Micrococcus gram stain: Introduction, Principle, Procedure and Result Inter

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

Introduction of Micrococcus

Micrococcus in gram stain is showing –

Violet color-positive
Round in shape-cocci
Arrangement in number of four-Tetrad
Therefore, gram-positive cocci (GPC) in tetrads as shown above image.

They are saprophytes and commensals and they can be an opportunistic pathogen, mainly in immunocompromised hosts e.g. HIV patients. They are difficult to identify Micrococcus as the cause of infection because the organisms are normally present in skin microflora, and the Microcococci are seldom linked to disease. In rare cases, the death of immuno-compromised patients has occurred from pulmonary infections caused by Micrococcus. They may be involved in other infections, including recurrent bacteremia, septic shock, septic arthritis, endocarditis, meningitis, and cavitating pneumonia (immuno-suppressed patients). Some species of this genus, Micrococcus are- Micrococcus luteus, Micrococcus mucilaginosis, Micrococcus roseus, etc.

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 staining of Micrococcus

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 Micrococcus on blood agar was used for Gram staining)

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 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.
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: Violet color, round in shape, and arrangement in a number of fours

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 in tetrads as shown above picture

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

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