Resazurin Reduction
Test (RRT)
Resazurin reduction t is another method of dye
reduction test and the principle of this test is nearly similar
to methylene blue reduction test. In MBRT the time for reduction of
the dye is measured, while in RRT, at a fixed period time, specific shade of
colour and its intensity is measured. There are two variations in RRT of
testing milk. One is 10 min RRT test that can be used as a rapid platform test
for quick assessment of milk at the raw milk reception dock. The other one is a
one hour RRT performed in the lab. In this we will talk about Rapid RRT.
Principle
Unlike methylene blue
the resazurin undergoes reduction through a series of colour shades
viz., blue, purple, and lavender, pink before completely getting reduced to
colourless.
Resazurin dye
which is blue in colour at the oxidation-reduction potential of + 0.3 volts
undergoes an irreversible change to pink colour compound (resorufin) when
the redox potential reduces to +0.2 volts.
When the redox potential is reduced
further to + 0.1 volts or less, the colour of dye changes to colourless
(dihydroresorufin), which is a reversible reaction. Usually, the degree of
reduction of the dye is measured after a fixed time of incubation of milk
sample in the presence of dye.
The reduction of dye to a particular shade of colour
is dependent upon the extent of depletion of oxygen by metabolic activity of
microbes. The colour change is measured with the help of a Lovibond colour
comparator and a standard resazurin disc.
Standard solution of resazurin
·
One tablet of Resazurin is
dissolved in 50 ml of cold sterile glass distilled water by gentle heating to
facilitate the dissolving. This is the bench solution for direct use and should
always be used as fresh.
·
Alternatively dissolve 0.05 g
of resazurin powder in 100 ml of distilled water and boil the
contents for 30 min. This will make a standard solution of 0.05%, which should
always be kept in a cool and dark place, stored in an amber coloured bottle.
·
The bench solution (0.005%) for regular
use should be prepared freshly by diluting the standard solution with distilled
water i.e. 1 ml of standard solution with 10 ml of distilled water.
Procedure
1. Take
10 ml of milk into a test tube and add 1 ml of working solution of Resazurin solution.
2. Put
air tight closure to prevent oxygen entry
3. Invert
the test tubes to mix the milk and Resazurin solution.
4. Place
the test tubes in a thermostatically maintained water bath at 37 ±5 °C and note
down the time of incubation (10 min).
5. At
the end of incubation match the colour of the milk with one of the colour
standards of Resazurin disc.
Resazurin chemical structures
Grading of milk
The
quality of raw milk is adjusted by using the following parameters.
Advantages of dye reduction test
Used
for estimating the suitability of milk for liquid consumption.
These tests
are cheaper and less time is required.
With the
help of these tests the activity is measured rather than the number of
bacteria.
Unlike the
artificial media used in SPC, in milk the natural environment for microbes is
present.
In
case of RRT, the results can be measured in a shorter time.
Some of
the bacteria capable of reducing the dye may not develop colonies on the medium
used in SPC.
Disadvantages
Rate
of reduction of dye varies considerably and is related to species and the rate
at which different micro-organisms grow at a particular temperature.
Inhibitory
substances like penicillin and other antibiotics prevent the growth of bacteria
and thus increase the reduction time.
Not suitable
for classifying milk with low bacterial counts of less than 105 /ml.
Reduction
capability may vary because of variation in proportion of bacteria carried into
cream layer by the rising fat globule.
These tests
do not give indication for the type of micro-organisms present.
Temperature
of incubation used during these tests is not the optimum for majority of the
micro-organisms present in milk.
Not suitable
for testing quality of pasteurized milk intended for processing because of the
low number of micro-organisms.
Require
continuous attention until reduction takes place.
