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

Introduction

Methylene Blue Stain, also known as methylene blue aqueous is an ordinary stain. It is used for capsule stain as well as simple staining for a rapid and easy way to determine cell shape, size, and arrangements of bacteria. It is also made easier to differentiate bacteria from yeast cells and presumptively identify the bacterial isolate by a simple wet mount method.

Principle of Methylene Blue Stain

The capsule is distinct from the slime layer that most bacterial cells form in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain takes on an acidic stain while a basic stain detects capsule production. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like methylene blue or safranin or crystal violet, 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 powder on a piece of clean paper (pre-weighed) and 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 is usable for several months.

Requirements for Staining Procedure

• Methylene blue (aqueous)
• Clean and grease-free glass slides
• Test organisms/ Sample (Clinical samples; food and 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

1. Transfer aseptically a loopful of microbial culture on a clean and grease-free dry slide.
2. Mix it with a loopful of aqueous methylene blue.
3. Prepare a smear by using a glass slide.
4. Leave the smear to air dry slowly.
5. Examine the entire smear systematically with the low power objective (10X ) and low light intensity.
6. If any suspicious capsule is encountered, examine it with the oil immersion objective (100X).

Result Interpretation of Methylene Blue Stain

• The capsule of Microbes: Colourless (hollow bodies)
• Background: Bluish
• Control strain: clear halos surrounding the cells

Limitations of  Methylene Blue Stain

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

Keynotes on Methylene Blue Stain

1. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating.
2. A drop of serum can be applied during smearing to enhance the size of the capsule which makes it easier observation with a typical compound light microscope.
3. A capsule is a gelatinous outer layer secreted by the cell of the organism ( bacteria/ fungi) that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides.
4. 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.
5. Simple staining has only one stain or dye. Methylene blue in simple staining gives up a hydroxide ion which leaves the stain positively charged. Since the surface of most bacterial cells is negatively charged, these positively charged stains adhere readily to the cell surface.
6. 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 (RNA), which is the important stainable constituent of bacterial bodies, lymphocytes, hemoprotozoal cytoplasm, and of nerve cell tigroid granules.
7. Loeffler’s alkaline methylene blue stain and Loeffler’s alkaline methylene blue are the modification of the original methylene blue stains. They are applied in In Ziehl-Neelsen Staining, and for the expression of metachromatic granules of C. diphtheriae.
8. New methylene blue is the preferred stain for the reticulofilamentous material and it is chemically different from methylene blue since methylene blue is a poor reticulocyte stain.

Methylene blue stained Footage

Klebsiella pneumoniae capsules in methylene blue stained smear

Bacillus species in Methylene blue wet mount microscopy

Live and dead yeast cells of Cryptococcus neoformans in Loeffler’s methylene blue

Giardia cysts in methylene blue wet mount of stool/ feces microscopy

White blood cell in new methylene blue wet mount of blood microscopy

Acid-fast bacilli of Mycobacterium tuberculosis in Ziehl-Neelsen stained smear of sputum with Loeffler’s methylene blue counterstain

Further Readings

1. Methylene blue -Sigma-Aldrich
2. Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
4. Downes F. P. and Ito K. (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th ed., APHA, Washington, D.C