Gram Stain: Introduction, Principle, Procedure, Result and Interpretation

Introduction of Gram Stain

Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

Principle of Gram stain

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.

Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram stain

a) Compound light microscope

b) Reagents and glasswares

Bunsen flame
Wire loop
Clean grease-free slides
Marker pen
Crystal violet (Basic dye)
Gram’s iodine(mordant)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen

Preparation of bacterial smear: from liquid culture

Take a clean, and grease-free slide for making a smear.
Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
Then with a circular movement of the loop, spread the cell suspension into a thin area.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Preparation of bacterial smear: from the solid medium

Take a clean, and grease-free slide for making a smear.
Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure of Gram Stain

Cover the smear with crystal violet and allow it to stand for one minute.
Rinse the smear gently under tap water.
Cover the smear with Gram’s iodine and allow it to stand for one minute.
Rinse smear again gently under tap water.
Decolorize the smear with 95% alcohol for 20-30 seconds.
Rinse the smear again gently under tap water.
Cover the smear again gently with safranin for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation of Gram Stain

Positive Control: violet color, round in shape in single, pairs and cluster

Test: red color and rod in shape

Negative Control: red in color and rod in shape

Result and Interpretation of Gram Stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-negative bacilli

Negative Control(NC): Gram-negative bacilli as shown above image.

E. coli under microscope|| Gram stain ||Gram Negative bacilli or Gram-negative rods as shown below-

Variety of bacteria under the microscope showing
Gram-positive bacteria, gram-positive cocci in singles, pairs, clusters i.e. Staphylococcus aureus
Gram-positive rods or bacilli
Bacillus species
Gram-negative bacilli or rods
Salmonella Typhi
Diphtheroids
Sputum gram stained smear
having ideal smear
Gram-positive cocci in pairs inside the pus cell
Gram-positive cocci in chains

A patient was 53 years old with Chronic Otitis Media (COM) having a failure of antibacterial drugs-
Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in the video.

Strongyloides stercoralis under Gram stained slide of sputum- a very very rare case-

Impression for this a rare case report from Gram stain of sputum While reporting gram stained slide, we always remember the nature of specimens and availability of organisms; and broaden our vision for report variety of causative agents without missing them because as you know laboratory diagnosis is the third eye of clinicians that makes treatment easier as well as short hospital stay and reduce patient economic burden.

Growth of Candida albicans on SDA and its Gram-stained smear under the microscope and germ tube test (GTT) positive as shown below-

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