HEMOLYTIC STREPTOCOCCUS DETECTION
BY THROAT CULTURE
©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
hemolysis, alpha and beta
Taking a throat swab,
light in position,
tongue depressed

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Hemolysis on blood agar,
alpha and beta

     The Streptococci cause more disease than any other single group of bacteria.  They are aerotolerant, but do not use O2 and lack catalase. Most streptococci are non-pathogenic, including lactic acid fermenters found in fermented milk products such as yogurt, buttermilk and sour cream.

(The image at the left is of Streptococcus pyogenes at 1000x, stained with methylene blue.)

   
Pathogenic strains of strep are characterized by two traits:

1.  HEMOLYSIS: Pathogenic strains produce the exotoxin streptolysin which causes the complete lysis of red blood cells.  When these strains are grown on blood agar, their colonies are surrounded by a yellowish halo of complete clearing on a background of the bright red agar, called beta hemolysis.   Some strains produce partial hemolysis on blood agar, and produce turbid halos with a greenish cast around the colonies, termed alpha hemolysis. Those strains which produce no lysis are termed (for some reason) gamma hemolytic.  Here is a labeled image displaying both alpha and beta hemolysis.
2. ANTIGENICITY: The M protein is part of the cell wall, and functions to mediate attachment and to resist phagocytosis. These M proteins have been serologically classified by Rebecca Lancefield into groups A through O. Pathogenic strains of strep are limited to those which carry the group A antigen.
Thus, pathogenic strep is Group A, beta hemolytic. Streptococcus pyogenes is most common member of this group. It causes a wide variety of diseases including:
Strep throat: beefy red pharynx, fever, sore throat (80% Strep infections are asymptomatic)

Puerperal fever: infection of the uterus following contamination during childbirth.

Rheumatic fever occurs in 3% of untreated strep-infected children with febrile exudative pharyngitis, and is thought to be an autoimmune manifestation. One to five weeks following a strep infection the sequelae may include rheumatoid arthritis, endocarditis, and/or pyelonephritis.

We will learn to perform a diagnostic test for beta hemolysis on blood agar in which a plate is inoculated with a throat swab (of the oropharynx and palatine tonsils) and incubated at 35C. Presence of nunerous colonies with the beta hemolytic reaction on the plate strongly suggests strep throat.
 


Illustrate the structure of the oropharynx, including palatine tonsils, soft palate, oropharynx and the uvula.  Review the subject in an anatomy text.  Click on the image to see a labeled view of the oral cavity .


EQUIPMENT AND
SUPPLIES:
well-focused light to illuminate oropharynx
Tongue depressors, sterile
Sterile swabs, 15 cm long
Blood Agar Plates
fresh culture of Streptococcus pyogenes
Incubator, 35C
  1. SETUP: For right handed persons: Position subject around the corner at the right end of desk, with the light to your left so light will project on rear of throat .
  2. Have subject comfortably open mouth, relax tongue, insert depressor all the way to the rear of tongue and depress entire tongue to hold out of way .
  3. TAKE SPECIMEN: Have subject say "Aaaah" and GENTLY swab across the rear of the oropharynx and tonsils (suspended on either side of the oropharynx) if present.
  4. APPLY TO BLOOD AGAR PLATE: Swab the specimen across one half of the surface of a Blood Agar Plate, rolling the swab as you sweep it across, then stab almost to the bottom of the agar several times at the origin of the streak. Swab a known culture of Streptococcus pyogenes on the other half of the plate as a positive control.
  5. INCUBATE: Place the plate agar side up at 35C for 24 hrs.
  6. SCORE THE PLATE: Examine the plate for hemolysis, record results. Handle with careful attention to aseptic technique, since known pathogens are growing on the plates.
Here are a few pictures of hemolytric reactions:


BLood agar plate with Streptococcus pyogenes streaked at the top and a throat swab on the bottom.  Note the clarity of the clearing around the S. pyogenes colonies, and the characteristic greenish turbidity of alpha hemolysis in the throat swab.
A mixed streak of non-hemolytic (larger colonies) and smaller Streptococcus pyogenes colonies showing zones of clearing around each colony.
Mixed streak showing the clearing around a concentration of S. pyogenes colonies.
Close up of a student's throat swab with alpha hemolysis surrounding the streak.
Here is a student's culture showing considerable beta hemolysis.  Streptococcus pyogenes is streaked at the top as a positive control.