_Procedure._—Add 0.4 cc. of the methylene blue solution to the sample in the 150 cc. bottle. As methylene blue has a slightly antiseptic property be careful to add exactly 0.4 cc. Add the methylene blue solution preferably below the surface of the liquid after filling the bottle with the sample. If the methylene blue is added first do not allow the liquid to overflow as coloring matter will thus be lost. Incubate the sample at 20° C. for ten days. Four days’ incubation may be considered sufficient for all practical purposes in routine plant-control work. If quick results are desired incubate the sample at 37° C. for five days using suitable stoppers[1a][2a] to prevent the loss and reabsorption of dissolved oxygen. The bacterial flora at 37° C. is different from that at 20° C. The lower temperature is more nearly the average temperature of surface waters and therefore the higher temperature should be used only when quick approximate results are essential. Observe the sample at least twice a day during incubation. Give a sample in which the methylene blue becomes decolorized a relative stability corresponding to the time required for reduction (see Table 15). For routine filter control ordinary room or cellar temperature gives fairly satisfactory results. For accurate studies, room temperature incubation is very undesirable, as the fluctuations in temperature which are ordinarily not noticed are responsible for appreciable deviations from the true values of relative stability. If the samples are incubated less than 10 days at 20° C. and are not decolorized place a plus sign after the stability value in order to indicate that the stability might have been higher if more time had been allowed. In applying this test to river waters it often happens that the blue coloring matter is removed either partly or completely through absorption by the clay which many rivers carry in suspension. True relative stabilities cannot be obtained for such waters except by determining the initial available oxygen at the start and the biochemical oxygen demand on incubation at 20° C. for 10 days (pp. 71–73). Germicides, such as hypochlorite of lime, if present in sufficient quantity, vitiate the results. If a sample contains free chlorine, therefore, store it about 2 hours, or until the chlorine is gone, and then add methylene blue. Table 15[78] gives the relation between the time in days to decolorize methylene blue at 20° C. (_t__{20}) and the relative stability number or ratio of available oxygen to oxygen required for equilibrium, expressed in percentage (S). Table 15.—RELATIVE STABILITY NUMBERS.