Methylene blue staining

Ⅰ.Introduction

In 1886, methylene blue was used for the first time in the medical field. Dr Paul Ehrlich discovered a strange phenomenon in an experiment: Methylene blue stained living neurons blue, and it has the same effect on Plasmodium (a parasite that causes malaria) in human blood.

Currently, it is widely used in chemical indicators, dyes, biological dyes and drugs

Methylene blue stain is a fast, economical and commonly used stain.

Ⅱ. Methylene blue: Properties

Methylene blue, or methylthioninium chloride, is an organic, odorless crystalline solid soluble in water and, to a lesser extent, in ethanol. In its pure state, it comes in the form of a dark green powder, it is also found commercially as a double salt with zinc chloride, brown in color.

Methylene blue is an oxidation-reduction agent (depends on its redox state: it is colorless in the reduced state, but is blue in the oxidized state).

Ⅲ. Preperation

◈ Methylene Blue solution according to Loeffler : Methylene blue: 0.30 g + Ethyl alcohol 95% : 30.0 ml + Distilled water 100.0 ml

Ⅳ. Principal

Since cells usually have negatively charged cell walls, the chromophores of basic, positively charged dyes tend to stick to cell walls, making them positive spots.

Methylene blue stain is a simple stain where a single dye is used to emphasize particular structures in the sample, shape (the size and arrangement of bacteria). The organisms in a sample will be the same color, even if the sample contains more than one type of organism.

◈ In some cases, the methylene blue stain gives better results than the Gram stain. Among these cases :

Ⅴ. How to stain with methylene blue

• After performing the bacterial smear (smearing, fixing)
• Place the slide on a staining rack and flood it with methylene blue. Leave on for 1-3 minutes
• Gently wash the slide with distilled water, drain off excess water, blot (do not rub) with absorbent paper and allow slides to air dry completely.
• Examine with the x100 immersion objective (with a drop of oil) with strong illumination (diaphragm open)

Checking for leukocytes in diarrheal stools

Place a small drop of faecal mucus or liquid stool on a microscopic slide. Add an equal volume of methylene blue (Loeffler blue). Mix with a small wooden stick; cover with a strip. Leave the coloring to take place for 2 to 3 minutes. Examine with the dry objective (× 40 or × 60).

Ⅵ. Interpretation

The stainable structures appear blue, the staining makes it possible to detect the morphology of these structures, their grouping mode and in some cases the cell type