Simple slide on the dye reduction test for testing quality of milk.

1. Presented by:
Richa Khatiwada

2. INTRODUCTION
 Test to find bacterial contamination of food (especially
milk).
 Based on utilization of oxygen by bacteria which
reduces dye and decolorizes it.
 Faster the decolorization, more inferior is the
bacteriological quality and vice-versa.

3. Methylene Blue Reductase Test(MBRT)
• Take 10ml of milk sample in a sterile test tube.
• Add 1ml of MBRT dye(dye concentration 0.005%).
• Close the tube with sterilized rubber stopper.
• Place in stand dipped in serological water bath
maintained at 37±1⁰C.
• Note time taken for decolorization.

4. FIGURE: Methylene Blue Dye Reduction

5. Grading Of Raw Milk Based On MBRT
As per BIS 1479 (Part 3): 1977 criterion for grading of raw
milk based on MBRT is as below:
TIME QUALITY
5 hours and above Very good
3 to 4 hours Good
1 to 2 hours Fair
Less than ½ hours Poor

6.  Rate of reduction depends on the nature of the organism present
in the milk. The rate of reduction by different microorganism is:
E.coli (coliforms) > Streptococcus lactis > faecal Streptococci >
micrococci> Thermoduric organisms > psychrotrophic organisms
 Light hastens reduction and therefore the tests should be kept
covered.
 Creaming causes bacteria to surface, so the tubes should be
inverted periodically.
Any manipulation that increases oxygen affects the test like cold
milk holds more oxygen, pouring milk back and forth and so on.
Factors Affecting

7. RESAZURIN TEST
• Same principle as MBRT
• More sensitive resazurin dye used
 Mix 10 ml milk sample with 1ml of resazurin dye
solution(0.05%).
 Place stopper and invert the tube to mix.Incubate at
37±1⁰C.
 Examine tubes at the end of an hour in the "one-hour
test" or at the end of three successive hourly intervals in
the "triplereading test."

8. FIGURE: RESAZURIN TEST

9. Contd…
Judgment of quality is based either on the color produced
after a stated period of incubation or on the time required
to reduce the dye to a given end-point.
COLOR QUALITY
Blue(no color change) Excellent
Blue to deep mauve Good
Deep mauve to deep pink Fair
Deep pink to whitish pink Poor
White Bad

10. OTHER FOODS
TESTED(EXPERIMENTAL)
• Maple sap
• Red meet
• Ground beef
• Eggs
• Seafood
• Vegetables, etc.

11. ADVANTAGES
• Simple, rapid, inexpensive
• Only viable cells actively reduce the dye
DISADVANTAGES
• Not all organisms reduce the dye equally
• Not applicable to food specimens that contain reducing
enzymes