INVESTIGATIONS ON THE RESAZURIN TEST FOR ASSESSING THE
HYGIENIC QUALITY OF MILK IN INDIA
An enquiry carried out under a scheme financed by the Imperial Council of Agricultural Research, New Delhi
By H. LAXMINARAYANA, Imperial Dairy Research Institute, Bangalore
(Received for publication on 6 May 1946)
T HE resazurin reduction test has recently come into great prominence in some of the western
countries as a rapid and discriminative method of judging the hygienic quality of milk. Resa-
zurin passes through a series of colour changes from the original blue to lilac, mauve, purple pink and
pink before being finally reduced to a colourless stage depending on the degree of activity of the
organisms present in milk [Davis, 1939 and 1942] and it is also highly sensitive to leucocytes [Thomas
and Davis, 1940]. For the routine testing of milk supplies, an one-hour resazurin reduction test
has been recommended in which milk is graded according to the colour shade to which resazurin is
reduced at the end of an hour's incubation at 37ºC.
Taking the reduction of resazurin to a purple pink, pink or colourless stage at one hour as an
indication of poor quality, Ramsdell et al [1935] and Barrett et al [1937] obtained a fairly good rela-
tionship between resazurin and methylene blue reduction tests. There was, however, a poor correla-
tion observed between the resazurin test and colony counts, although milks which did not reduce
resazurin appreciably within one hour generally had low counts whilst high count milks usually
reduced it within one hour. These workers also claim that the one hour resazurin test gives as much
information as the longer methylene blue reduction test gives in 5 to 7 hours and that the test is very
valuable in detecting physiologically abnormal and pathological milk on account of its great sensi-
tiveness to leucocyte action. Davis et al [1939] found that of 422 samples graded 'good' (blue)
by the one hour resazurin test, 59 failed methylene blue test and of 396 samples graded 'good' by
methylene blue test, only 5 graded 'poor' (pink) and 28 'doubtful' (purple pink) by the resazurin
test. Thomas and Davies [1940] found that 81 per cent of the samples examined by them passed or
failed both resazurin test (purple pink in one hour) and methylene blue test (accredited standards—
4½ hours in summer and 5½ hours in winter). Microscopic examination confirmed that milk with a
high leucocyte content reduced resazurin quickly and had a much less marked and more variable
action on methylene blue. Thomas. [1943] also found a close relationship between one hour resazurin
readings and methylene blue reduction times in half hours. The relationship between the colony
count and stage of reduction of resazurin was good in the case of milks with really high counts (over
10 millions per ml.) and with milks of very low bacterial counts (under 10,000 per ml.), but at inter-
mediate stages the relationship was poor. Thus market milks with high colony counts reduced
resazurin actively within one hour at 37ºC.
The resazurin test would, therefore, appear to possess considerable advantages over other known
methods for the determination of the bacteriological condition and hygienic quality of milk and its
adoption for testing milk in India would prove of great value in the efforts to improve the condi-
tions of milk production and supply in the country. Several modifications of the test have been
recommended for different purposes but owing to the high temperatures and wide climatic variations,
as well as the present state of milk production and supply in the country, the one-hour resazurin
test is likely to be found most useful for immediate application. Before introducing the test in India,
however, it is necessary to determine how it compares with the more familiar methylene blue-reduc-
tion and plate count tests when applied to the three types of milk—cow, buffalo and mixed (cow and
buffalo) milks—marketed in this country, and how far it is useful and reliable in assessing the hygienic
quality of milk produced and handled under Indian conditions. With the above object in view the
present investigation was undertaken.
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