Written and maintained by CASRAI Editorial Board
Last updated
This page describes products and general use; it is not medical advice.
Alpha hemolysis on a blood agar plate is a greenish or brownish discoloration of the medium around a bacterial colony. The red blood cells in that zone are discolored, not destroyed. Reading it correctly depends on knowing what the plate is, how to light it, and what separates alpha from beta and gamma reactions.
What a blood agar plate is, and its purpose
A blood agar plate is a general-purpose enriched medium. The American Society for Microbiology (ASM) protocol states its purpose as growing fastidious organisms and differentiating bacteria by their hemolytic properties. “Blood agar” is not a consistently defined medium: the term generally means an enriched base medium to which defibrinated mammalian blood has been added.
Who uses it: microbiology laboratory staff and students who need to grow fastidious organisms or read hemolytic patterns.
How the plate is built
In the United States, blood agar is usually prepared from Tryptic Soy Agar (TSA) or Columbia agar base with 5% sheep blood. ASM gives one commonly used base formula, soybean-casein digest agar (also called trypticase soy agar, TSA or blood agar base), adjusted to pH 7.3 and sterilized by autoclaving. The blood is added after sterilization: the melted base is cooled to 45 to 50 °C, then 5% (vol/vol) sterile defibrinated blood warmed to room temperature is swirled in, avoiding bubbles. ASM notes why the order matters: hot agar can damage red blood cells, and cold blood can cause the agar to gel before pouring.
For the plain, blood-free medium and how plates are classed and stored, see what an agar plate is. For sterilizing the base, see what an autoclave is.
Types and variants
| Variant | Typical use | Key difference |
|---|---|---|
| TSA base with 5% sheep blood | General-purpose enriched, differential plate in US laboratories | The usual US preparation (ASM) |
| Columbia agar base with 5% sheep blood | Same purpose as above | Different base medium, same 5% sheep blood (ASM) |
| Rabbit or horse blood in place of sheep blood | Growth of NAD-requiring organisms such as Haemophilus species | Hemolytic patterns may be inconsistent with those on sheep blood (ASM) |
| Human blood | Discouraged | Greater chance of exposure to blood-borne pathogens such as HIV or hepatitis (ASM) |
What alpha hemolysis looks like
ASM defines alpha hemolysis as the conversion of red blood cell hemoglobin to methemoglobin in the medium surrounding the colony, which produces a green or brown discoloration. Microscopic inspection of alpha-hemolyzed red cells shows an intact cell membrane, so it is not true lysis. ASM warns against two confusions: textbooks that call alpha “partial hemolysis,” and mixing it up with the weak, subtle lysis of Streptococcus agalactiae or Listeria monocytogenes. Beta hemolysis never includes the brown or green discoloration.
ASM’s examples of alpha-hemolytic organisms are the viridans group streptococci (including the S. mutans, mitis and salivarius groups) and Streptococcus pneumoniae. On prolonged incubation many alpha-hemolytic organisms begin to look clearer, but if the surrounding medium still holds any shade of brown or green, the reaction is still alpha.
What causes the green halo
A 2020 study in mSphere (McDevitt et al., 5(6):e01117-20) tested S. pneumoniae on blood agar plates containing 5% sheep blood, incubated aerobically and in 5% CO2. Hydrogen peroxide-deficient mutants lacked the halo, while mutants lacking the pneumolysin gene still produced it. Adding catalase eliminated the halo, and wild-type culture supernatants oxidized oxy-hemoglobin to met-hemoglobin within 30 minutes. The authors concluded that the halo is hydrogen peroxide oxidizing hemoglobin, and described the idea that it comes from a hemolysin as a misconception. Anaerobic cultures of alpha-hemolytic streptococci lacked the greenish discoloration, which means incubation atmosphere affects what you see.
Alpha vs beta vs gamma hemolysis
| Pattern | What the plate shows (ASM) | Example organisms named by ASM |
|---|---|---|
| Alpha | Green or brown discoloration around the colony; red cell membranes intact | Viridans group streptococci; S. pneumoniae |
| Beta | Complete lysis: a clear zone approaching the color and transparency of the base medium around the colony | Streptococcus pyogenes (weaker, subtler beta in S. agalactiae and L. monocytogenes) |
| Gamma | No hemolysis: no reaction in the surrounding medium | Enterococcus faecalis at 24 hours |
Gamma is not always final. ASM shows an Enterococcus strain that is non-hemolytic at 24 hours but shows alpha hemolysis at 48 hours in transmitted light, and notes that many so-called gamma streptococci eventually display weak alpha hemolysis.
How to read the plate
ASM instructs that the plate must be held up to a light source and observed with the light coming from behind (transmitted light). Under reflected light alone, subtle reactions can be missed: ASM’s own photographs of S. agalactiae show no obvious hemolysis in incident light and subtle hemolysis in transmitted light. For very weak reactions, ASM suggests removing the colony with an inoculating loop to view the cells directly beneath it. Incubation time and atmosphere both matter, because the appearance can change with each. For incubator basics, see what a lab incubator is.
Safe use and handling basics
ASM advises that students should be able to explain and practice safe laboratory techniques, and points to its laboratory safety guidance and its biosafety guidelines for teaching laboratories. ASM discourages human blood in these plates because of the risk of exposure to blood-borne pathogens. For general containment background, see biosafety levels BSL-1 to BSL-4.
Common mistakes
- Calling alpha “partial hemolysis” and treating it as a weak form of beta. ASM says the cells are discolored, not lysed.
- Reading the plate only under reflected light, which can miss subtle reactions.
- Calling a plate gamma at the first read when later incubation may show weak alpha.
- Assuming rabbit or horse blood plates will show the same pattern as sheep blood. ASM says the patterns may be inconsistent.
- Pouring blood into hot agar, which can damage the red cells, or cold blood into agar that then gels early.
FAQ
What is the blood agar plate test?
It is the use of a blood agar plate to see how an organism affects the red blood cells in the medium. The reaction is classed as alpha, beta or gamma, and read in transmitted light. A related stain-based look at one organism is covered in Haemophilus influenzae Gram stain.
What is the purpose of a blood agar plate?
According to ASM, it is a general-purpose enriched medium used to grow fastidious organisms and to differentiate bacteria by their hemolytic properties.
What does alpha hemolysis look like on a blood agar plate?
A green or brown discoloration of the medium around the colony, with the red cell membranes still intact.
Is alpha hemolysis true hemolysis?
No. ASM says microscopic inspection shows intact cell membranes, so it is not true lysis. The mSphere study attributes the S. pneumoniae halo to hydrogen peroxide oxidizing hemoglobin.
Sources
- American Society for Microbiology, “Blood Agar Plates and Hemolysis Protocols” (author Rebecca Buxton), asm.org/protocols/blood-agar-plates-and-hemolysis-protocols, including the protocol PDF, checked 2026-10-29.
- McDevitt E, Khan F, Scasny A, Thompson CD, Eichenbaum Z, McDaniel LS, Vidal JE. “Hydrogen Peroxide Production by Streptococcus pneumoniae Results in Alpha-hemolysis by Oxidation of Oxy-hemoglobin to Met-hemoglobin.” mSphere 2020;5(6):e01117-20, PMC7729260, DOI 10.1128/mSphere.01117-20, checked 2026-10-29.
