Bile sample gram stain: Introduction, Procedure and Result Interpretation

Bile Sample Gram Stain: Introduction, Procedure and Result Interpretation

Introduction of Bile Sample

Bile sample in Gram-stained smear has Gram-positive bacteria as shown above picture. Bile is a dark-green-to-yellowish-brown fluid that is made and released by the liver and stored in the gallbladder and it helps with digestion. It breaks down fats into fatty acids, that can be taken into the body by the digestive tract and it is free from of microorganisms i.e. sterile fluid.

Composition of Bile

Cholesterol ( mostly)
Bile acids/salts
Bilirubin
Water
Potassium
Sodium
Copper and
other metals

Bile sample gram stain showing gram-positive and gram-negative bacteria as shown above picture. 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 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 decreasing 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 counter stain uses, they take up the color of the stain and appear pink.

Requirements for bile sample Gram stain

a) Compound light microscope

b) Reagents and glass wares

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 ( bile sample)

Preparation of smear for bile sample

Take a clean, and grease-free slide for making a smear.
Take one or two loopful of bile sample and place 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 Bunsen burner two to three times.

Procedure of Gram stain for bile sample

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 low power (10X) objective, and then under oil immersion (100X) objective.

Observation

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

Test: Red color,rod in shape while other organisms violet color, round in shape in single, pairs and cluster

Negative Control: red in color and rod in shape

Result and Interpretation

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 and Gram positive cocci in single, pairs and cluster

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

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