METHODS OF PHYSIOLOGICAL ANALYSIS 69 



oxidized dye, the most readily observed and most conclusive evidence of 

 oxidation-reduction pattern is obtained by intracellular reduction of the 

 oxidized dye. This can be made to occur rapidly or slowly as desired. The 

 material can be brought at once, after staining, into anaerobic conditions, 

 where reduction will take place rapidly or decrease in oxygen may be 

 brought about gradually by the oxygen consumption of the material itself 

 in a sealed container of small volume. It is necessary to make certain that 

 injury from lack of oxygen or accumulation of carbon dioxide or other 

 products of metabolism does not occur before reduction. 



Suitable concentrations and periods of exposure to the dye must be de- 

 termined by trial for the organism concerned, with both oxidized dye and 

 leucobase. Too high concentration or too long exposure results in differen- 

 tial injury and retardation or absence of reduction of dye in the injured 

 region; consequently, the reduction pattern becomes different from that of 

 the uninjured individual. An axial reduction gradient may even be the re- 

 verse of the normal, because the region which normally reduces most rap- 

 idly is also most susceptible to injury by the dye and, when injured to a 

 certain degree, though still living, reduces least rapidly. Examples are 

 given in the following chapter. 



If a true picture of oxidation-reduction pattern is to be obtained, it is 

 also highly important, with gradual oxygen decrease by the living mate- 

 rial, that equal oxygen distribution in the medium be maintained; other- 

 wise differences in reduction which have no relation to physiological pat- 

 tern may occur. For example, when two or more eggs or embryos lie close 

 together without change of position after staining, reduction usually oc- 

 curs first in the adjoining regions of each, because oxygen decreases more 

 rapidly between them than elsewhere. A region of an embryo remaining 

 continuously in contact with the bottom of the container is likely to re- 

 duce before others. Free-swimming organisms in a sealed container pro- 

 vide more or less completely for equal oxygen distribution; but if they 

 tend to aggregate, unequal distribution usually results. An aggregation of 

 Paramecium reduces much more rapidly after staining with methylene 

 blue than isolated individuals swimming free in the same preparation, and 

 at a certain oxygen-level an individual which has become colorless in the 

 aggregation becomes blue at once on leaving it. With nonmotile forms — 

 cleavage stages, for example — in a small sealed container equal distribu- 

 tion of oxygen may be maintained by frequent change in position of the 

 preparation with respect to gravity. 



It is not to be assumed that a dye-reduction pattern — for example, an 



