9.7: Blood Agar Plate Media, BAP
- Demonstrate proficiency in aseptic techniques required for the proper inoculation and handling of blood agar plates.
- Differentiate hemolytic patterns (alpha, beta, and gamma) exhibited by microorganisms when grown on blood agar.
- Understand the clinical relevance of blood agar media in microbiology, connecting laboratory findings to real-world applications such as the diagnosis of infectious diseases.
Blood Agar
Blood agar is an enriched medium used for the isolation and cultivation of fastidious bacteria . Fastidious organisms require specific nutrients and don’t grow well on general media like nutrient agar. Blood agar is prepared from a tryptic soy agar base enriched with 5% sheep blood (Fig.1).
Several species of gram-positive cocci produce exotoxins called hemolysins , which are able to destroy red blood cells (RBCs) and hemoglobin. Blood agar can differentiate bacteria based on their hemolytic properties. The three major types of hemolysins are beta hemolysis (β), alpha hemolysis (α), and gamma hemolysis (γ).
Beta hemolysis is the complete lysis of red blood cells. It is defined by a clear zone of hemolysis surrounding the colonies grown on a blood agar plate (Fig.2). The clear zone is a result of the complete denaturation of the hemoglobin protein. β-hemolytic bacteria include group A streptococci like Streptococcus pyogenes . Infections caused by S. pyogenes include strep throat, scarlet fever, bacteremia, pneumonia, necrotizing fasciitis, myonecrosis, and Streptococcal Toxic Shock Syndrome (StrepTSS)
Figure 2: Clearing of blood agar by beta-hemolytic Streptococcus pyogenes .
Alpha hemolysis is the partial destruction of RBCs. Microscopic inspection of alpha-hemolyzed RBCs shows that the cell membrane is intact. During α-hemolysis, hemoglobin is reduced to methemoglobin which produces a greenish discoloration of the agar around the colonies (Fig.3). Some of the α-hemolytic species are a part of the human normal flora (e.g., Escherichia coli ), but some species like Streptococcus pneumonia are pathogenic.
Figure 3: Normal upper respiratory biota demonstrating alpha-hemolytic activity. The greenish discoloration is a result of incomplete red blood cell lysis.
Gamma hemolysis is a lack of hemolytic activity and results in simple growth with no change to the medium present (Fig.4). Enterococcus faecalis , a common human urinary tract pathogen, frequently exhibits gamma hemolytic activity.
Figure 4: This streak plate of Enterococcus faecalis on blood agar illustrates no hemolytic activity.
The Diagnosis of Strep Throat
Streptococcus pyogenes is a potentially pathogenic bacterium known to cause strep throat, scarlet fever, pneumonia, bacteremia, and Streptococcal Toxic Shock Syndrome. An identifiable characteristic of S. pyogenes is beta-hemolysis, or complete destruction of red blood cells. This results in a clear zone around S. pyogenes grown on blood agar. When a patient is showing symptoms of strep throat, a throat swap is taken. The throat swap is then used to inoculate blood agar media. If results a clear zone around the bacterial growth, there is a high probability S. pyogenes is the cause of infection. Therefore, if a culture from a throat swab shows beta hemolysis, there is high likelihood that patient is infected has strep throat.
Figure 5. The image on the left depicts tonsillitis, which is a common symptom of strep throat. The image on the right depicts S. pyogenes colonies on blood agar. The clear zones around the bacterial growth indicate beta hemolysis. Throat swaps from a patient with strep throat will have similar results.
Laboratory Instructions
- On the bottom of your blood agar plate, use a sharpie to divide the plate into three sections.
- Label the plate with your name/group name, date, course section, species initials, and plate type (e.g., blood).
- With a sterile cotton swab, aseptically inoculate each section of the plate with the corresponding bacterial species. Make sure to use a new cotton swab with each bacterial species.
- Seal the plate with parafilm.
- Invert the petri plate and incubate for 24 hours at 37 °C.
- Observe, record, and interpret results.
Resources
- Rebecca Buxton. 2005. Blood agar plates and hemolysis protocols. Web. 1 October 2016 Rebecca Buxton. 2005. Blood agar plates and hemolysis protocols. Web. 1 October 2016 http://www.asmscience.org/content/education/protocol/protocol.2885
- Stevens, D. L., & Bryant, A. E. (2016). Severe group A streptococcal infections. https://www.ncbi.nlm.nih.gov/books/NBK333425/
Attributions
- 22.4: Blood Agar Plates (BAP) is shared under a CC BY license and was authored, remixed, and/or curated by Kelly C. Burke .
Figure 5 Modified from:
- "S. Pyogenes Infection (Sketch).jpg" by Sam-Bullo, Wikimedia Commons is licensed under CC BY 4.0
- "Streptococcus pyogenes agar sangre.jpg" by Micronerdbox , Wikimedia Commons is licensed under CC BY 2.0