LAB 3
Bacterial Staining Techniques II
I. Differential Stains: Gram Stain and Acid-fast Stain
II. Morphological Unknown
I. DIFFERENTIAL STAINS
A. Gram Stain
B. Acid-fast Stain
A. Gram Stain
The previous lab introduced 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:
1) 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.
2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer
membrane beyond the plasma membrane. The space between the plasma membrane and the outer
membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in
triggering the body’s immune response and contributing to the overall negative charge of the cell.
Spanning the outer membrane are porin proteins that enable the passage of small molecules.
Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.
This is The Most Important staining technique in Bacteriology.
Cell wall structure of Gram+ and Gram-
21
, GRAM STAIN
Cell Color
Procedure Reagent Gram Positive Gram Negative
Fixed cells on slide COLORLESS COLORLESS
Primary stain Crystal Violet PURPLE PURPLE
Mordant Iodine PURPLE PURPLE
Decolorizer Alcohol PURPLE COLORLESS
Counterstain Safranin PURPLE RED
An easy way to remember the steps of the Gram stain is...
22
Bacterial Staining Techniques II
I. Differential Stains: Gram Stain and Acid-fast Stain
II. Morphological Unknown
I. DIFFERENTIAL STAINS
A. Gram Stain
B. Acid-fast Stain
A. Gram Stain
The previous lab introduced 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:
1) 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.
2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer
membrane beyond the plasma membrane. The space between the plasma membrane and the outer
membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in
triggering the body’s immune response and contributing to the overall negative charge of the cell.
Spanning the outer membrane are porin proteins that enable the passage of small molecules.
Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.
This is The Most Important staining technique in Bacteriology.
Cell wall structure of Gram+ and Gram-
21
, GRAM STAIN
Cell Color
Procedure Reagent Gram Positive Gram Negative
Fixed cells on slide COLORLESS COLORLESS
Primary stain Crystal Violet PURPLE PURPLE
Mordant Iodine PURPLE PURPLE
Decolorizer Alcohol PURPLE COLORLESS
Counterstain Safranin PURPLE RED
An easy way to remember the steps of the Gram stain is...
22
