What is Gram Stain?

• Gram staining, a differential staining technique, is one of the most important

methods used by microbiologist. The technique allows us to separate bacteria into
two fundamental groups; Gram positive and Gram negative. The differences
between these two groups rests in the chemistry of their cell wall. Gram positive
cells have a cell wall composed of a very thick layer of peptidoglycan, a uniquely
bacterial molecule. While, Gram negative bacteria have a cell wall composed of a
thin layer of peptidoglycan surrounded by an outer membrane.

• Gram stain or Gram staining, also called Gram's method, is a method

of staining used to distinguish and classify bacterial species into two large groups
(gram-positive and gram-negative). The name comes from the Danish
bacteriologist Hans Christian Gram, who developed the technique.

Who is the founder of Gram Stain?

• The Gram stain was devised by the Danish physician, Hans

Christian Joachim Gram, while working in Berlin in 1883.
• He later published this procedure in 1884. At the time, Dr.
Gram was studying lung tissue sections from patients who had
died of pneumonia.

• Based on this reaction, bacteria classified into Gram positive

and Gram negative bacteria.
• The cell wall compostion differences makes difference.
What procedures in doing Gram Stain?

The Gram Stain Procedure

Staining procedure
1. Flood slide with crystal (or gentian) violet- 60 seconds.
2. Flood with Gram's iodine - 180 seconds.
3. Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless.
(Wash with water).

This third step is the most critical and also the one most affected by
technical variations in timing and reagents.

4. Flood with safranin (pink color) (10% Fuchsine) - 60 seconds. (Wash with water).
5. Air dry, or blot with absorbent paper.
Results
As shown below, organisms that retain the violet-iodine complexes after washing in
ethanol stain purple and are termed Gram-positive, those that lose this complex
stain red from the safranin counter stain are termed Gram-negative.

Typical Gram stain

Negative

Positive

Mix
Procedure in Gram Stain
Step 1 - Prepare a Smear
• Suspend some of the material to be stained in a drop of water on a microscope
slide, spread the drop to about the size of a nickel.
• Allow to air dry. Heat fix by gently warming above a flame or other heat source.

Step 2 - Apply the Primary Stain

• Flood the Smear with Crystal Violet

• Allow to stand 30 sec to 1 min
• Rinse with water to remove excess stain
Step 3 - Apply the Fixing Agent
• Flood the Smear with Iodine solution
• Allow to stand 30 sec to 1 min

Step 4 - Rinse
Rinse with water to remove excess Iodine

Step 5 - Decolorize
• Drip 95% Alcohol across the slide about 5 sec
• The effluent should appear pale or clear
Step 6 - Rinse
• Rinse with water to remove excess alcohol

Step 7 - Counterstain
• Flood the slide with Safranin solution
• Let stand 30 sec

Step 8 - Rinse, Dry and Observe

• Rinse with water to remove excess stain
• Blot dry
• Observe under Oil Immersion
What is to be Gram stain?
 Gram staining is a differential staining technique that differentiates bacteria into
two groups: gram-positives and gram-negatives. The procedure is based on the
ability of microorganisms to retain color of the stains used during the gram stain
reaction.

 Gram staining is a common technique used to differentiate two large groups of

bacteria based on their different cell wall constituents. The Gram
stain procedure distinguishes between Gram positive and Gram negative
groups by coloring these cells red or violet

 The Gram stain differentiates bacteria into two fundamental varieties of cells.
Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-
positive," whereas those that are decolorized and stain red with carbol fuchsin (or
safranin) are said to be "gram-negative." This staining response is based on the
chemical and structural makeup of the cell walls of both varieties of bacteria.
Gram-positives have a thick, relatively impermeable wall that resists
decolorization and is composed of peptidoglycan and secondary polymers.
Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein
bilayer known as the outer membrane, which can be disrupted by decolorization.
Some bacteria have walls of intermediate structure and, although they are
officially classified as gram-positives because of their linage, they stain in a
variable manner. One prokaryote domain, the Archaea, have such variability of
wall structure that the Gram stain is not a useful differentiating tool.
Typical Gram-positive bacteria:

1. Staphylococci such as Staphylococcus epidermidis and Staphylococcus

aureus which is a common cause of boils.
2. Streptococci such as the many species of oral streptococci, Streptococcus
pyogenes which causes many a sore throat and scarlet fever
and Streptococcus pneumoniae which causes lobar pneumonia.
3. Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).
4. Actinomyces such as Actinomyces odontolyticus which is found in mouth.
5. Species of the genus Bacillus such as Bacillus subtilis which are common
microbes living in soil.

Typical Gram-negative bacteria:

1. Bordetella pertusis, the causative agent of whooping cough

2. Salmonella typhi, the causative agent of typhoid
3. Vibrio cholera, the causative agent of cholera
4. Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria

Generally cocci are Gram-positive but there are exceptions. The most significant from a
clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears
as a Gram-negative diplococcus looking very much like a pair of kidney bean.
Importance of a Gram Stain:

The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria
based on staining characteristics, enabling the bacteria to be examined using a light
microscope. The bacteria present in an unstained smear are invisible when viewed
using a light microscope. Once stained, the morphology and arrangement of the
bacteria may be observed as well. Furthermore, it is also an important step in the
screening of infectious agents in clinical specimens such as direct smears from a
patient.

The Gram stain procedure enables bacteria to retain color of the stains, based on the
differences in the chemical and physical properties of the cell wall.

1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called
Crystal Violet in the cell wall.
Example: Staphylococcus aureus

Fig: Gram positive bacteria

 2. Gram negative bacteria: Stain red or pink due to retaining the counter staining
dye called Safranin.

Example: Escherichia coli

Fig: Gram negative bacteria

Bacterial Morphology:

Bacteria are very small unicellular microorganisms ubiquitous in nature. They are
micrometers (1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan
and reproduce by binary fission. Bacteria vary in their morphological features.

The most common morphologies are:

Coccus (pleural: Cocci):

Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in

grape-like clusters (Staphylococci), in chains (Streptococci) or in cubical arrangements
of eight or more (sarcinae).
For example: Staphylococcus aureus, Streptococcus pyogenes

Bacillus (pleural: Bacilli):

Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs
(diplo-bacilli) or chains (streptobacilli).
For example: Bacillus cereus, Clostridium tetani
Spirillum (pleural: Spirilla):

Spiral-shaped bacteria

For example: Spirillum, Vibrio, Spirochete species.

Some bacteria have other shapes such as:

Coccobacilli: Elongated spherical or ovoid form.

Filamentous: Bacilli that occur in long chains or threads.
Fusiform: Bacilli with tapered ends.