Dye Reduction tests are used to check the microbial load present in the milk sample. It is convenient and quick to use.
AIM:- To carryout Microbiological analysis of milk samples by Dye Reduction Tests.
Milk is one of the important food that is being consumed, utilized and traded across the globe. Milk is composed of carbohydrates (lactose), fats, proteins, enzymes, vitamins and minerals. It is also a source of energy for consumers and has various health benefits. Due to its nutritional value, it has become part of our daily diet. On the other side, Milk being rich in nutrients is highly susceptible for contamination by various microorganisms. Hence, milk become an efficient carrier for variety of pathogen and causing food borne diseases.
Milk gets easily contaminated from various sources like air, infection of cattle, feed of cattle, milk handlers, surround environment etc. If contaminated milk and its products are not processed or sterilized properly then the pathogens persisting in the product can lead to various diseases in consumers. To prevent such food borne disease, certain standards of milk grades have been defined that are safe and acceptable for consumption.
Quality of milk is determined based on the microbial load indicating the hygienic practices during milking, storage, transportation and processing. Milk obtained by hygienic practices from healthy cattle should not contain more than 5×105bacterial cells/ml. This load can be decreased by pasteurization of milk which is an important step in processing of milk. If the load is higher in the pasteurized milk then the shelf life of the milk will decrease and will also cause food borne diseases to consumers.
According to Bureau of Indian Standards (BSI), there are few following Rapid Platform Tests (RPT) for microbiological analysis of milk samples including both raw and pasteurized milk.
- DMC – Direct Microscopic counting.
- LPC – Laboratory Pasteurisation Count.
- SPC – Standard Plate Count.
- DRT – Dye Reduction Tests.
- Thermophilic, Pyscrophilic counts.
- Coliform count on violet red bile lactose agar.
- Presumptive coliform count on Mac Conkey’s broth.
In this article, we will be discussing about DRT.
DYE REDUCTION TESTS (DRT):-
DRT is the most used rapid platform test to grade the quality of milk in short time. Dye reduction test is done using redox dyes such as Methylene blue, Resazurin. Therefore, DRT can be done by two methods based on usage of the redox dye. Two dye reduction tests for milk samples are MBRT (Methylene Blue Reduction Test) and RRT (Resazurin Reduction Test) where MBRT requires minimum 30 minutes and RRT requires minimum 10 minutes for the results.
The test works on the principle of color change of dye like methylene blue, resazurin when incubated with milk or dairy products. The change of color is due to the oxidation and reduction reactions caused by bacterial Dehydrogenase enzyme present in the milk or dairy sample. Dehydrogenase is flavin enzyme that transfers hydrogen atom to electron acceptors in the electron transport chain.
Bacteria consumes dissolved oxygen for its electron transport chain (oxygen acts as a final electron acceptor). When the oxygen level declines in the milk sample, the dye acts like electron acceptor. When dye gains more and more electrons its redox potential declines causing change in color. The time required to change the color is called the reduction time. Higher the dehydrogenase enzyme activity lower is the reduction time. The enzyme activity is directly proportional to the bacterial load in the sample. Hence, this method can be used to estimate the bacterial load and determine milk quality.
MBRT – Milk in udder of cattle has low redox potential to reduce dye methylene blue immediately. Whereas milk obtained after milking has high redox potential [>0.3 volts] due to interference of oxygen from surrounding. In such high redox potential, the dye remains in blue color. When bacterial load increases and oxygen decreases, the redox potential decreases [0.06 to 0.01 volts] causing de-colorization of dye. This colour change occurs when one atom of hydrogen is taken up by the double bonded nitrogen of methylene blue, leading to conversion of dye to reduced colorless state. Blue color can reappear when sample is interfered with oxygen again.
RRT – Resazurin is a redox dye which is blue in colour at oxidized state but when redox potential decreases from 0.2 to 0.05 volts, this blue resazurin gets converted to pink resorufin through shades of purple and lavender. Resorufin state is an intermediate state which is irreversible. Further decrease in the redox potential [0.15 to 0.0 volts] leads to formation of a reduced state of irreversible resorufin that is a colouless reversible dihydroresorufin. This final state can turn back to pink resorufin when oxygen interfere again into the sample.
- Methylene blue solution.
- Resazurin solution.
- Sterile saline solution.
- Test tubes.
- Test tube rack.
- Water bath.
- Milk samples.
- Cotton plugs.
- Prepare standard solutions of methylene blue and resazurin and store it in a air tight container.
- Mix the milk samples thoroughly by shaking up side down the sample container to distribute the contents of milk evenly and add aseptically 10 ml of the sample each in six sterile test tubes (three tubes for MBRT and three tubes for RRT) and plug them with cotton plugs.
- Label three of the tubes as color control (oxidized state), color control (reduced state) and test for both MBRT and RRT.
- Add 1ml of freshly prepared methylene blue solution in two test tubes labeled color control (oxidized state) and test where as add 1ml of sterile saline in tube labeled color control (reduced state). Follow the same for RRT using freshly prepared resazurin solution.
- Mix all the tubes thoroughly and place them in a water bath at 37°C for 30mins for MBRT and 10mins for RRT.
- After incubation remove all the tubes from water bath and check for the color discoloration and note the results.
- Continue the incubation till 5 hrs and note the discoloration record at intervals of 30min for MBRT till the discoloration is attained [Regard the milk is discolored when the whole column of milk is decolorized].
- For RRT record the color variations in the incubation period of 10 min and report the final color attained without disturbing the tube as a result.
- The time at which complete reduction takes place is noted as a reduction time of dye for the milk sample used.
- Interpret the quality of milk sample by comparing the reduction time of discoloration for MBRT and color obtained at result for RRT with standard charts given by FSSAI. [NOTE: shake the tubes labeled color control (oxidized state) while recording the results.
Interpretation of Dye Reduction Test:-
FOR MBRT- Grade the quality of milk sample based on time of discoloration of methylene blue completely in the tube labeled test. Tube labeled color control(oxidized state) will be blue in color due to presence of oxygen by shaking and tube labeled color control(reduced state) will has no color due to absence of dye. More the time taken to attain discoloration of dye for the sample better will better will be the quality of milk.
|Time of discoloration in tube labeled test||Grade for quality of milk sample|
|5 and above 5 hrs||Very good|
|3 and 4 hrs||Good|
|1 and 2 hrs||Fair|
FOR RRT- Grade the quality of milk sample based on the end color change of resazurin dye in the test tube labeled test. Tube labeled color control (oxidized state) will in blue or lilac in color due to presence of oxygen by shaking, tube labeled color control (reduced state) will be colorless due to absence of dye or it will be pink if dye is added.
|Color change in test tube labeled test||Grade for quality of milk sample|
|Blue / Lilac||Good|
|Mauve / pink||Poor|
NOTE: Color obtained in the color control tubes are only for the indication of colour expected and is not reported as results for the milk sample.
Advantages of Dye Reduction test:-
- It is rapid test and takes less time to interpret quality of milk. RRT is takes less time than MBRT.
- In RRT resourfin production is irreversible hence even shaking the tube will not hamper the result.
- Method is easy and inexpensive.
Limitations of Dye Reduction test:-
- Dye used in the process being a redox dye, and interference of oxygen by shaking or other activity and light can lead to false results mainly in MBRT.
- Concentration of dye used relatively effects the reduction time.
- Milk having large number of leucocytes may interfere with the results as the cells may take up the color leading to false results.