Isolation and Identification of Staphylococcus and Streptococcus

Staphylococcus

Overview of Staphylococci

Staphylococci are Gram-positive cocci that are catalase positive and halophilic. They are commonly found on the skin and mucous membranes and are responsible for a variety of infections, including abscesses, boils, septicemia, endocarditis, cystitis, osteomyelitis, and pyelonephritis. The most clinically significant species include Staphylococcus aureus, S. epidermidis, and S. saprophyticus.

• S. aureus: Most virulent; can survive for months on dry surfaces; spread by contact with pus, skin, or objects; MRSA is resistant to most β-lactam antibiotics.

• S. epidermidis: Normal skin flora; not usually pathogenic; major virulence factor is biofilm formation (especially on catheters); distinguished from S. saprophyticus by novobiocin sensitivity.

• S. saprophyticus: Normal flora of female genitalia; associated with community-acquired urinary tract infections.

Isolation and Identification of Staphylococci

Selective and differential media are used to isolate and identify Staphylococci. Mannitol Salt Agar (MSA) is a common medium containing beef extract, peptone, 7.5% sodium chloride (selective for Staphylococci), mannitol, and phenol red (pH indicator). S. aureus ferments mannitol, producing acid and turning the phenol red indicator yellow, while other Staphylococci do not ferment mannitol, leaving the medium red.

• Selective agent: 7.5% NaCl inhibits most bacteria except Staphylococci.

• Differential agent: Mannitol fermentation is indicated by a color change in phenol red.

Coagulase Test

The coagulase test is the definitive method for confirming the pathogenicity of S. aureus. Nearly all pathogenic S. aureus are coagulase positive, meaning they can clot plasma. Other Staphylococci are coagulase negative. The test involves inoculating plasma with the test organism and incubating at 37°C. Clot formation indicates a positive result.

• Procedure: Inoculate plasma, cover, incubate, and observe for clotting.

• Interpretation: Any clot formation is a positive result for S. aureus.

Streptococcus

Overview of Streptococci

Streptococci are Gram-positive cocci that are catalase negative and usually form chains. They inhabit a wide range of hosts and environments, including the mouth, nares, pharynx, skin, and various tissues. Pathogenic species include Streptococcus pneumoniae and S. pyogenes.

• S. pneumoniae: Alpha-hemolytic, causes pneumonia, inhibited by optochin.

• S. pyogenes: Beta-hemolytic, Group A, causes pharyngitis and other infections, sensitive to bacitracin.

Hemolysis on Blood Agar Plates (BAP)

Blood agar is used to differentiate Streptococci based on their hemolytic properties. Hemolysins produced by Streptococci cause different types of hemolysis:

• Alpha (α) hemolysis: Partial lysis, greenish-brown color (e.g., S. pneumoniae).

• Beta (β) hemolysis: Complete lysis, clear zone (e.g., S. pyogenes).

• Gamma (γ) hemolysis: No hemolysis, red color remains (non-hemolytic).

Identification of Streptococci Using Disks

Specific disks are used to identify Streptococcal species on blood agar:

• A Disk (Bacitracin): Used to identify Group A Streptococci (S. pyogenes), which are sensitive to bacitracin.

• P Disk (Optochin): Used to identify S. pneumoniae, which is sensitive to optochin.

Isolation from the Upper Respiratory Tract

To isolate Streptococci from the respiratory tract, a swab is taken from the throat and inoculated onto a blood agar plate. The plate is incubated in a capnophilic environment (rich in CO2), often using a Brewer's jar.

Blood Agar Plate (BAP) Composition and Use

Blood agar plates are composed of general-purpose media (e.g., nutrient agar or tryptic soy agar) with 5% sheep red blood cells added at 50-55°C to keep RBCs intact. Growth on BAP allows observation of hemolysis, aiding in bacterial identification.

Summary Table: Hemolysis Types and Identification

Hemolysis Type	Appearance on BAP	Example Species	Key Test
Alpha (α)	Greenish-brown, partial lysis	S. pneumoniae	Optochin sensitivity (P disk)
Beta (β)	Clear zone, complete lysis	S. pyogenes	Bacitracin sensitivity (A disk)
Gamma (γ)	No hemolysis, red color remains	Non-pathogenic Streptococci	None

Rapid Strep Test

Rapid Strep tests are used in clinical settings for quick detection of Group A Streptococcal antigen from throat swabs. A positive result should be confirmed by culturing on blood agar.

Additional info:

• Lancefield grouping: Streptococci are classified into groups (A-V) based on cell wall antigens, with Group A being the most clinically significant for human disease.

• Capnophilic incubation: Many Streptococci grow best in elevated CO2 environments, which is why Brewer's jars or CO2 incubators are used.