SIMPLE STAINING

TECHNIQUES

BY: GLINDA V. GOMEZ

The use of a single stain or dye to create contrast between the bacteria and the background is referred to as simple staining.

The simple stain is a very simple staining procedure involving only one stain.

Its chief value lies in its simplicity and ease of use. Simple staining is often employed when information about cell shape, size, and arrangement is desired.

In a simple stain, the smear is stained with a solution of a single dye which stains all cells the same color.

Differentiation of cell types or structures is not the objective of the simple stain.

However, certain structures which are not stained by this method may be easily seen, for example, endospores and lipid inclusions.

SIMPLE STAINING depends on the fact that bacterial cells differ chemically from their surroundings and thus can be stained to contrast with their environment so they are more readily visible.

SIMPLE STAINING depends on the fact that bacterial cells differ chemically from their surroundings and thus can be stained to contrast with their environment so they are more readily visible.

WHAT IS A DYE?

Dyes are generally salts in which one of the ions is

colored. A salt is a compound of a positively

charged ion and a negatively charged ion.

METHYLENE BLUE +MBC CHLORIDE

The color of the stain is in the positively charged METHYLENE BLUE ion.

+ -

Simple dye methylene blue is actually the salt methylene blue chloride, which dissociates as

Bacterial cells have a slight negative charge when the pH of their surroundings is near neutrality, which it generally is.

The negatively charged bacterial cell combines with the positively charged methylene blue ion, with the result that the cell is stained.

It is the difference in charge that produces an affinity between the dye and the bacterial cell.

Dyes may be divided into two groups – basic and acidic.

If the color is in the positive ion of the dye, we call it a basic dye.

If the color is in the negatively charged ion, we call it an acidic dye or stain.

Staining with basic dyes

For staining you may use the basic dyes methylene blue, crystal violet, and carbol fuchsin.

While this these are all basic dyes, they differ in the rate and degree to which they will stain a cell.

• Methylene blue reacts with the negatively charged cell at the lowest rate taking 30-60 seconds to stain a microbial preparation properly.

• Crystal violet is more reactive and normally requires only about 10 seconds.

Carbol fuchsin is an even more powerful dye, normally requiring only 5seconds.

Its reactivity is so great that you may have difficulties from over staining, especially in preparations that contain large amounts of organic material and debris.

Carbol fuchsin is a mixture of the basic dye fuchsin and phenol.

Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. 

Negative stain technique.

An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained.  

• The predominant charge on a bacterial cell (or a protein) is a function of the acidity of its environment.

• Decreasing the acidity (raising the pH) increases the net negative charge on the cell, providing a stronger attraction to basic dyes.

• The reverse holds true for acidic dyes. Therefore, basic dyes stain poorly at a high pH.

1. Provide appropriate contrasting background so that the object can be observed with relative ease. Most of the objects, tissue cells, microbes are transparent and light can pass through them . If this object is stained, the light is reflected and the object can be visualised easily. 

2. Simple staining is useful to study the shape of the object. 

3. Simple staining is useful to study the size of the object. 

4. Simple staining is useful to study the arrangement of the cells eg. chains, bunch, paired arrangement in bacteria. 

5. Only one type of dye (stain) is used in simple staining.

Advantages

Simple stains cause all cells in a smear to appear more or less the same color regardless of the type of cells

DISADVANTAGE