Gram positive cocci(GPC): in single, pair, chain and cluster

Gram Positive Cocci(GPC): In Single, Pairs, Chain and Cluster

Gram Positive Cocci (GPC)

Gram-positive bacteria in Gram stain are showing Gram-positive cocci singly, in pairs, chains, and in groups/clusters that indicate the possibility of an organism is Staphylococcus aureus as shown above image. 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

Principle of Gram stain on the various basis, like permeability of cell wall of bacteria and formation of dye-iodine complex, affinity, pH of the cytoplasm of organisms and presence of magnesium ribonucleate as follows:

Permeability of cell wall of bacteria and formation of the dye-iodine complex (lake): The permeability of the cell wall of Gram-negative bacteria is more porous compared to the Gram-positive bacterial cell wall. The formation of the lake takes place in the cytoplasm of bacteria. The decolorizer ( alcohol) can not pass through the cell wall of Gram-positive bacteria and hence these cells become purple or violet whereas the Gram-negative bacterial cell wall permits the above decolorizer into the cell. Since the complex is soluble in organic solvent and insoluble in water, the dye-iodine complex will be washed away from the cell. As a result, these cells, now, are colorless and will take the color of counterstain, i.e. safranin or neutral red or dilute carbol fuchsin and appear pink/ red as shown in negative control as shown above picture.

Affinity

Acidic substances have an affinity to basic dye and basic substances have an affinity to acidic dye.

the pH of the cytoplasm of organisms

pH could be another factor that researchers have shown to describe the principle of Gram stain. The pH of the cytoplasm of Gram-positive bacteria is 2-3 whereas that of Gram-negative bacteria is 4-5. The acidic nature of the cytoplasm is further increased after the addition of iodine. Thus, it is the acidity of the cytoplasm of Gram-positive bacteria, which has an affinity to basic dye.

Presence of Magnesium ribonucleate

Magnesium ribonucleate in the Gram-positive bacteria has an affinity to basic due. As a result, Gram-positive bacteria take the color of crystal violet. It has also been proved that these bacteria become Gram-negative after removal of Magnesium ribonucleate .

Requirements for staining Staphylococcus aureus

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 (Overnight culture of Staphylococcus aureus on blood agar was taken for Gram staining.)

Preparation of Staphylococcus aureus smear

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 blood agar 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

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

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

Test: Purple color, round in shape and arrangement in singles, 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-positive cocci in single, pairs, and cluster

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

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Gram Positive Cocci(GPC): In Single, Pairs, Chain and Cluster