344 
Notes . 
BUFFER EFFECTS OF TAP-WATER IN THE ESTIMATION OF 
CARBON DIOXIDE BY CHANGE IN HYDROGEN-ION CONCENTRATION.— 
The hydrogen-ion concentration method of measuring respiration, as described 
by Haas (1), involves a consideration of the buffer effects of the reagents employed. 
In this method the respiring material is placed in water containing an indicator, and 
the time taken to change the medium from one known P H value to another is noted. 
The P H is determined by comparison with standard buffer solutions having the same 
concentration of indicator and contained in tubes of the same dimensions as the tube 
with the respiring material. After several closely agreeing determinations have been 
made, a solution of the reagent is substituted for the water and the effect noted. In 
this way the times taken to do the same amount of work are compared : that is, 
a measure of the reaction velocities is obtained. The normal rate of respiration 
is taken as the reciprocal of the time required to effect the standard change of P H in 
water alone, and the rate under the influence of the reagent is expressed as per cent, 
of the normal. 
Owing to the known injurious effect upon organisms of prolonged exposure to 
distilled water, tap-water is frequently used in these experiments. As a preliminary 
to a study of the effect of the hydrogen-ion concentration of the medium on the 
respiration of Wheat seedlings, the writer has made an investigation of the buffer 
effects of tap-water which brings out some points of interest. 
Method. Water from the Edinburgh main supply was boiled in a £ Resistance ’ 
glass flask until free from carbon dioxide. The flask was closed with a clean stopper 
while still boiling, and allowed to cool to room temperature before being used. The 
temperature at which the experiments were performed was i6°c. + [ * This boiled 
water was titrated with a standard solution of carbon dioxide, prepared as follows : 
distilled water was charged with carbon dioxide under pressure in a ‘ Sparklet’ 
siphon; io c.c. of this solution were mixed with 30 c.c. distilled water. This 
standard solution was approximately saturated (a saturated solution of C 0 2 at 25 0 C. 
has a P H of 4-8) and showed a P H of 5 + 0-05 with methyl red, and did not vary 
appreciably in four hours’"exposure to the laboratory air in an open beaker. As each 
determination occupied about twelve minutes, the C 0 2 solution may be assumed to be 
constant for that period. With a pipette of fine bore, graduated to 0 01 c.c., and 
provided with a rubber bulb, it was possible to drop accurately 0-05 c.c. 
10 c.c. of the boiled tap-water were taken in a Jena glass test-tube (4" X %") fitted 
with rubber tubing at the mouth to enable it to be clamped off easily. Three drops 
each of phenol red (1 in 10,000) and thymol blue (1 in 1,000) were added, and the 
resulting colour matched with a set of borate buffers made up according to Palitzsch’s 
directions (2). These gave a range of P H values from 9-24 to 6-77, and with the 
above mixture of indicators a well-graded and easily read series of colours was 
obtained. The boiled tap-water gave a very constant upper limit of P H 9-24. 
One drop of the C 0 2 solution was added, and the tube at once clamped off and 
quickly inverted. A small bubble of air included acted as stirrer. (Any error 
introduced in this way was a constant one.) This was repeated until the next (more 
acid) buffer standard in the series was matched, and the procedure continued until 
P H 6-77 was reached 
