Demonstration of bacterial pigmentation

 

Demonstration of bacterial pigmentation

Aim:

To observe pigmentation by various bacteria when grown on nutrient agar.

Principle:

When a single bacterial cell is placed on a solid or liquid medium, it begins to divide and eventually, a colony appears in the place. Microorganisms exhibit visible physical differences in appearance in their colonies such as colonial shape and appearance, pigmentation and smell and their growth pattern when grown on different types of media. These differences are called cultural characteristics or morphology and may be used as an aid in identifying and classifying them. Cultural characteristics or morphology are determined by observing microorganisms cultured in nutrient broth and on nutrient agar plates, after incubation.

Chromogenic bacteria produce intracellular pigments that are responsible for the color of the colonies on the agar surface. Some bacteria produce extracellular soluble pigments that are excreted into the medium and that also produce a colored colony. Most microorganisms are nonchromogenic and will appear cream, white, or gray.  Pigmented bacteria are also known as chromobacteria. Bacterial pigments are water soluble or insoluble; water soluble pigments are diffused in the growth medium. Chemically, bacterial pigments are pyrrole, phenazine, carotenoid, xanthophylls and quinine or quinone derivatives. The pigment molecules are synthesized in cell wall or periplasmic space. Only aerobic and facultatively aerobic bacteria are pigmented because, molecular oxygen is essential for pigmentation. Pigment synthesis is also dependent on light, pH, temperature and media constituents like indicator dyes.  In bacteria, pigment formation is associated with morphological characteristics, cellular activities, pathogenesis, protection and survival.

Pigment                                              Bacteria

Purple                                                  Spirillum rubrum

Violet                                                  Chromobacterium violacein

Yellow                                                Xanthomonas campestris

Orange                                                Sarcina aurentiaca

Red                                                     Serratia marcescens

Black                                                   Prevotela melaninogenica

Golden                                                Staphylococcus aureus

Pink                                                     Micrococcus roseus

Fluorescent blue/green                        Pseudomonas aeruginosa

Fluorescent yellow                              Pseudomonas fluorescens

S. aureus is a facultatively anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope, and has round, usually golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacterium's name; aureus means "golden" in Latin.   Some strains of Staphylococcus aureus are capable of producing staphyloxanthin - a golden coloured carotenoid pigment. This pigment acts as a virulence factor, primarily by being a bacterial antioxidant which helps the microbe evade the reactive oxygen species which the host immune system uses to kill pathogens.

Serratia marcescens is a motile,short rod-shaped, Gram-negative, facultative anaerobe bacterium.  S. marcescens produces a reddish-orange tripyrrole pigment called prodigiosin.  Prodigiosin is made up of three pyrrole rings and is not produced at 37°C, but at temperatures below 30°C.

Pseudomonas aeruginosa is a common gram-negative, rod-shaped bacterium.  P. aeruginosa can secrete a variety of pigments, including pyocyanin (blue-green), pyoverdine (yellow-green andfluorescent), and pyorubin (red-brown). The species name aeruginosa is a Latin word meaning verdigris ("copper rust"), referring to the blue-green color of laboratory cultures of the species. This blue-green pigment is a combination of two metabolites of P. aeruginosa, pyocyanin (blue) and pyoverdine (green), which impart the blue-green characteristic color of cultures.

Materials required:

Routine microbiological facilities

Nutrient agar plates

Overnight cultures of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus

Procedure:

Using an inoculation loop nutrient agar plates were inoculated with Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus.  Two plates were kept for Serratia marcescens culture.

All plates were kept at 37oC for overnight incubation.  One set of Serratia inoculated plate were kept overnight at room temperature.

After incubation, the plates were observed for colony morphology.

Observation:

S. aureus formed medium sized, round, golden yellow pigmented colonies

Pseudomonas aeruginosa colonies were large, translucent, with a diffused greenish blue pigment.

Serratia marcescens formed medium sized, round, bright red pigmented colonies

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