EFFECT OF INVERSION OF TUBES AT DIFFERENT INTERVALS ON
THE REDUCTION OF METHYLENE BLUE IN MILK
By E. V. SESHACHARYULU and ZAL R. KOTHAVALLA, Imperial Dairy Research Institute, Bangalore
(Received for publication on 11 January 1943)
DUCLAUX [1887] was the first to observe the
reducing action of milk on dyes and Neisser and
Wechsberg [1900] made use of the methylene blue
reduction in milk for judging the hygienic value
and keeping quality of milk. Barthel and Orla
Jensen [1912] devised a technique to carry out this
test which was used on the continent and in Eng-
land. In the United States and Canada, the
standard technique suggested by the American
Public Health Association [1934] was employed
for the grading of milk.
Wilson [1935] suggested a modification in per-
forming the methylene blue reduction test which
involves the inversion of test-tubes once at half-
hourly intervals. This modified method has now
been generally accepted. Frayer [1937] and
Johns [1938] have supported Wilson's modifica-
tion, whereas Thornton [1937] has opposed its
adoption on the basis that the inversion of the tubes
at half-hourly intervals complicated the technique.
Johns [1939] has shown that since there was satis-
factory agreement in the reduction times when the
tubes were inverted at intervals of ½, 1 or 2 hours,
a two-hourly inversion of methylene blue tubes
gave the same result as half-hourly inversion, thus
answering Thornton. Davis [1939], commenting
on Johns's results, has remarked 'if this is generally
true, one of the chief objections to the methylene
blue test largely vanishes'. No further work
appears to have been carried out either supporting
Johns's results or contradicting them.
The authors of this article, therefore, undertook
the investigation in question to ascertain whether
the findings of Johns have a general applicability
to different kinds of milk. The kinds of milk
examined in this investigation were; (a) individual
cow's and buffalo's milk, (b) cow herd and buffalo
herd milk, (c) mixed milk (cows and buffaloes),
(d) milk handled by town dairies, (e) village pro-
duced milk, and (f) pasteurized milk. On the whole
218 samples were examined.
Experimental
The technique employed for carrying out the
methylene blue reduction test was just the same
as suggested by Wilson with the exception that the
methylene blue tubes containing the same sample
of milk were inverted once at ½, 1, 1½, 2, 2½ and 3
hour intervals. Complete decolorization of the
methylene blue was taken as the end point. All
the experiments were done in duplicate. No
difference in reduction time between the dupli-
cates was found.
The average methylene blue reduction times in
minutes and their differences at different intervals
of inversion are given in Tables I and II.
TABLE I
Average methylene blue reduction times in minutes
at different intervals of inversion
TABLE II
Differences in reduction times in minutes
at different intervals of inversion from
those at half-hourly intervals
Kinds of milk
Individual cow's milk (I. D.I.
Farm)
Individual buffalo's milk (I. D.
I. Farm)
Cow curd milk (I. D. I. Farm) .
Buffalo curd milk (I. D. I. Farm)
Mixed milk (cows and buffaloes,
I. D. I. Farm)
Milk handled by town dairies
Village produced milk
Pasterisized milk (I. D. I. Farm)
I. D. I. Imperial Dairy Insti-
tute
Number
of
samples
30
30
34
31
31
20
20
22
½ hour
456.9
396.0
326.8
306.1
326.8
301.3
227.9
448.8
1, 1½
and 2
hours
456.9
396.0
326.8
306.1
326.8
301.3
227.9
448.8
2½ hours
470.7
410.9
339.1
318.8
341.1
313.0
240.5
457.5
3 hours
475.8
415.9
343.5
323.8
346.2
316.8
248.6
457.5
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
Average .
Maximum .
Minimum .
1, 1½
and 2
hours 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2½ hours
13.8
24.0
9.0
14.9
19.0
10.0
12.3
18.0
6.0
12.7
20.0
8.0
14.3
25.0
7.0
11.7
22.0
4.0
12.6
18.0
10.0
8.7
10.0
5.0
3 hours
18.9
32.0
14.0
19.9
25.0
14.0
16.7
25.0
12.0
17.7
31.0
18.0
19.4
35.0
11.0
15.5
25.0
8.0
20.7
34.0
13.0
8.7
10.0
5.0
277