_Reagent._—Methylene blue solution. A 0.05 per cent aqueous solution of
methylene blue, preferably the double zinc salt or commercial
variety.[60b]
_Collection of sample._—Collect the sample in a glass-stoppered bottle
holding approximately 150 cc. If the dissolved oxygen is low observe
precautions similar to those used in collecting samples for dissolved
oxygen (p. 66).
_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.
───────────────────────────────────┬───────────────────────────────────
Time required for decolorization at│ Relative stability.
20° C. │
───────────────────────────────────┼───────────────────────────────────
_Days._ │ _Percentage._
0.5│ 11
1.0│ 21
1.5│ 30
2.0│ 37
2.5│ 44
3.0│ 50
4.0│ 60
5.0│ 68
6.0│ 75
7.0│ 80
8.0│ 84
9.0│ 87
10.0│ 90
11.0│ 92
12.0│ 94
13.0│ 95
14.0│ 96
16.0│ 97
18.0│ 98
20.0│ 99
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