66 L. N. M. ])x:ysens 



2. The pretreatment of the culture. Factors such as age, growth 

 medium, and Hght intensity may be varied. 



3. The intensity and wavelength of the exciting light and the 

 lengths of light and dark periods. 



Further data are obtainable by following the time course of changes 

 in optical density caused not by light but by alterations in the me- 

 dium. These measurements are not discussed in this paper. 



EVALUATION OF THE DATA 



Among the more useful results which can be derived from measure- 

 ments of the time course at various wavelengths are the difference 

 spectra. These spectra often allow the identification or characteri- 

 zation of the substances changing their absorption. 



The difference spectrum can be obtained as follows. All external 

 conditions are kept approximately constant, and a periodic sequence 

 of Ught and dark periods is given. It is often found possible to select 

 the external conditions and the patterns of illumination in such a 

 way that at each wavelength the (change in) optical density is a periodic 

 and reproducible function of time. A difference spectrum is then ob- 

 tained by plotting the changes in optical densities which occur be- 

 tween two corresponding times t and t' of the time course graph as a 

 function of the wavelength. This difference spectrum is equal to the 

 difference of the absorption spectra of the cells at the times t and t' . 

 It is the sum of the difference spectra of the intermediates and other 

 substances, which change their absorption spectra. If t is the time 

 at which the illumination period starts and t' is a time between the 

 beginning and end of the illumination period, the difference spectra 

 at various times t' may give a clue to the time sequence in which the 

 intermediates change their absorption spectrum. If, e.g., one inter- 

 mediate changes much more rapidly at the onset of illumination than 

 the others, the difference spectrum obtained for a time t' shortly 

 after the start of illumination is caused only by this intermediate. 



Another family of difference spectra can be obtained selecting a 

 different value of a parameter, such as the intensity of the exciting 

 light or the composition of the medium. The comparison of difference 

 spectra obtained under various conditions may greatly facilitate the 

 analysis of these spectra in terms of the difference spectra of single 

 intermediates. After such an analysis has been carried out, it is pos- 

 sible to plot the (relative) concentrations of the intermediates (or 



