Chapter V —37— Physical Ch emistry 



measuring cells which have not been injured, for the very delicate 

 protoplasm modifies its pK the moment that there is any alteration 

 in the cell. Vles has insisted, on the other hand, on the fact that 

 the measurement has no value unless during the entire experiment 

 the cells keep their normal CO2 pressure. Now the altered proto- 

 plasm in contact with air, unless special precautions are taken, 

 loses its CO2, which raises its pU, and it is difficult in the extreme 

 to realize ideal conditions. 



Electrometric methods or Clark indicators are used to deter- 

 mine pB. values. The electrometric method has been used on 

 crushed cells, extracts of plant juices, and finally on living cells. 



Vles, Reiss and Wellinger have advocated a technique which 

 consists of crushing the cells after instantaneous freezing and 

 effecting a measurement at the precise moment when the crushed 

 mass returns to the cryoscopic point. In this way all escape of 

 CO2 seems to be avoided. But more often one is limited to meas- 

 uring the pB. of plant juices obtained from crushed tissues by the 

 electrometric method, sometimes even in a pressure of 100 atmos- 

 pheres (Kappen). Another method consists of introducing micro- 

 electrodes into living cells (Crozier, Ellis, Peterfi, Small) but 

 this is applicable only in cases of large cells. This method likewise 

 causes injuries to the cells and undoubtedly brings about a mixture 

 of cytoplasm with vacuolar sap. It is moreover a difficult opera- 

 tion in plant cells because of the resistance of the cellulose walls. 



As the electrometric method is extremely delicate and necessi- 

 tates costly apparatus, the method most used is the Clark indicator 

 method. This presents great disadvantages, for the point of color 

 change may be modified by the presence of protein matter and its 

 products of disintegration, such as the amino acids. Thus in col- 

 loidal gels the color change is aberrant in consequence of chemical 

 reactions. This is called the protein error. Other causes of error 

 result from the fact that the indicators dissolved in the lipides are 

 not dissociated and that then their color does not correspond to any 

 indicator. This causes a lipide error. The point of color change 

 may also be displaced by the presence of a strong concentration of 

 neutral salts : the salt error. It may also be changed by a modifica- 

 tion in the concentration of the indicator: concentration error. 

 Finally, the indicators do not penetrate living cells. Vles has used 

 the method of crushing cells which consists of producing a sudden 

 yet careful pressure on cells in an indicator solution between cover 

 slip and slide, causing a break limited to the cell wall. An immedi- 

 ate release in pressure leads then to the taking into the interior of 

 the cell of a small quantity of the indicator, the color of which can 

 thus be noted before any effusion of CO2. 



Botanists, however, have usually been limited here also to the 

 use of indicators either on juices extracted from tissues or on 

 sections of tissues which, in being sectioned, were necessarily 

 injured. 



A more precise method for measuring pH has been to introduce 

 dyes by micro-injection with the aid of a micromanipulator. The 



