion concentration that might bring about changes in the 

 Huid. although all of them are not suited for living cells. All 

 these methods prevent the luminescence of bacteria (Harvey and 

 Morrison, 1923) and Cypridina, which certainly means less oxygen 

 than io~ 5 atmospheres, and most of them involve nascent 

 hydrogen. 



1. By addition of cells actively using oxygen such as muscle, 

 yeast or bacteria. If the fluid is a cell extract requiring oxygen, 

 it will use up its own oxygen on standing in a narrow tube. The 

 reducing power of cells varies but practically all will reduce 

 methylene blue and most will reduce indigo-carmine. For the 

 significance of this in terms of oxidation-reduction potential the 

 reader is referred to the papers of Clark (1923-25). 



2. Nascent hydrogen can be generated in the fluid by adding 

 aluminium amalgam or magnesium plus some neutral ammonium 

 salt. In the first case A1(OH) 3 is formed and in the second an 

 ammonium-magnesium salt. In either case the PH does not 

 change much, and the oxygen is quickly washed out. 



3. Small amounts of sodium hydrosulphite (or hyposulphite, 

 Na 2 S 2 O 4 , not thiosulphite) may be added to the fluid. It is 

 best to dissolve the hydrosulphite in a powerful buffer and to 

 buffer the fluid under investigation as the hydrosulphite is acid. 

 To determine when the right amount of hydrosulphite has been 

 added methylene blue can be used as indicator. It is decolorized 

 when reduced by the hydrosulphite, which also absorbs all 

 oxygen in the solution. Shaking with air or addition of potassium 

 ferricyanide [KsFe(CN)e] will oxidize the hydrosulphite with 

 return of the blue color to methylene blue. The advantage of 

 hydrosulphite is its practically instantaneous production ot 

 anaerobic conditions. The disadvantage is a possible injurious 

 effect on living cells, which I believe should be investigated 

 rather carefully. My own observations show that the ciliated 

 cells of the gills of Mytilus, lightly stained in methylene blue, 

 stop beating and the blue color disappears (reduction) in hydro- 

 sulphite sea water very quickly. When fresh aerated sea water 



