Blood Agar – Its Composition, Principle and uses

Blood agar (BA) is an enriched media that provides multiple nutrients to those bacteria that require special nutrients to grow which is also called fastidious bacteria. Its unique composition and properties make it an indispensable medium in the laboratory. In this article, we will explore the history, composition, appearance, principle, procedure, applications, and limitations of BA, shedding light on its significant role in microbiological research and diagnostics.

History of Blood Agar

The use of BA dates back to the late 19th century when microbiologists recognized the need for a solid culture medium that supports the growth of various bacteria. It was originally developed by a German bacteriologist, Richard Julius Petri, in collaboration with his mentor, Robert Koch. Petri’s invention of the Petri dish, a flat, cylindrical dish with a lid, facilitated the development of blood agar as a reliable medium for bacterial cultivation.

Composition of Blood Agar

Blood agar is a complex medium composed of BA base which contains several basic ingredients to provide essential nutrients for bacterial growth and blood. The primary components of BA include:

1. Agar: Agar, derived from seaweed, serves as the solidifying agent that transforms the liquid medium into a gel-like consistency. It provides a solid surface for bacterial colonies to develop and facilitates the isolation and enumeration of different bacterial species.
2. Peptones: Peptones, derived from animal or plant proteins, serve as a source of nitrogen and amino acids. They provide the necessary nutrients to support bacterial growth and metabolism.
3. Tryptose: Tryptose, a pancreatic digest of casein, contains a mixture of peptides and amino acids that serve as an additional nutrient source for bacteria.
4. Sodium chloride: Sodium chloride, commonly known as table salt, helps maintain the osmotic balance of the medium, ensuring optimal bacterial growth.
5. Sheep or horse blood: 5% defibrinated mammalian blood is a crucial component of blood agar, providing additional nutrients, growth factors, and indicators for bacterial metabolism. Sheep or horse blood is often used due to their compatibility with most bacterial species.

Appearance of Blood Agar

Blood agar typically appears as a dark red, transparent medium with a gel-like consistency. The medium’s color and transparency arise from the presence of blood components, particularly hemoglobin, which impart a distinctive appearance.

Principle of Blood Agar

The principle of blood agar relies on the ability of bacteria to exhibit different hemolytic patterns when cultured on this medium. Hemolysis refers to the breakdown or lysis of red blood cells, and it can be categorized into three types:

1. Alpha-hemolysis: Some bacteria produce enzymes, such as alpha-hemolysins, that partially break down hemoglobin, resulting in a greenish discoloration of the agar around the bacterial colonies. This zone of incomplete hemolysis is known as the alpha-hemolytic zone.
2. Beta-hemolysis: Other bacteria produce potent exotoxins, known as beta-hemolysins, that completely lyse red blood cells. This leads to a clear zone around the colonies where the red blood cells have been lysed, creating a translucent appearance.
3. Gamma-hemolysis: Some bacteria do not exhibit any hemolytic activity, resulting in no changes to the medium surrounding the colonies. This is referred to as gamma-hemolysis.

By observing the hemolytic patterns on blood agar, microbiologists can make preliminary identifications of bacterial species or groups. For example, Streptococcus pneumoniae typically displays alpha-hemolysis, while Streptococcus pyogenes exhibits beta-hemolysis.

Procedure

The preparation of blood agar involves several steps to ensure the proper incorporation of ingredients and sterilization of the medium. The detailed procedure is as follows:

1. Prepare the nutrient agar or blood agar base according to the desired formulation and dissolve it in 1 liter of distilled water by gentle heating while stirring. The temperature should be sufficient to dissolve the components completely but not exceed the agar’s gelation temperature.
2. Adjust the pH of the medium to the desired level (usually around 7.4) using a suitable pH indicator and a base or acid solution.
3. Autoclave the medium at 121 C for 15 mins to ensure sterilization and remove any potential contaminants.
4. Allow the medium to cool until it solidifies, but maintain a temperature that prevents the solidification of blood components.
5. Aseptically introduce 5% (v/v) aseptically collected sheep or horse blood into the cooled medium, ensuring proper mixing to distribute the blood evenly.
6. Pour the blood agar into sterile Petri dishes and allow it to solidify completely before use.

List of Bacteria with Colony Characteristics 

The following table presents a list of common bacteria encountered in blood agar cultures, along with their corresponding colony characteristics:

Bacterial Species	Colony Characteristics
Staphylococcus aureus	Large, golden-yellow colonies with a clear zone of beta-hemolysis
Streptococcus pyogenes	Small to medium-sized, grayish-white colonies with a zone of beta-hemolysis
Escherichia coli	Medium-sized, mucoid colonies with no hemolysis
Pseudomonas aeruginosa	Large, white – grey colonies with beta-hemolysis
Enterococcus faecalis	Small, tan to light brown colonies with alpha-hemolysis
Neisseria meningiditis	Round , smooth grey colonies with no hemolysis

Uses of Blood Agar

Blood agar finds extensive applications in various areas of microbiology, including:

1. Bacterial Identification: Blood agar enables the identification of bacterial species especially pathogens such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria species based on their characteristic colony morphology, hemolytic patterns, and other growth characteristics. These observations aid in the diagnosis of infectious diseases and guide appropriate treatment strategies.
2. Hemolysis Testing: The different hemolytic patterns exhibited by bacteria on blood agar, such as alpha, beta, and gamma hemolysis, provide valuable information about their pathogenic potential. This testing is particularly useful in differentiating streptococcal species.
3. Antibiotic Susceptibility Testing: Blood agar serves as a suitable medium for performing antibiotic susceptibility testing. By inoculating bacterial isolates onto blood agar plates containing different antibiotic discs, researchers can determine the effectiveness of various antimicrobial agents against specific bacterial strains.
4. Research and Epidemiology: Blood agar is an essential tool in research studies and epidemiological investigations. It allows researchers to study the prevalence, distribution, and characteristics of bacterial pathogens, contributing to our understanding of infectious diseases and the development of preventive measures.

Limitations of Blood Agar

While blood agar is a valuable medium, it has certain limitations:

1. Haemophilus hemolyticus is inhibited on blood agar due to the presence of inhibitors present in blood.
2. Results need confirmations tests
3. Results may vary due to the types of blood used  

Conclusion

Blood agar stands as a cornerstone in the field of microbiology, offering a versatile medium for bacterial cultivation, identification, and diagnostic purposes. Its carefully formulated composition, interaction of ingredients, and distinct appearance contribute to its efficacy in promoting bacterial growth and enabling the differentiation of various species. While blood agar has limitations, its widespread applications in research, diagnostics, and epidemiology make it an indispensable tool in the study of infectious diseases and microbial ecology.

References:

1. Forbes, B. A., Sahm, D. F., Weissfeld, A. S., & Bailey, W. R. (2007). Bailey & Scott’s diagnostic microbiology (12th ed.). Mosby.
2. MacFaddin, J. F. (2000). Media for isolation-cultivation-identification-maintenance of medical bacteria (Vol. 1). Williams & Wilkins.
3. Atlas, R. M. (2010). Handbook of Microbiological Media. CRC Press.
4. https://asm.org/Protocols/Blood-Agar-Plates-and-Hemolysis-Protocols