Methylene Blue as a Stain | What is it used for with Example

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Methylene Blue as a stain

Methylene Blue is a cationic stain. In simple terms, it is a blue dye that is charged positively and binds the negatively charged components of the cells such as the nucleus, DNA or RNA present in the cytoplasm. These cells usually have a low affinity. It is a popular stain used for a variety of activities, including bacterial identification and cellular structure in both plant cells and animal cells. Methylene Blue solution is used to stain the cells. The purpose of staining with methylene blue is to determine cell mortality. When methylene blue stain is given to a sample, a healthy cell causes the stain to become colorless.

Methylene blue USP is used for several purposes, including as an antidepressant, a treatment for cyanide poisoning, and a titration indicator in a laboratory. It turns dark blue when it is dissolved in water. It is utilized in a number of scientific investigations for a variety of procedures. It is an efficient redox indicator in chemistry because it turns clear when it comes into touch with reducing substances. It is used as a titration aid in solutions. It’s also employed as a pigment in microbiology to analyze nucleic acid chains. It can be added to a solution to colour RNA and DNA for visual analysis.

Methylene Blue can be used as a counterstain. For using counterstain you have to decolourize the primary stain using a decolorizing agent (an acid-alcohol solution)

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What does methylene blue stain

One of the most popular uses of methylene blue today is in chemistry, biology, medicine and microbiology for staining or highlighting the sections specimens for the animal, bacterial, and blood tissues. It can be used to examine the forms, structures, and locations of dead cells, cell structures and other tissues in plants as well as animals in great detail.

Methylene blue solution contains a blue dye that stains acidic animal cell components (such as the nucleus) blue, allowing to readily examine and analyze them during microscopy, histology, haematology, morphology, and other biological research. Methylene blue works well as a counter-stain for Eosin Y, a red stain used to stain connective tissue and cytoplasm. It is also used in Gram staining to identify gram-positive and gram-negative bacteria.

Uses of Methylene Blue as a stain

Methylene Blue is used for various purposes in biology. These purposes are:-

Uses of Methylene Blue as a stain

  • For determining cell damage:  When methylene blue stain is introduced to a sample of cells or tissue, a healthy cell causes the stain to become colourless. This is because the enzymes in the cell degrade the methylene blue, causing it to lose its colouration. Because the enzymes in the cell have been inactivated by the dye, there will be no response if the cell is dead. Refer here for more details about this use.
  • Identification of Microorganisms: Methylene blue is a dye frequently employed by biologists to aid in the identification of microorganisms. As the bacteria are nearly colourless, a biologist can detect bacterial forms and structures by adding a drop or two of methylene blue to a microscope slide. In biology, a stain is a dye such as methylene blue. It acts by attracting chemicals to biological tissues and binding to them.
  • Identifying Nucleic Acids: When methylene blue comes into contact with acids, it turns a vivid shade of blue. This characteristic makes it extremely valuable for identifying nucleic acids such as DNA and RNA. It can also be used in cellular structure, gram’s staining techniques as an alternative to chemical crystal violet.
  • Identifying RNA Sequences: Because of its affinity for nucleic acids, methylene blue has been employed in specialized procedures such as “northern blotting” to identify RNA sequences (also called “northern hybridization”). Furthermore, methylene blue is a safer alternative to another chemical known as ethidium bromide, which is frequently employed in the viewing of DNA on gels in procedures known as “western blots.
  • Calculating viable cells in yeast sample: Methylene blue has also been used to calculate the proportion of viable cells in a yeast sample quickly. Dead yeast cells lack an enzyme that decolorizes methylene blue, but living yeast cells do. As a result, when yeast cells are suspended in a dye-containing solution, the dye colours the dead cells blue while leaving the living cells uncoloured. Refer here for more details about this use.
  • Identifying distinctions between bacterial, viral and fungal diseases: A technique used to identify and distinguish between bacterial, viral, and fungal diseases is the basic staining approach used by Löffler in alkaline methylene blue staining. Because the positively charged methylene blue dye is attracted to negatively charged particles such as DNAs, RNAs and polyphosphates, it is a cationic dye that stains cells blue. Swabbed specimens from patients are spread onto microscope slides, and the methylene blue solution is placed on the surface. The slide is viewed under a microscope after being covered with a glass coverslip.
  • Methylene blue can also be used as a surgical and medical marking stain (albeit it can induce localized tissue inflammation), as a diagnostic agent in renal function testing, and as a vital nerve staining agent.
  • Furthermore, methylene blue is employed to enhance the visibility of the interaction between Fehling’s solution and reducing sugars. It is also used as a reagent in volumetric redox titrations.
  • methylene blue stain is used in the experiment to increase the visibility of biological components. It selectively stains certain tumour cells of the head, neck and mouth in cancer operations.

Methylene Blue Staining Procedure

Methylene blue is used in gram staining. Gram staining is one of the most important staining methods in microbiology. The predominant colour in gram staining is methylene blue. Gram-positive organisms are organisms that preserve the primary colour and look purple-brown under a microscope. Gram-negative organisms that do not take up primary stains look red under a microscope.

Step 1: The first step in gram staining is to stain the slide with Methylene Blue dye.

Step 2: The following step, often known as fixing the dye, requires employing iodine to produce a Methylene blue-iodine combination to inhibit dye removal.

Step 3: Following that, a decolourizer, usually ethanol and acetone, are used to remove the dye. The capacity of the bacterial cell wall to retain the methylene blue after solvent treatment is the underlying concept of gram staining. Gram-positive bacteria contain more peptidoglycans, whereas gram-negative organisms have more lipids.

Step 4: Initially, all bacteria absorb the methylene blue stain; however, when a solvent is used, the lipid layer of gram-negative organisms is dissolved. Gram-negative bacteria lose the main stain as the lipid layer dissolves. In contrast, solvent dehydrates the gram-positive cell walls, limiting the passage of the methylene blue-iodine combination, and therefore the bacteria stay stained. The duration of decolourization is an important stage in gram staining because extended contact to a decolorizing chemical can eliminate all stains from both kinds of bacteria.

Step 5: The last phase in gram stain consists of using basic fuchsin stain to decolorize gram-negative bacteria to provide them with a pink colour for better identification. It is sometimes referred to as counterstain. Safranin or basic fuchsin stains are used as a counterstain to help differentiate gram-positive and gram-negative bacteria.

How to remove MB Stain

Removal from Clothing

Methylene blue is a tough stain to get out of clothing. It generally leaves a lasting discoloration. We recommend treating the stain with full strength detergent, allowing it to sit for a few minutes, massaging the area with a bristle brush, and washing with clean water. Repeat steps until the spots are no longer visible.

Removal from Skin

To remove methylene blue stains from the skin, quickly wash them with a soap solution. Methylene blue is easily removed from the skin with pure ethyl alcohol and 70% isopropyl alcohol. Repeat these steps and you will get rid of the stains


Why is methylene blue necessary ?

Methylene Blue is necessary in microbiology because it has the capability to bind to nucleic acids, thus enhances visibility by staining cellular structures.

Why do we use stains like methylene blue for viewing cells, such as human epithelial cells, under the microscope?

Stains like methylene blue are commonly used in microscopy to enhance the visibility of cells and cellular structures. It selectively binds to specific organelles or components, aiding in the visualization of fine cellular details. Thus it helps in studying cell morphology, behavior, and diagnosing diseases.

What is the purpose of adding methylene blue to the specimen ?

The purpose of methylene blue staining is to determine cell viability. When methylene blue stain is administered to a sample, a healthy cell renders the stain colorless.

Why do we use methylene blue to stain cheek cells?

Methylene blue being a basic dye, binds to the negatively charged components of cells, such as nucleic acids (DNA and RNA). When applied to cheek cells, it imparts a blue color to the cellular components making it easier to observe their morphology, highlighting their structure including the cell membrane, nucleus, and cytoplasm. It also helps in identification of abnormalities.

What type of dye stain is methylene blue?

Methylene blue is a blue cationic thiazine dye that is widely used in chemistry, biology, and medicine to diagnose and treat a variety of illnesses and diseases.

Can we stain clothes with methylene blue?

Yes, methylene blue can stain clothes and can also leave stubborn marks on clothes. When reduced (with a reducing bleach), methylene blue turns colorless but reverts to blue when oxidized (exposed to air).

Why does methylene blue stain the nucleus?

Methylene Blue is a cationic stain and it is used to stain the nucleus because it has a positive ionic charge, it will interact with tissue cell nuclei that have a negative ionic charge. This dye may react with anionic groups as well as the phosphate groups of nucleic acids (DNA and RNA), making it useful as a nuclear stain.

What is methylene blue stain is used mostly for?
Methylene blue is used In microbiology for staining acidic animal cell components such as the nucleus, bacteria, and blood tissue specimens. Use it to examine the forms, structures, and locations of dead cells and other tissues in detailed observation.

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The information provided here is based on general knowledge, articles, research publications etc and we do not claim the authenticity of any of the information provided above. We do not claim or suggest/advise any medical, therapeutic, health or nutritional benefits of MB. We do not supply or promote our MB product for the applications which are covered by valid patents and which are not approved by the FDA.

Macsen Labs is a manufacturer and supplier of several grades of Methylene Blue such as:-

Macsen Labs group’s Methylene Blue Synthesis Process granted a patent from the Indian patent office

The Indian Patent Office granted a patent to Mr Achal Agrawal, CEO of Macsen Labs, Udaipur. The title of the patent is Novel Improved Method for Synthesis of Diaminophenothiazine Compounds and it concerns a novel process for synthesising the compound Methylthioninium Chloride or Methylene Blue. Macsen Labs has now achieved a unique position by this patent and from now nobody will be able to copy this process. Read more

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