Blood Agar and Types of Hemolysis

Last updated on June 26th, 2021

Blood agar is an enriched, bacterial growth medium. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media but grow on blood agar. Blood agar is a type of growth medium with trypticase soy agar base enriched with 5% sheep blood.

Beta Hemolysis in Sheep Blood Agar.
Beta hemolysis in sheep blood agar.

Blood agar consists of a base containing a protein source (e.g. Tryptones), soybean protein digest, sodium chloride (NaCl), agar, and 5% sheep blood. 

Blood contains inhibitors for certain bacteria such as Neisseria and Haemophilus genera and the blood agar must be heated to inactivate these inhibitors and to release essential growth factors (e.g., V factor). Heating of blood agar converts it into chocolate agar (heated blood turns a chocolate color) and supports the growth of these bacteria.

Composition of Blood Agar Base

  • Pancreatic digest of casein
  • Papaic digest of soy meal
  • NaCl
  • Agar
  • Distilled water

Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize by autoclaving.

Choice of the Blood

Sheep blood is the first choice to prepare blood agar plates followed by horse, rabbit or goat blood.

Human blood, particularly expired citrated donor blood, should not be used because this may contain substances inhibitory to the growth of some pathogens such as antibiotics, and antibodies like ASO or anti-M protein that could interfere with the growth of S. pyogenes. Citrate inhibits the growth of beta-hemolytic streptococci. Human blood may also contain infectious agents.

Preparation of Blood Agar

  1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by autoclaving at 121°C for 15 minutes.
  2. Transfer thus prepared blood agar base to a 50°C water bath.
  3. When the agar base is cooled to 50°C, add sterile sheep blood aseptically and mix well gently. Avoid the formation of air bubbles.  You must have warmed the blood to room temperature at the time of dispensing to the molten agar base.
    (Note:  If you are planning to prepare a batch of blood agar plates, prepare few blood agar plates first to ensure that blood is sterile).
  4. Dispense 15 ml amounts to sterile Petri plates aseptically
  5. Label the medium with the date of preparation and give it a batch number (if necessary).
  6. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture.  The shelf life of thus prepared blood agar is up to four weeks.

Quality control of Blood Agar

Optochin and Bacitracin Sensitivity of the isolates in Blood Agar
Optochin and bacitracin sensitivity of the isolates in Blood agar
  1. The pH of the blood agar range from 7.2 to 7.6 at room temperature.
  2. Inoculate the plates with 5-hour broth cultures of Streptococcus pyogenes and S. pneumoniae. Inoculate also a plate with H. influenzae and streak with S. aureus (i.e. Satellitism Test).
  3. Incubate the plates in a carbon dioxide enriched atmosphere at 35-37°C overnight.
  4. Check for the growth characteristics of  each species
    1. S. pyogenes: Beta-hemolysis
    2. S. pneumoniae: Alpha-hemolysis
    3. Satellitism of H. influenzae

Uses of Blood Agar

Blood agar has two major uses:

  1. Isolation, identification (with the use of either Optochin disc or Bacitracin disc and testing the sensitivity of the isolate), and antimicrobial susceptibility of Streptococci.
  2. Determine the type of hemolysis, if any. 

Hemolysis

Types of hemolysis (α, β and γ)
Types of hemolysis (α, β and γ)

Certain bacterial species produce extracellular enzymes that lyse red blood cells in the blood agar (hemolysis).  These hemolysins (exotoxin) radially diffuses outwards from the colonies causing complete or partial destruction of the red cells (RBC) in the medium and complete denaturation of hemoglobin within the cells to colorless products.

Four types of hemolysis are produced in sheep blood agar namely; alpha (α) hemolysis, beta (β) hemolysis, gamma (γ) hemolysis, and alpha prime or wide zone alpha hemolysis.

Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting sub-surface colonies. To know the type of hemolysis, the blood agar plate must be held up to a light source and observed with the light coming from behind (transmitted light).

If either type of hemolysis is present, then one will observe a zone of hemolysis surrounding a growing colony.

Various types of Hemolysis
Various types of Hemolysis

Alpha (α) hemolysis

Partial lysis of the RBC to produce a greenish-grey or brownish discoloration around the colony. Alpha hemolysis is due to the reduction of RBC hemoglobin to methemoglobin in the medium surrounding the colony. Many of the alpha-hemolytic streptococci are part of the normal body flora. But Streptococcus pneumoniae which is also alpha-hemolytic causes serious pneumonia and other deadly infectious diseases.

Viridans group of streptococci also gives alpha-hemolysis.

Beta (β) Hemolysis

Beta-hemolysis is the complete lysis of RBCs, resulting in a distinct, clear,
colorless zone surrounding and under the colony. The RBC membrane is destroyed. Organisms of Group A beta-hemolytic streptococci-Streptococcus pyogenes and Group B, beta-hemolytic streptococciStreptococcus agalactiace are beta-hemolytic. The maximal activity of both the hemolysins (oxygen labile (SLO) and oxygen stable (SLS) hemolysins) of group A streptococci, is observed only in anaerobic conditions so beta-hemolytic colonies are better observed when plates are incubated in increased Co2 concentration.

Other beta-hemolytic organisms are Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus.

Double Zone hemolysis produced by Clostridium perfringens
Double zone hemolysis produced by Clostridium perfringens

Gamma (γ) or non-hemolysis

Gamma-hemolysis indicates no hemolysis of RBCs.There is no change in the medium under and surrounding the colonies.

Alpha prime or wide zone alpha hemolysis

A small zone of intact erythrocytes immediately adjacent to bacterial colony, with a zone of complete red-cell hemolysis surrounding the zone of intact erythrocytes. This type of hemolysis may be confused with β-hemolysis.

Target Hemolysis

 Clostridium perfringens are readily identified in the laboratory by its characteristic “double zone” hemolysis also known as target hemolysis.

About Acharya Tankeshwar 473 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.