Gram Staining Procedure and its Details

Gram Staining Procedure and its Details

Please watch the video given below

Introduction

  • Gram staining procedure was discovered by Han’s Christian Gram in 1884.
  • Gram staining is a universal staining technique used for identification and classification of organisms.
  • In this staining, method bacteria are classified into two groups that are-

                                                                        1. Gram-positive bacteria

                                                                        2. Gram-negative bacteria

  • This classification of bacteria depends upon the property of a cell to retain or lose the primary stain after the treatment of decolourising agent. 
  • Gram staining is a basic and widely used technique.
  • This technique was modified by many scientists but the best result was obtained by Hucker and Conn’s modification.

Requirements

  1. A clean grease free slide.
  2. Bacterial cell suspension.
  3. Nichrome Wire loop.
  4. Primary stain – Crystal violet.
  5. Mordant- Gram’s Iodine.
  6. Decolourising agent- 95% alcohol ( 95% Ethanol).
  7. Counterstain- Basic fuschin or Safranin.

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Negative Staining Method and its Mechanism

Negative Staining Method and its Mechanism

Introduction

  • Negative staining procedure is also called as Relief staining.
  • Some bacteria have non-ionic charge or slime layer on their surface.
  • Because of which the stain doesn’t penetrate inside the cell and the bacterial cells are not easily stained.
  • In case of such bacterial cells negative method is used for observing the morphological characters of the cell.
  • In negative staining method actually, the background is stained and the bacterial cell remains colourless.
  •  This is the only staining method in which bacterial cells are not stained but they are made visible against a dark background.
  • Generally, the acidic stain such as 10 % Nigrosin, Eosin, India ink and Congored is used.

Procedure

  1. A grease-free slide is taken and a smear is made on the slide by using Nichrome wire loop.
  2. The slide is allowed to air dry.
  3. Here there is no Heat fixation step.
  4. After air drying a drop of stain is added on an end of a slide and a thin film of stain is made by using another slide.
  5. These film is allowed to air dry and observed under oil immersion.

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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.

Please check out the below given video on Simple Staining procedure and mechanism

 

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.

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