Streptococcus Identification



Identification of Streptococcus spp can cause a range of infections from throat infections, toxic shock an a number of other skin and organ infections that can be mild to deadly. So it is important to be able to identify and differentitate between the different Streptococcus spp for treatment.   After a sample has been isolate and a culture grown a Gram Stain is preformed. Gram stains are the first test performed and  Gram reaction and cell morphology dictates the next test performed.  Streptococcus spp are Gram (+) and have a rope/chain like appearance.  




S. pyogenes (causes Strep throat).  The coccuc/ovoid Gram (+) shaped cells form long distinctive chains ("strepto" greek for twisted chain).  Once the Gram stain is done and Gram (+) coccus are seen then the catalase test is done to differentiate between Staphylococcus and Streptococcus (see blog on Staphlyococcus Identification).  Since Streptlococcus spp are Catalase (-) you proceeded with the following identification methods to differenitate within the Streptolococcus spp.  Once the spp is determine they are grouped into groups based on the carbohydrate antigens found on the cell wall, Lancefield grouping.  Group A, B, and D are thought to be medically significant.


Blood Agar



Streak plate Streptococcus pyogenes (above)
Streptococcus bovis (below)

                 Differential media containing 5% sheep blood used to distinguish between species of Streptococcus.  Species that produce the protein hemolysin O and S and  can lyse red blood cells.


Beta hemolysis, complete break down of the red blood cell creating a colorless, clear zone around the growing colony.





Alpha hemolysis, only the hemoglobin is broken down, reduction of the hemoglobin to methemoglobin producing a cloudy/green apperance around the growing colon




Enterococcus faecalis

Gamma hemolysis, growth of the bacteria with no breakdown of red blood cells.
















 

 Streptococcus pyogenes is beta hemolytic, complete lysis of the red blood cells in the media, resulting in clear zones around the colony growth.


Streptococcus bovis:  alpha, incomplete lysis of red blood cells, green area around colony growth,


Enterococcus faecalis:  gamma, growth with no blood cell lysis

Bacitracin Test

The Bacitracin test is done on Streptococcus spp to test for antibiotic susceptibility and can differentiate between Group A beta hemolytic Streptococcus pyogenes from the rest of the Streptococcus spp.
Streptococcus mitis:  

  There is no zone of inhibition indicating resistance to Bacitracin.  S. mitiis is alpha 

 hemolytic and has a greenish hue around the growing colonies due to incomplete lysis, 

 hemoglobin borken down to methemoglobin.


Streptococcus pyogenes:
Zone of no growth, inhibition, seen around the Bacitracin disc.






S. pyogenes:  The zone of inhibition around the bacitracin disc, approx 14mm measuring the entire length of the zone, indicates sensitivity. The zone of inhibition is red because the red blood cells did not lyse. Grown for 24 hrs at 37 degree's C



Bile Esculin Agar (BEA)

BEA is used to differentiate Group D Streptococcus sp, Lancfield grouping, from the rest of the Streptococcus sp.
Contains:
-bile
-the carbohydrate esculin
-iron salts
Differential media:
Group D Streptococcus sp. will convert esculin into 6,7-dihydroxy-coumarin which reacts with the iron salts to form a black precipitate.
Non Group D Streptococcus sp. do not produce the black color.

 

Upper Left: Enterococcus faecalis
Upper Right:  Streptococcus mitis
Lower Left:  Streptococcus pyogenes
Lower Right:  Streptococcus bovis

 6.5% NaCl 
This test is used to differentiate Enterococcus from non-Enterococcus Group-D Streptococcus.  Enterococcus is a halophile that can grow in high salt concentrations.  Growth is indicated by a drop in the pH which trns the media from purple to yellow with bromcreol blue as the pH indicator
Important ingredients: 6.5% salt, dextrose, bromcresol purple
Differential/Selective: used to test for salt tolerance and can be used to identify enterococcal group D streptococci. NaCl is a selective agent and those organisms that can grow in higher salt concentrations also ferment the dextrose in the media. This causes acid formation causing the bromcresol purple indicator to turn yellow. 
(A)                                 (B)

(A) Entercoccus faecalis: Color change from purple to yellow indicating fermentation of the dextrose and salt tolerance.
(B) Streptococcus bovis: No color change or growth.


All Images copyright T.Sturm

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