Simple Staining- Principle, Procedure and Result Interpretation

True to its name, the simple stain is a very simple staining procedure involving only one stain. You may choose from methylene blue, Gram safranin, and Gram crystal violet. Basic stains, such as methylene blue, Gram safranin, or Gram crystal violet are useful for staining most bacteria.

Objectives of Simple Staining

  1. To perform a simple staining procedure.
  2. To compare the morphological shapes and arrangements of bacterial cells.

What is the Principle of Simple Staining?

The simple stain can be used as a quick and easy way to determine cell shape, size, and arrangements of bacteria. Since the surface of most bacterial cells and cytoplasm is negatively charged, these positively charged stains adhere readily to the cell surface.

The principle of simple staining is based on the principle of producing a marked contrast between the organism and its surrounding, by the use of basic stain. A basic dye consists of a positive chromophore that strongly attracts negative cell components and charged molecules such as nucleic acids and proteins.

With simple staining, the bacterial smear is stained with a single reagent, which creates a clear contrast between the organism and its background. Basic staining with a positively charged chromogen is preferred, as bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. The purpose of simple staining is to clarify the morphology and arrangement of bacterial cells. The most commonly used basic colors are methylene blue, crystal violet, and carbon fuchsin.

Reagents and Equipment’s for Simple Staining

Methylene blue, crystal violet, and carbol fuchsin, Microincinerator or Bunsen burner, inoculating loop, staining tray, microscope, lens paper, bibulous (highly absorbent) paper, and glass slides.

Procedure of Simple Staining

  1. Place a slide on the staining tray and flood the smear with one of the indicated strains, using the appropriate exposure time for each: carbon fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue, 1 to 2 minutes.
  2. Gently wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way, you can reduce the loss of organisms from the preparation.
  3. Using bibulous paper, blot dry, but do not wipe the slide.
  4. Examine all stained slides under oil immersion.

Result Interpretation of Simple Staining

Simple staining
simple staining

Bacilli and diplobacilli: Rod-shaped bacteria, purple
Spirilla: spiral-shaped bacteria, purple
Cocci: spherical-shaped bacteria, purple