348 WATER HYCIENE 
required oxygen. He stated that available oxygen is used up according 
to the following order: Dissolved oxygen, nitrates, nitrites, sulphates, 
phosphates, etc. Since all streams are not stable it is necessary to use 
other terms to fit their condition. For such conditions the term relative 
stability is used. Stable streams have a relative stability of 100 since 
they have all of the oxygen that is demanded by the organic matter. A 
relative stability of 60 means that a stream has 60 per cent of the oxygen 
necessary to make it stable. This is determined in the following manner: 
Reagent. Methylene blue solution. A 0.05 per cent aqueous solu- 
tion of methylene blue, preferably the double zine salt or commercial 
variety. . 
Collection of Sample. Collect the sample in a bottle holding approxi- 
mately 150 c.c. If the dissolved oxygen is low, observe precautions 
similar to those used in collecting samples for dissolved oxygen (p. 162). 
Use rubber stoppers or a cork stopper of good grade which has been 
boiled in water. 
Procedure. Add 0.4 c.c. of the methylene blue solution to the sample 
in the 150 c.c. bottle. As methylene blue has a slightly antiseptic prop- 
erty be careful to add exactly 0.4.¢.c. 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 as 
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 to prevent the loss and reabsorption of dissolved 
oxygen. The bacterial flora at 37° C. is different from the flora at 20° C. 
The lower temperature is more nearly the average temperature of sur- 
face 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 XXXIV). For routine filter 
control ordinary room or cellar temperature will give 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 
ten 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 
