350 WATER HYGIENE 
The theory of the test is unique. Methylene blue is very sensitive 
to products of putrefaction. Such compounds as hydrogen sulphide 
mercaptans, etc., and other reducing substances quickly reduce it to its 
colorless leuco base. Consequently when this is mixed with a polluted 
water, it will change from blue to white as soon as any of these com- 
pounds are formed. They are present when putrefaction sets in and, 
therefore, cause the change in the color of the dye. Phelps has stated 
that this occurs when the nitrites are used up. This reducing time rep- 
resents the time “ required for the exhaustion of available oxygen.” 
Lederer (1914, 1915) who, as bacteriologist and chemist for the 
Sanitary District of Chicago has had ample opportunity to study this 
subject, has pointed out that great care must be used in the test to 
secure comparable results. He has devised some unique methods for 
the study of required oxygen. They are given in Standard Methods as 
follows: 
Biochemical Oxygen Demand of Sewage and Efluents (Lederer, 
1914). Relative Stability Method. The relative stability method may 
be employed to obtain a measure of the putrescible material in sewages 
and cffluents in terms of oxygen demand. 
Procedure for Effluenis. Divide the total available oxygen, including 
the oxygen of nitrites and nitrates, by the relative stability expressed 
as a decimal. 
Procedure for Sewages. Make one or two dilutions with fully aerated 
distilled water of known dissolved oxygen content. Tap water may be 
employed if it is free from nitrates. Vary the relative proportions of 
sewage and water to be employed to give a relative stability of from 50 
to 75. Unless proper seals are employed bring the water as well as the 
sewage to the temperature at which the mixtures are to be incubated 
before preparing the dilutions. During the manipulation avoid aeration. 
Having made the proper dilutions, determine the relative stability of each. 
Calculate the oxygen demand in parts per million by the following 
formula: 
. OC1-p) 
Oxygen demand is Rp 
In this formula O is the initial dissolved oxygen of the diluting water; 
0 is the proportion of sewage; and & is the relative stability of the mix- 
ture. Ordinarily the available oxygen in crude sewages, septic tank 
effluents, settling tank effluents, and tradewastes can be neglected. 
Sodium Nitrate Method. For the determination of the biochemical 
oxygen demand the sodium nitrate method may be used. The method 
