Question

how is streptococcus pneumoniae isolated? what are the steps and tests required?

Answer 1

ANSWER

Streptococcus pneumoniae is a fastidious bacterium, growing best at 35-37°C with ~5% CO2. Usually, it is cultured on media that contain blood, but can also grow on a chocolate agar plate. On blood agar plate, colonies of Streptococcus pneumoniae appear as small, grey, moist colonies and characteristically produce a zone of alpha-hemolysis (green).

The following specialized tests are used to identify colonies on a Blood Agar Plate. S. pneumoniae can be identified using Gram stain, catalase, and optochin tests simultaneously, with bile solubility as a confirmatory test. If these tests indicate that the isolate is S. pneumoniae, serological tests to identify the serotype can be performed.

1) Catalase test:

Catalase is an enzyme which breaks down hydrogen peroxide (H2O2) into H2O and O2. The O2 is given off as bubbles in the liquid. This test is primarily used to differentiate between gram-positive cocci. Members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.

A) Procedure:

• Grow the isolate(s) to be tested for 18-24 hours on a Blood Agar Plate at 35-37°C with ~5% CO2
• From overnight growth on the Blood Agar Plate, use a disposable loop to carefully remove a colony and place it on a glass slide
• Add 1.0 ml of 3% H2O2 to the slide and mix with the bacteria.
• Observe the bacterial suspension on the slide immediately for vigorous bubbling

B) Result:

• Any bubbling from a transferred colony indicates a positive test.
• The absence of bubbling from a transferred colony indicates a negative test.

2) Optochin test:

S. pneumoniae strains are sensitive to the chemical optochin. Optochin sensitivity allows for the presumptive identification of alpha-hemolytic streptococci as S. pneumoniae.

A) Procedure:

• Grow the strain(s) to be tested for 18-24 hours on a Blood Agar Plate at 35-37°C w ith ~5% CO2
• Use a disposable loop to remove an isolated colony from the overnight culture on the Blood Agar Plate and streak onto one half of a Blood Agar Plate.
• Place a P disk (Optochin disk) within the streaked area of the plate and incubate the Blood Agar Plate overnight at 35-37°C with ~5% CO2
• Observe the growth on the Blood Agar Plate near the Optochin disk and measure the zone of inhibition, if applicable.

B) Result:

• Using a 6 mm, 5 μg disk, a zone of inhibition of 14 mm or greater indicates sensitivity and allows for presumptive identification of pneumococci.
• Zones of inhibition should be measured from the top surface of the plate with the topremoved.

3) Bile solubility test:

The bile (sodium deoxycholate) solubility test distinguishes Streptococcus pneumoniae from all other alpha-hemolytic streptococci. S. pneumoniae is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant.

A) Procedure:

• Grow the isolate(s) to be tested for 18-24 hours on a Blood Agar Plate at 35-37°C with ~5% CO2
• Add bacterial growth from the overnight Blood Agar Plate to 1.0 ml of 0.85% saline to achieve turbidity in the range of a 0.5-1.0 McFarland standard.
• Divide the cell suspension equally into 2 tubes (0.5 ml per tube).
• Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube. Add 0.5 ml of 0.85% salineto the other tube. Mix each tube well.
• Incubate the tubes at 35-37°C in CO2.
• Vortex the tubes.
• Observe the tubes for any clearing of turbidity after 10 minutes. Continue to incubate the tubes for up to 2 hours at 35-37°C in CO2 if negative after 10 minutes. Observe again for clearing.

B) Result:

• A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test.