Proteus Gram stain: Introduction, Principle, Procedure and Result Interpre

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

Introduction of Proteus

Proteus mirabilis images in Gram stain showing Gram-negative rods small to large in size as shown above picture. They are Gram-negative rods having size of 1-3 µm × 0.5 µm. They are non-encapsulated, non-sporing, and actively motile. They possess peritrichate flagella. They may show as short cocobacillary forms, long and filamentous type, especially in young culture. Many strains possess fimbriae. They are commensals of our intestine. Proteus species are also saprophytic and widely distributed in nature. They are opportunistic pathogens and may cause the following infections-

Urinary tract infection (UTI)
Pyogenic lesions
Infection of the ear ( otitis media)
Respiratory tract infections (RTIs)
Pyelonephritis

Note: Some strains are also multi-drug resistant due to having R plasmids.

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

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

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 Proteus on MacConkey agar was used for Gram staining)

Preparation of bacterial 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 MacConkey 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: red color and short and long rod in shape and also some filamentous forms

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 short cocobacillary forms, long and filamentous as shown in picture

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

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