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

Introduction of Loeffler Methylene Blue Stain

Loeffler methylene blue stain is a metachromatic stain and thus it is preferred for metachromatic staining especially for Corynebacterium diphtheriae and also as a counterstain in Acid-fast staining.

Principle of Loeffler Methylene Blue Stain

Loeffler Methylene Blue is used in the presumptive identification of the etiological agent of diphtheria, Corynebacterium diphtheriae. Metachromasia is a phenomenon or the property of certain biological materials of staining a different color from that of the stain used and such condition characteristically seen in Corynebacterium diphtheriae, is due to an accumulation of polymerized polyphosphates in high concentration inside the cell. This state appears as polyphosphate granules stained deeply blue, surrounded by lighter blue-stained cytoplasm. These granules and are often called Babes-Ernst bodies or metachromatic granules. The cell itself is distinguished by a banded or beaded appearance. This stain is also a counterstain in Acid-fast staining; where Non-Acid Fast bacteria and background material has appeared as blue in color.

Composition of Loeffler Methylene Blue Stain

• Methylene blue:0.5 gm
• Ethanol (ethyl alcohol), 95%: 30 ml
• Distilled water: 100 ml

Loeffler Methylene Blue Stain Preparation

• Weigh the methylene blue on a piece of clean paper (pre-weighed). Dissolve the stain in about 30 ml of water.
• Transfer the stain to a clean brown bottle.
• Add the alcohol and the remainder of the water. Mix well.
• Label the bottle and store it in a dark place at room temperature. The stain for several months.

Requirements

• The test sample, suspected C. diptheriae in throat swab smear
• Loeffler Methylene Blue Stain
• Bunsen burner
• Microscope
• Cedarwood oil
• Control stain (Corynebacterium diphtheriae ATCC 13812)

Procedure of Loeffler Methylene Blue Stain

• Allow suspected C. diptheriae in throat swab smear to air dry and fix with gentle heat.
• Flood the smear with Loeffler methylene blue stain for 1 minute.
• Rinse in the tap water.
•  Blot dry, then examine under oil immersion objective

Result and Interpretation

Metachromatic granules of bacteria: Deep blue

Cytoplasm: Pale blue

Control Strain: Metachromatic granules are deep blue while the cytoplasm is pale blue.

Limitations of the Loeffler Methylene Blue Stain

1. Overstaining is no preferred since it may reduce the contrast between the bacteria and background, or between the cytoplasm and granules.
2. Some strains of bacteria like Propionibacterium, Actinomyces, and pleomorphic forms of streptococci may copy the characteristic stained appearance of C. diphtheriae.

Keynotes on Loeffler Methylene Blue Stain

• In Ziehl-Neelsen Staining, this stain is used as a counterstain that provides background and non-acid fast structures blue.
• Loeffler’s medium is the best for the expression of metachromatic granules of C. diphtheriae.
• This stain is not Loeffler’s alkaline methylene blue stain since it is lacking potassium hydroxide.

Further Readings

1. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
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. , et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C. 9.
4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA. 10.
5. Lillie, R.D. 1977. H.J. Conn’s Biological Stains, 9th ed. Williams & Wilkins Company, Baltimore, MD.