Methylene Blue Stain: Introduction, Principle, Composition, Preparation

Methylene Blue Stain: Introduction, Principle, Composition, Preparation, Staining Procedure, Result Interpretation and Keynotes

Introduction of Methylene Blue Stain

Methylene Blue Stain, also called methylene blue aqueous is a simple stain and is used for capsule stain as well as simple staining for a quick and easy way to determine cell shape, size, and arrangements of bacteria. It is also applicable to differentiate bacteria from yeast cells and presumptively identify the bacterial isolate by a simple wet mount technique.

Principle of Methylene Blue Stain

The capsule is different from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain takes on an acidic stain and a basic stain to detect capsule production. A positive capsule stain needs a mordant that precipitates the capsule. By counterstaining with dyes like methylene blue or crystal violet or safranin, the bacterial cell wall takes up the dye. Capsules of organisms appear colorless with stained cells against a dark background.

Composition of Methylene Blue Stain

Methylene blue 0.5 gm

Distilled water 100.0 ml

Preparation of Methylene Blue Stain

Weigh 0.5 gm methylene blue on a piece of clean paper (pre-weighed). Dissolve the stain in about 30 ml of water.
Finally, add the remaining water.
Transfer the stain to a clean brown bottle.
Label the bottle and store it in a dark place at room temperature. The stain for several months.

Requirements for Staining Procedure

Methylene blue ( aqueous)
clean and grease-free glass slides
Test organisms/ Sample ( Clinical samples; food & dairy samples, Water samples)
Bunsen burner/ Sprit lamp
Inoculating loop or sterile bamboo stick
Compound Microscope
Cedarwood oil
Waste bin
Gloves
Control strain(Encapsulated bacteria/ fungus)

Staining Procedure of Methylene Blue Stain

For Capsule staining

Transfer aseptically a loopful of culture on a clean and grease-free dry slide.
Mix it with a loopful of aqueous methylene blue.
Make a smear by using a glass slide.
Allow the smear to air dry slowly.
Examine the entire smear systematically with the low power objective (10X ) and low light intensity.
If any suspicious capsule encounter, examine with the oil immersion objective (100X).

Result Interpretation of Methylene Blue Stain

The capsule of Organism: Colourless (hollow bodies)

Background: Bluish

Control strain: clear halos surrounding the cells

Limitations of Methylene Blue Stain

Capsules are more delicate. That is why using heat during the staining process should be avoided given that capsules would be easily destroyed.
Washing the slide with water is also avoided in capsule staining since it would dislodge the capsule.

Keynotes on Methylene Blue Stain

Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating.
A drop of serum can be used during smearing to enhance the size of the capsule which makes it more easily observed with a typical compound light microscope.
A capsule is a gelatinous outer layer secreted by the cell of the organism ( bacteria/ fungi) and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides.
The capsule stain begins as a negative stain. Cells are spread in a film of the acidic stain and are not heat-fixed. Heat-fixing causes shrinkage of the cells, leaving an artefactual white halo around them that might be falsely interpreted as a capsule.
Simple staining has only one stain or dye. Methylene blue in simple staining gives up a hydroxide ion which leaves the stain positively charged. Because the surface of most bacterial cells is negatively charged, these positively charged stains adhere readily to the cell surface.
Methylene blue is a good nuclear stain, the staining solutions are always either aged or alkalized, if not both. More importantly, it is an excellent stain for ribonucleic acid, which is the important stainable constituent of bacterial bodies, lymphocyte, and hemoprotozoal cytoplasm, and of nerve cell tigroid granules.

universe84a