Staphylococcus Aureus Detection Methods

In this Staphylococcus Aureus detection methods post we have briefly explained about Staphylococcus Aureus Cultural Characteristics and biochemical reactions for detection of Staphylococcus aureus.

They are spherical cocci, approximately 1 μm in diameter, arranged characteristically in grapelike clusters. Cluster formation is due to cell division occurring in three planes, with daughter cells tending to remain in close proximity.

They may also be found singly, in pairs and in short chains of three or four cells, especially when examined from liquid culture. Long chains never occur.

They are non-sporing, non-motile and usually non-capsulate with the exception of rare strains. They stain readily with aniline dyes and are uniformly gram-positive but old and phagocytosed organisms may be gram-negative.

Staphylococcus aureus

Detection of Staphylococcus aureus: Staphylococcus aureus colony structure 

Detection of Staphylococcus aureus

Cultural Characteristics

They are aerobes and facultative anaerobes. Optimum temperature for growth is 37°C (range being 12-44°C). Optimum pH is 7.5. They can grow readily on ordinary media.

Nutrient Agar

After aerobic incubation for 24 h at 37°C, colonies are 1-3 mm in diameter and have a smooth glistening surface, an entire edge, a soft butyrous consistency and an opaque, pigmented appearance.

Most strains produce golden-yellow (aureus) pigment, though some strains may form white (non-pigmented) colonies. These white colonised strains of S. aureus are fully virulent.

Pigmentation is characteristic of this species when grown aerobically. Pigmentation is enhanced on fatty media such as Tween agar, by prolonged incubation, and by leaving plates at room temperature.

Non-pigmented strains are not uncommon. The pigment is believed to be lipoprotein allied to carotene. Grown anaerobically, colonies are often smaller and grayish in color.

Blood Agar

The colonies have the same appearance as on nutrient agar, but may be surrounded by a zone of b-hemolysis.

Hemolysis is more likely to be present if sheep, human or rabbit blood is used instead of horse blood and if incubation is in air with 20 percent added carbon dioxide. Hemolysis is weak on horse blood agar.

MacConkey Agar

Colonies are smaller and are pink due to lactose fermentation. MacConkey agar indicates lactose fermenting property. LF produce pink colonies and Non-lactose fermenters (NLF) produce Colorless colonies due to neutral red indicator.

Milk Agar

This medium is prepared by mixing 200 ml of sterile nutrient agar containing 2 percent agar and 100 ml of fresh or sterilized milk and poured into plates.

On this medium, after overnight incubation, the colonies of S. aureus are larger than those on nutrient agar and pigmentation is well developed and easily recognized against the opaque white background.

Phenolphthalein Agar

This is an indicator medium and assists in the identification of S. aureus in mixed cultures. The culture plate is inoculated with the test culture and incubated aerobically at 37°C for 18 hours or for 3 days at 30ºC.

Appearance of the colonies of S. aureus on this medium is similar to those on nutrient agar. All strains of S. aureus produce phosphatase which liberates phenolphthalein from sodium phenolphthalein diphosphate.

Selective Salt Media

Selective medium may be useful for the isolation and enumeration of staphylococci from materials, such as feces, food and dust, likely to contain a predominance of other kinds of bacteria.

Therefore, 7 to 10 percent of sodium chloride may be added to nutrient agar (salt agar) or milk agar (salt milk agar); mannitol salt agar containing 1 percent mannitol, 7.5 percent NaCl, and phenol red in nutrient agar; and Ludlam’s medium containing lithium chloride and tellurite; and salt cooked meat broth (10% NaCl).

Biochemical Reactions

Sugar fermentation test

Aureus ferments a range of sugars (glucose, maltose, lactose, sucrose, including mannitol) producing acid but no gas.

Sugar fermentation is of no diagnostic value except for mannitol, which is usually fermented anaerobically by Staph. aureus but not by other species.

Catalase test

Staphylococcus aureus is a gram positive, catalase and coagulase positive coccus and by far the most important pathogen among for the detection of Staphylococcus aureus. It produces enzymes such as catalase which are considered to be virulence determinants.

Lipolytic test

When grown on media containing egg yolk, produce a dense opacity because most strains are lipolytic.

Phosphatase test

They also produce phosphatase. This is a useful screening procedure for detection of Staphylococcus aureus from Staph. epidermidis in mixed cultures, as the former gives prompt phosphatase reaction, while the latter is usually negative or only weakly positive.

DNAase test

It produces a deoxyribonulease (DNAase), and a heat-stable nuclease (thermonuclease, TNAase) helps in detection of Staphylococcus aureus.

Other biochemical tests

Indole negative, MR positive, VP positive, urease positive, hydrolyzes gelatin and reduces nitrates to nitrites are some other biochemical tests helps in the detection of Staphylococcus aureus.

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