Gram Stain Technique on the Basis of Differences in Cell Wall

Christian Gram developed the technique of gram stain on the basis of differences in the cell wall.

On the basis of this technique bacteria can be divided into two groups:

Gram Positive Bacteria:

These are the group of bacteria that stained purple. They retain the primary dye due to the formation of CV-I complex in the cell wall.

Gram Negative Bacteria:

These bacteria, are stained pink because they retain secondary dye in the cell wall.

Comparison of Gram Positive and Gram-Negative Cell Wall:

Difference in staining is due to difference in structure of cell walls of two groups. It is clear from the following table:

Characteristics Gram Positive Gram Negative
Number of Major Layers
Chemical makeup. (a)    Peptidoglycan (50% of dry weight in some bacterial cells)

(b)   Techoic acid

(c)    Lipotechoic acid



(d)   Lipids (1-4%)

(a)    Lipopolysaccharides


(b)   Lipoproteins

(c)    Peptidoglycan

10% dry weight of some bacterial cells

(d)   Lipids (11-12%)

Overall thickness 20-80 nm 8-11 nm
Outer membrane No Yes
Periplasmic space Present in some Present in all
Permeability More permeable Less permeable


Chemical Composition of Cell Wall:

The cell walls of most bacteria have a macromolecule called peptidoglycan.

Its amount varies in different types of bacteria.

Peptidoglycan is composed of long glycan chains cross-linked with peptide fragments.

The cell wall also contains sugar molecules, techoic acid, lipoproteins and lipopolysaccharides. These are linked to peptidoglycan.

Cell Walls of Archaeobacteria:

Cell walls of Archaeobacteria are different from Eubacteria. They do not contain peptidoglycan. Their cell walls are composed of proteins, glycoproteins and polysaccharides.

Cell Walls of Other Bacterial Groups:

Many bacterial groups have no cell wall structure which is characteristic of gram positive or gram negative bacteria.

No Cell Wall in Some Bacteria:

Some bacteria have no cell wall (like Mycoplasmas).

Cell Membrane


Beneath the cell wall is the cell membrane or plasma membrane.


It is thin, delicate, and flexible and completely surrounds the cytoplasm. Any damage to it

Chemical Composition:

Bacterial membrane differs from eukaryotic membrane because it does not contain sterols (such as cholesterol).


Cell membrane regulates the transport of proteins, nutrients, sugar and electrons or other metabolites.

It also contains enzymes for respiratory metabolism.

Cytoplasmic Matrix


The cytoplasmic matrix is the substance present between the plasma membrane and the nucleoid.

The plasma membrane and everything present within is known as protoplast.

Properties of Cytoplasmic Matrix:

It is a major part of protoplast.

It has gel like consistency.

Small molecules can move through it rapidly.

Large separate structures such as chromatin/nuclear body, ribosomes, mesosomes, granules and nucleoid are present in this matrix.

The cytoplasm of prokaryotic cell has no membrane bound organelles and cytoskeleton (microtubules).



The DNA of bacteria is a single, circular and double stranded molecule. It aggregates as an irregular shaped dense material called as nucleoid.

Other names for nucleoid are nuclear body, chromatin body and nuclear region.

In bacteria chromosomes and nuclear membrane absent.


Nucleoid is present near the center of cell.

Visible in the Light Microscope:

It is visible in the light microscope after staining with Feulgen stain.


It is very long molecule of DNA which is tightly folded so as to fit in the cell component. The closed circle chromosome (DNA) of Escherichia coli is about 1,4000 µm.

Haploid Organisms:

Because of the presence of a single chromosome, bacteria are haploid organisms.


In to the single circular DNA molecule found in all bacteria, some species also contain one or more plasmids.

A plasmid is a small, self-replicating circle of extra DNA. It possesses only a few genes, which generally give extra survival advantage. They contain drug, heavy metals, disease and insect resistant genes.

Some give resistance to antibiotics. For example, some staphylococci contain a plasmid which includes a gene for the enzyme penicillinase. This breaks down penicillin, thus making the bacteria resistant to penicillin.

Other plasmid genes are known which give resistance to disinfectants, cause disease or responsible for the fermentation of milk to cheese by lactic acid bacteria

In the modern genetic engineering techniques plasmids are important vectors. Genes are inserted in them to get complex chemicals as food, hydrocarbons, with applications in clearing oil spills and producing protein from petroleum.



They are mostly free in the cytoplasm. Sometimes they are loosely attached to the membranes.


They are smaller (70S) than eukaryotic (80S) ribosome.

Number of Ribosomes:

There are thousands of ribosomes in each healthy growing cell.

Chemical Composition:

Ribosomes are composed of RNA and proteins.


They arc protein factories.



The cell membrane, invaginates into the cytoplasm forming a structure called as

Shapes of Mesosomes:

Mesosomes are in the form of vesicles, tubules or lamellae.


Mesosomes are involved in DNA replication.

They play a role in cell division.

Some mesosomes are involved in the export of exocellular enzyme.

Respiratory enzymes are also present on the mesosomes.

Granules and Storage Bodies


Bacteria live in competitive environment and the nutrients become short. Therefore they store extra nutrients when possible.

Storage Materials:

Storage materials may be glycogen, sulphur, fat and phosphate.

Waste materials are generally excreted in the form of alcohol, lactic acid and acetic acid.


Some bacteria, mainly of the genera Clostridium and Bacillus, form endospores (spores produced inside cells). They are thick-walled, long-lived and extremely resistant, particularly to heat, drought and short-wave radiations. Their position in the cell is variable and is of importance in recognition and classification.

Certain Species of Bacteria Produce Spores:

Formation / Production:

Spores are metabolically dormant bodies and are produced at a later stage of cell growth.

Types of Spores:

Spores are of two types:

Exospores: These are formed external to the vegetative cells.

Endospores: These are formed within the vegetative cells.

Properties / Functions:

Spores are resistant to adverse physical environmental conditions. For example light,) high temperature, desiccation, pH and chemical agents.

Under favorable conditions they germinate and form vegetative cells.



Cysts are dormant, thick-walled, desiccation resistant forms. However they are not heat resistant.


They are developed during differentiation of vegetative cells.


These can germinate under suitable conditions.

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