Blood Agar and Types of Hemolysis

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 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, so 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

Beed heart peptone10 gm
Tryptose10 gm
Sodium chloride5 gm
Agar15 gm
Sheep blood5%
Final pH at 25°C 7.3 ± 0.2 

Protein sources may differ among manufacturers and may be pancreatic digest of casein, papic digest of soy meal, neutralized peptone, yeast extract, or a combination of them. Please check the paper insert in the purchased media.

Choice of the Blood

Sheep blood is the first choice to prepare BA 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. Residual antibiotics in host blood and antibodies like ASO or anti-M protein could interfere with the growth of S. pyogenes. Citrate inhibits the growth of beta-hemolytic streptococci. Infected human blood may also contain infectious agents.

Preparation of Blood Agar

Preparation of blood agar from dehydrated blood agar base

  1. Prepare the BA base as instructed by the manufacturer.
  2. Sterilize by autoclaving at 121°C for 15 minutes.
  3. Transfer thus prepared BA base to a 50°C water bath.
  4. 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.
  5. Dispense 15 ml amounts to sterile Petri plates aseptically
  6. Label the medium with the date of preparation and give it a batch number (if necessary).
  7. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture.  The shelf life of thus prepared BA is up to four weeks.

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.

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. 


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 diffuse 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. Hold the BA plate must be up to a light source and observed with the light coming from behind (transmitted light) to know the type of hemolysis.

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

Alpha hemolysis is the partial lysis of RBCs to produce a greenish-grey or brownish discoloration around the bacterial 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 flora of humans 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 streptococci-Streptococcus 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.

Modifications in Blood Agar

Adding dyes and antibiotics helps make the blood agar selective to specific pathogens. The most common modification is the addition of the dye crystal violet, antibiotics kanamycin and neomycin, and heating of the blood agar to form chocolate agar. 

Crystal Violet Blood Agar (CVBA)

CVBA can isolate and identify Group A, Beta-hemolytic streptococci.

Streptococcus pyogenes causes numerous infections in humans, including strep throat (Streptococcal pharyngitis), rheumatic fever, post-streptococcal glomerulonephritis, wound/skin infections (cellulitis, erysipelas, necrotizing fascitis, myonecrosis) is a beta-hemolytic Group A Streptococcus.

Adding crystal violet to the blood agar prevents the growth of Staphylococcus aureus and other oral commensals. For the preparations of CVBA, add 1 ml of 0.02% w/v crystal violet to every 1000 ml of Blood agar. 

Neomycin Blood Agar

This modification is done by adding the neomycin stock solution to the blood agar. This agar acts as selective media for isolating group A streptococci (S pyogenes) and group B streptococci (S agalactiae). 

Neomycin suppresses the growth of oral commensals and Gram-negative rods, facilitating the growth of obligate anaerobes. In 250 ml of blood agar, add 1 ml of working neomycin sulfate to prepare 70 mg/ml of neomycin blood agar. 

The inoculated agar plate should be incubated under anaerobic conditions for 24-48 hours at 36 ±1℃. Group A streptococci (S pyogenes) colonies appear white to gray, small (1-2 mm), translucent or opaque with a zone of beta-hemolysis. In contrast, the growth of S agalactiae or Group B streptococci in neomycin blood agar is moderate to high with a clear zone of beta-hemolysis.  

Laked Blood Kanamycin and Vancomycin Agar (LKV)

This agar is helpful for the isolation and partial identification of obligate anaerobic gram-negative bacilli, especially Prevotella species. It is an enriched, differential, and selective medium. 

The medium comprises casein, meat peptone, soy peptone, dextrose, and yeast extract for the nutritional compounds required to grow gram-negative bacilli. 

Kanamycin and Vancomycin are selective agents that prevent the growth of most obligate gram-positive and facultative anaerobic bacteria. Laked sheep blood and vitamin K1 facilitate the recovery and pigment production of Prevotella melaninogenica and Porphyromonas species. 


  1. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
  3. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  4. Facklam and Washington. (1991). In Balows, Hausler, Hermann, Isenberg and Shadomy (ed.), Manual of clinical microbiology, 5th ed. American society of microbiology, Washington, D.C.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

45 thoughts on “Blood Agar and Types of Hemolysis

  1. when an incubate blood agar .it turns to black colour… every thing is like preparation. plz take out from this problem.

  2. I have a friend who claims that adding Blood at 50 degrees centigrade causes heamolysis in the final product i.e. Blood Agar. The microbiologist using the product complain that they cannot differentiate the type of bacterial growth if its beta or alpha. Any suggestions on this issue? thank you in advance.

    1. Thank you for your comment. There are two possibilities..
      1. The temperature of Blood agar base is more than 50°C; You can test adding the blood after it’s temperature is 45°C
      2. The problem might be in the Blood you are using. Do you use sheep blood or human blood?

  3. I have a query with what you explained on the gamma hemolysis. Actually you have said no hemolysis. please if possible help me understand on my email.

  4. My teacher says blood agar should be sterilized in an inspissator. Should it be as it has protein in it?

    1. Your teacher might be saying media containing eggs such as Lowenstein Jensen medium uses inspissator. For Blood Agar, we sterilized blood agar base, and later you mix the blood. As blood is sterile body fluid, you do not need to sterilize it.

    2. Jiskirat, you might have mistaken, your teacher might not have said so. He/She might have says, you have to sterilize egg containing medium such as Lowenstein Jensen medium using inspissator. In the case of Blood Agar, you sterilize and prepare blood agar base, in which you add required amount of Blood (Blood is sterile body fluid, so you need not to sterilize it). For procedure, please go through the post again.

  5. Can you please help me with easy steps on analysis of plant extracts on anti-bacteria activity against Heliobacter pylori, thank you

    1. Dear Sam
      Culturing Helicobacter pylori in the laboratory is not a easy process and we use other tests, mainly Urea breathe test to diagnose if the patients admitted in our hospital have H.pylori infection. As i have not grown H.pylori and also not involved in extraction of plant products to check their antibacterial effects, I am sorry, i am not able to guide you with easy steps you have to follow. I am hopeful you will find it in pubmed or other similar portals of scientific interests .

  6. thank you this very good work. please could include the preparation of layered blood agar and the advantages it has over blood agar?

  7. 1. why do one need to pour water unto the nutrient agar imside the erlenmeyer flask and not the other way/
    2. what is the medium colour brfore and after adding the blood/

  8. Hello Mr. Acharya,
    Your blog is useful. I saw it for preparation of Manuals for NABL accreditation of laboratory.

    Could you please help me:
    1. You mentioned about shelf life of blood agar as 4 weeks. Can you give a standard reference for this
    2. Do we have to pack this blood agar in single packing…..
    3. Sterility Check of blood agar should be done for how many percent of plates prepared.

    If the same principles can be applied for other media like MHA

  9. I need your help sir, am writing a project on hydrocarbon degrading bacteria, what are the procedure sir.

    1. Dear Abdul Wasiu
      Thank you for your query, but my areas of expertise is only on pathogenic bacteria, So i may not be the right person to guide on the project you are planning to begin.

  10. Sir why they named delta hemolysis as one of the type of hemolysis. They are not mentioned properly in any books . Could you help me??

    1. In some papers, it is mentioned that Staphylococcus has delta hemolysin, but we do not use delta hemolysis much often so i do not have much idea about this type of hemolysis. Where you find about delta hemolysis?

  11. Thank you sir..can you help me out comparion between sheep blood agar, horse blood agar , human blood agar

  12. VERY CLEAR and helpful info and tech for every one who want to learn from; Sincerely. Thanks

  13. What anticoagulant should be used while collecting sheep blood please ?
    Does any can be harmful to the media ?

  14. If blood agar was to be used to culture S. aureus and P. aeruginosa will there be a possibility that another type of bacteria will grow???

    1. Yes, if the sample contains other bacteria, they will also grow on it. Blood agar is an enriched culture medium and supports growth of most of the pathogenic bacteria except few fastidious bacteria like Haemophilus influenzae and obligate intracellular bacteria.

We love to get your feedback. Share your queries or comments

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Recent Posts