Simple Staining Procedure and its Mechanism

Simple Staining Procedure and its Mechanism

Introduction

  • Simple staining is a method of staining in which bacteria are stained by using a single stain.
  • Simple staining is also called as monochrome staining or positive staining.
  • Examples of simple stain are Methylene blue, Safranin, Malachite green, Basic fuchsin and crystal violet etc.
  • In simple staining procedure cell are uniformly stained.

Procedure of simple staining

  1. A clean grease free slide is taken .A grease free slide is is made by first washing the slide with detergent wiping the excess water and the slide is passed through flame.
  2. On these grease free slide smear is made by using a sterile wireloop and cell suspension.
  3. These slide is allowed to air dry .
  4. After air drying these slide is rapidly passed through a flame for three to four times for heat fixation.
  5. After heat fixation the slide is placed on the staining rack and flooded with a particular stain and these stain is allowed to react for three minutes.
  6. Futher the slide is washed under running water.
  7. The slide is air dried and washed under oil immersion.

Flow chart of simple staining procedure

Flow chart of Simple staining procedure
Flow chart of Simple staining procedure

Mechanism of simple staining

  • A stain has a ability to bind a cellular component .These abilites depend upon the charges present on cellular component and charges present on chromophore group of stain.
  • Bacteria has large number of carboxyl group on its surface and these carboxyl group has negative charge.
  • When these carboxyl group carry out ionization reaction it shows COO and H
  • That is COOH = COO + H+.
  • In nature these H+ ions are present on cell surface and further replaced by other positively charged  ions like Na+ or k+.
  • Now when these simple stains are used it has chloride group
  • Further these stain carry out dissociation for example if the stain is  Malachite green it will  carry out dissociation and give free radicals
  • That is MgCl = Mg+ and Cl
  • Now these free Mg + ions give positive charge on chromophore group.
  • When these stain is applied to a cell these positively charged Mg + ions replace the K + or Na + present on cell surface.
  • Thus a ionic bond is formed in between positively charged Mg + ions and cell surface.
  • Thus it result in staining of cell.

Application 

  • Simple staining procedure stain bacteria easily and helps in observation under microscope.
  • It is useful in preliminary studies of morphological characters of cell that is its size, shape and arrangement.

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