Blood Agar: Composition, Preparation, Uses and Colony Morphology of Common Organisms

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.

Figure: 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.

Blood agar is the single most important and universally used primary plating medium in clinical microbiology. It is included in virtually every specimen workup — from throat swabs and wound cultures to blood cultures and CSF — because it supports the growth of nearly all clinically significant bacteria while simultaneously providing hemolysis patterns that give immediate presumptive identification clues within 18–24 hours of incubation.

The key diagnostic value of blood agar lies not just in growing organisms but in revealing how each organism interacts with red blood cells — a genetically determined characteristic that directly reflects bacterial virulence factors and narrows identification rapidly before any biochemical testing is performed.

Composition of Blood Agar

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 Blood Agar 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

Figure: 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

S. pyogenes: Beta-hemolysis S. pneumoniae: Alpha-hemolysis 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

Figure: 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.

Figure: 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 agalactiae 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.

Figure: 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 (Alpha') Hemolysis

A fourth type of hemolysis — less commonly described but clinically important — is alpha-prime (α') hemolysis, also called wide-zone alpha hemolysis or target hemolysis.

Alpha-prime hemolysis produces a small, inner zone of complete (clear) beta-hemolysis immediately around the colony, surrounded by a wider outer zone of partial (green) alpha-hemolysis. This creates a distinctive "target" or "bull's-eye" appearance.

Clostridium perfringens produces the most classic alpha-prime hemolysis, sometimes called double-zone hemolysis, due to the combined action of its theta-toxin (producing the outer alpha zone) and alpha-toxin/lecithinase (producing the inner beta zone). This double-zone appearance on blood agar is a strong presumptive identifier for C. perfringens in anaerobic cultures.

Summary of hemolysis types:

Target Hemolysis

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

Colony Morphology of Clinically Important Organisms on Blood Agar

Gram-positive cocci

Gram-positive rods

Gram-negative organisms

Clinically Important Modifications of Blood Agar

Blood agar can be modified by adding selective agents, changing the blood source, or altering preparation to create specialist media:

→ Full article: Chocolate Agar

→ Full article: Columbia CNA Agar

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.

Where Techs Get Confused

• Bacillus anthracis vs. Bacillus cereus. Nearly identical colony size and "ground-glass" appearance. The one reliable bedside distinguisher is hemolysis: B. anthracis is non-hemolytic (gamma); B. cereus produces a wide, beta-hemolytic zone. Given the biosafety stakes, this is the single most important hemolysis reading on this entire page.
• Streptococcus pneumoniae vs. viridans streptococci. Both produce visually identical green, alpha-hemolytic colonies. Hemolysis type alone cannot tell them apart; optochin sensitivity and bile solubility are what actually distinguish them, S. pneumoniae is optochin-sensitive and bile-soluble, viridans strep is neither.
• Streptococcus pyogenes (GAS) vs. Streptococcus agalactiae (GBS). Both are beta-hemolytic but GAS produces a wide beta zone, often 2 to 4 times the colony's own diameter; GBS produces a narrow zone that barely extends past the colony edge.
• True alpha vs. alpha-prime. A colony with a wide green halo might still be hiding a small, true clear zone immediately around it, the alpha-prime, or "target," pattern. Missing that inner clear zone means missing the classic presumptive signature of Clostridium perfringens.

References

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.