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Investigations on Resazurin Test
EXPERIMENTAL
The one-hour resazurin test, methylene blue reduction test and plate counts were carried out
on some 650 samples of cow, buffalo and mixed (cow and buffalo) milks, drawn from different sources,
namely ;
(i) farm produced milk (herd samples from Imperial Dairy Research Institute and a private
dairy herd nearby),
(ii) village produced milk (from producer-retailers and street hawkers) and
(iii) milk handled by private dairies (individual producers' supplies and bulk tank samples).
In the case of farm produced milk, the samples were obtained at the time of production (morn-
ing) and examined within 2 to 3 hours after keeping them in the refrigerator (40º-50ºF.) during the
interval. Village produced milk samples and those from private dairies were brought to the laboratory
in ice boxes and examined immediately. The latter class of milk probably included both morning
and previous evening's productions and came to the dairies from city stables as well as from moffusil
centres (10 to 12 miles). The interval between the times of production and examination in the case
of village samples was from 5 to 6 hours while in the case of private dairies it varied from 5 to 16
hours. During this interval, the milk was exposed to atmospheric temperatures varying from 68º
to 95ºF. (average morning temperatures in the late winter and early summer months in Bangalore).
Methods. The plate counts were determined on milk agar at 37ºC. [cf. Ministry of Agriculture
1934] and the methylene blue reduction test was carried out according to Wilson's modified technique
[Wilson, 1935] The method recommended by Davis [1939] and further described by Davis [1943]
and Davis and Newland [1944] was followed for the one hour resazurin test. One ml. of 0.005 per
cent resazurin solution (prepared from Eastman Kodak resazurin powder) was added to 10 ml. of
milk and the tubes gently inverted twice to ensure proper mixing and placed in a water-bath at a
temperature of 37º±0.5ºC. The methylene blue reduction tubes were also prepared and incubated
and the samples plated out at the same time. At the end of one hour the resazurin tubes were gently
inverted and the stage of reduction (colour of resazurin) noted.
The colour shades shown by resazurin were quite distinct and well-defined and after a little
experience it was possible to record them subjectively. In view of the wide differences in fat contents
of Indian milks (from 3.5 per cent in cow's milk to 7.5 per cent in buffalo's milk), the exact matching
of the colours presented some difficulties. Samples of milk having a low fat content showed deeper
tints than those with high fat content and in addition it is possible that the slight yellow tinge in
cow's milk due to carotene may affect the shades to some extent. As the colour shades observed in
the case of mixed (cow and buffalo) milk with 5 to 5.5 per cent fat were found to agree fairly satis-
factorily with those shown by all types of milk, they were taken as standards and painted on glass
slides for reference in cases of doubt.
It was observed in several cases that the resazurin reduction was very rapid at first but the cor-
responding methylene blue reduction time was fairly long. In such anomalous cases, a microscopic
examination of the milk smears was carried out.
RESULTS
The results of the one hour resazurin test, methylene blue reduction times and plate counts
obtained for the different types of milks examined are summarized in Tables I to IV below.
DISCUSSION
The object of the investigation, as stated in the introduction, was to find out the applicability
of the resazurin test for determining the hygienic quality of Indian milks. Accordingly the results
have been interpreted so as to give indications on :
(i) the stages of resazurin reduction to be used for grading Indian milks ;
(ii) the comparative grading efficiency of the resazurin test ; and
(iii) the effect of the conditions of production on the resazurin test.