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Gram Stains

Background and Introduction

In Lab 2 you were introduced to simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria.  Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement.  The Gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure.  Most bacteria can be divided into two groups based on the composition of their cell wall: Gram+ and Gram- This is The Most Important staining technique in Bacteriology. 

Gram Positive Organisms 

Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane. Characteristic polymers called teichoic and lipoteichoic acids stick out above the peptidoglycan and it is because of their negative charge that the cell wall is overall negative. These acids are also very important in the body’s ability to recognize foreign bacteria. Gram-positive cell walls stain blue/purple with the Gram stain. 

diagram of a Gram positive cell wall

´┐╝Gram Negative Organisms

Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer membrane (lipopolysaccharide) beyond the plasma membrane. The space between the plasma membrane and the outer membrane is called the periplasm. The outer leaflet of the outer membrane is composed mainly of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in triggering the body’s immune response. Gram-negative cells will stain pink with the Gram stain. 

diagram of a Gram negative cell wall


Procedure Step


Gram Positive Cell color

Gram Negative Cell Color

Fixed cells on slide



Primary stain

Crystal Violet 
















If your staining procedure was successful you will see Gram positive cocci and Gram Negative bacilli in your stain

Gram stain with Gram positive cocci and Gram Negative bacilli Gram stain with Gram positive cocci and Gram Negative bacilli

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