CALVIN B. COULTER 313 



of the halves. The ends of the side arms are then hooked into the 

 electrode tubes containing buffer solution, and the cocks carefully 

 opened. The fluid in the middle portion is brought to the same level 

 on each side by adding a small amount, one or two drops, of buffer 

 solution to the buffer chamber on one side or the other. With this 

 type of apparatus the fall in electrical potential across the U-tube is 

 relatively great, and the change in hydrogen ion concentration in the 

 test fluid during the passage of current is reduced to a minimum. 

 The determinations were made at 19 ± 1°C. without a thermostat 

 in a room of even temperature. The current used was the Edison 

 direct street current of 220 volts, giving a drop of almost exactly 7 

 volts per cm. in the U-tube. 



Fresh defibrinated sheep blood was washed with two or more 

 changes of saline solution and then freed from electrolyte by washing 

 with four changes of ten volumes each of isotonic saccharose solution 

 (m/4), and finally made up to 10 per cent concentration of cefls in 

 saccharose. It was found that the reaction of the saccharose solu- 

 tion should not be more alkaline than pH 6.5 as at more alkaline reac- 

 tions the viscidity of the sedimented cell mass is so great that it is 

 reemulsified only with difficulty and with the development of consid- 

 erable hemolysis. 



To determine the direction and rate of movement of red cells at 

 varying hydrogen ion concentrations, a titration was first carried out 

 to determine the H concentration resulting from the addition of 

 graded amounts of HCl to the cell suspension at hand. 5 cc. of cells 

 were introduced into twelve or more tubes, in two series. To the 

 first series of tubes were then added 5 cc. of saccharose solution and 

 increasing amounts of m/10 or m/100 HCl, the latter dilution made up 

 in saccharose. Between manipulations the tubes and the stock flask 

 were kept tightly stoppered to exclude atmospheric CO2 as far as pos- 

 sible. After gentle agitation the tubes were allowed to stand at room 

 temperature for 10 to 15 minutes, then centrifugated and the super- 

 natant fluid was drawn off. Of this, one portion of 5 cc. with indicator 

 added was compared with a standard series for the colorimetric deter- 

 mination of hydrogen ion concentration, the remaining portion 

 serving as a color screen for the standard tube. The value thus 

 obtained represents the H concentration with which the cells were 

 in equilibrium. 



