METHYLENE BLUE REDUCTION TEST (MBRT) To Check The Quality of Milk In a Microbiology Lab

Introduction

Ensuring the quality of milk is crucial in the dairy industry, and one effective method to assess this is the Methylene Blue Reduction Test (MBRT). This simple yet informative test measures the microbial activity in milk, providing insight into its quality and safety for consumption. In this post, we’ll delve into the MBRT procedure and understand how to interpret the results.

Procedure

The MBRT is straightforward and requires minimal equipment:

1. Preparation: Add 1 ml of methylene blue solution (concentration 1: 25,000) to 10 ml of milk in a sterile test tube.

2. Mixing: Seal the tube with a rubber stopper and gently invert it three times to ensure the methylene blue is thoroughly mixed with the milk.

3. Incubation: Place the test tube in a water bath maintained at 35°C.

4. Observation: Examine the tube at regular intervals over a period of up to 6 hours.

Methylene Blue Reduction Time (MBRT)

The key observation in this test is the time it takes for the methylene blue to become colorless. This is known as the Methylene Blue Reduction Time (MBRT). The underlying principle is that methylene blue loses its color in the presence of active microorganisms, which consume the oxygen, reducing the dye.

Interpretation of Results

The MBRT provides a clear indication of milk quality:

• Excellent Quality: Decolorization time is more than 5 hours. This indicates minimal microbial activity, signifying that the milk is fresh and of high quality.

• Good Quality: Decolorization time is between 4 to 5 hours. This suggests moderate microbial activity, indicating the milk is still of good quality but should be consumed relatively soon.

• Fair Quality: Decolorization time is between 3 to 4 hours. This level of microbial activity means the milk is starting to spoil and should be used promptly.

• Poor Quality: Decolorization time is less than 2 hours. High microbial activity indicates significant spoilage, and the milk is likely unfit for consumption.

Table: Grading of Milk Samples Based on MBRT

Effects of Genetic Manipulation

Genetic manipulation is an un-natural practice by which the scientists can produce any organism with desired traits. Along with some advantages, this manipulation can have several harmful effects over the society which are discussed as follows:

1.    1. An organism which is born through un-natural means can have an untested genetic build up and if such genes become a part of the society can lead to many disasters.

2.  2.  It can also cause an ecological imbalance. The new organism can be resistant to certain drugs and antibiotics which can lead to the introduction of certain resistant variety of a disease causing harmful microbe.

3.   3. There are high chances that the information of changing the genetic make up could be misused.

4.    4. The genetically modified foods can cause an allergy among certain people who are allergic to a particular chemical. Moreover they will become the part of out genetic structure and can cause mutations at small level in our DNA.

5.  5.   The organisms which are used for testing die an un-natural death or have to undergo a lot of pain so they are given “mercy killing” in the end (As in case of Sheep clone Dolly). This practice of torturing the animals is unacceptable.

6.    6. There is also a risk of generating a superior human species which can be misused. 

7.   7.  There is a small but significant risk of the transmission of usually fatal zoonotic diseases. The introduction of these diseases to the human population could have devastating consequences.

8.  8.   There are other unknown risks related to environment. 

9.  9.  The genetically modified crops will prove expensive to the farmers because the seeds of such crops cannot be used to sow the crop again. So the farmer has to buy the seed every time. (As in case of BT cotton)

1. One of the major factor of concern is Bioterrorism where a deadly pathogen is used as a weapon. Bioterrorism can lead to huge disasters and loss of several lives across the globe.

WESTERN BLOT

A Western Blot (immunoblotting)is an analytical technique used for detection of specific proteins. The protein from the sample is first separated according to size by SDS-PAGE (Polyacrylamide Gel Electrophoresis) and then transferred to a nitrocellulose or Polyvinylidene Fluoride (PVDF) membrane where they become immobilized. Then they are detected first by a specific primary antibody and then by enzyme linked secondary antibody. The enzyme linked to the secondary antibody forms a colored band after conjugation which shows the presence of specific protein. It is used as a confirmatory test in many medical diagnostic tests like HIV, because it gives information if a specific antibody is been secreted against the pathogen.

Alpha Complementation method for Bacterial Screening

Alpha complementation is a method for bacterial screening that have been transformed with a plasmid vector, carrying the N-terminal coding sequence for β-galactosidase of the lac operon. The lac Z gene is required for galactosidase metabolism. The pUC plasmid carries the alpha fragment of the lac Z gene which is the amino-terminus of the protein and is non functional alone. The omega fragment is found in E. coli chromosome, which is the carboxyterminus of the protein and  is nonfunctional alone. When the alpha and omega fragments interact, they become functional and beta-galactosidase protein can be produced. This interaction is known as alpha complementation. It is useful during the screening of beta-galactosidase assay. Artificial galactosides like X-Gal which acts as a substrate for beta-galactosidase and leaves a blue precipitate when hydrolyzed therefore pUC8-transformed E. coli colonies appear blue. They contain plasmids that do not have inserts interrupting the lac Z gene. White colonies consist of bacteria that carry plasmids that have insert-interrupted lac Z genes. (Blue/White Cloning of a DNA Fragment and Assay of β-Galactosidase)