226
Resazurin Test for Assessing the Sterility of Dairy Utensils
[ XVII
The washed or sterilized utensils as mentioned below were thoroughly rinsed with quarter-
strength Ringer's solution in quantities indicated against each.
Half pound bottles . . . . . . . . . . . . .
One pound bottles . . . . . . . . . . . . .
Milk pails . . . . . . . . . . . . . . .
Churns . . . . . . . . . . . . . . .
Receiving tank . . . . . . . . . . . . .
Cooler . . . . . . . . . . . . . .
10 ml.
20 ml.
150 ml.
500 ml.
500 ml.
500 ml.
In the case of the cooler, the whole surface was thoroughly scrubbed with a sterile brush using
500 ml. solution for the purpose. The utmost precautions were observed so as to carry out the opera-
tions under aseptic conditions and absolutely sterile apparatus was used for performing the tests.
Slight exposure to outside contamination could not, however, be avoided in the case of the receiving
tank and cooler. In all the trials controls were employed and wherever extraneous contamination
was suspected, the results of such experiments were discarded.
Plate counts. Portions of the rinsings (1 ml. and 3 ml. in the case of sterile utensils and suitable
dilutions in other cases) were plated in triplicate on milk agar. The plates were incubated at 37°C.
for 48 hours and averages of the colony counts taken excluding those showing more than 20 per cent
discrepancies.
Presumptive coliform test. Portions of the rinsings in appropriate serial dilutions were tested
for the presence of coliform bacteria in McConkey broth tubes (duplicate) after 48 hours' incubation.
Negative tubes were incubated for another 48 hours and the reactions noted.
Resazurin test. One ml. portion of the rinse solution was added to 10 ml. sterile separated milk
(aged for not less than two weeks) and then incubated at 22°C. for 24 hours. The incubated sample
was then thoroughly shaken, one ml. of 0.005 per cent resazurin solution added and the resazurin
colour changes at 37°C. after 10, 20, 30, 60, 120 and 180 minutes observed using the standard Com-
parator for comparing the colours [Davis and. Watson, loc. cit.].
In the course of the preliminary trials with bottles, it was found that the incubated samples in
respect of both the washed and partially sterilized series, invariably showed a rapid resazurin reduc-
tion (pink or colourless) in 30 minutes. This was probably due to the long incubation of 24 hours
allowing an undue development of the initial flora. Furthermore, any substantial shortening of this
period would help in considerable saving of time in obtaining the results. Accordingly other time-
temperature combinations for incubating the rinse samples were tried out. Of these, incubation
for 16 hours at 22°C. was found to give most satisfactory results and was also convenient as it corres-
ponded with the farm routine. Hence the resazurin test was carried out on rinse samples incubated
for 16 hours at 22°C.
RESULTS
The mean colony counts (per ml. of rinse solution) obtained in respect of different utensils and
the corresponding resazurin reduction stages at the end of 30 minutes, one hour and two hours are
given in Table I. The resazurin colour shades (blue, lilac, mauve, pink mauve, purple pink, pink
and white) are expressed in terms of disc numbers 6 to 0 (indicated on the resazurin. comparator)
for facilitating interpretation. There was no appreciable colour change at the end of 10 minutes
due to the interval being too short and the reduction after three hours had invariably advanced to
either a pink or colourless stage in almost all cases. Hence these observations are not included in
the data. The results of the coliform test for each group of samples are also given.
It is seen that the resazurin colour changes (disc numbers) are fairly comparable with plate
counts in bringing out the marked difference between washed and sterilized utensils in their standards
of cleanliness. There is, however, some discrepancy observed in the case of one lb. bottles since
the washed series also show high disc numbers comparable to those of the sterilized ones. By increas-
ing the period of sterilization from one hour to two and four hours slightly higher disc numbers are
obtained. The plate counts for all the groups of sterilized bottles, with the exception of half pound
bottles subjected to sterilization for one hour, are below 10 per ml. of rinse solution [satisfactory
standard according to Chalmers, 1945] and the corresponding resazurin disc numbers are above