Blood Agar- Composition, Principle, Preparation, Uses and Hemolysis

Blood agar is a type of bacterial growth medium that contains blood as an additional source of nutrients. Blood agar is used to culture fastidious organisms, which are bacteria that have specific nutritional requirements and do not grow well on ordinary media. Blood agar is also used to differentiate bacteria based on their ability to hemolyze (break down) red blood cells in the blood.

Blood agar consists of a base medium, such as tryptic soy agar or Columbia agar, that provides the basic nutrients for bacterial growth, such as proteins, carbohydrates, salts, and vitamins. To this base medium, 5% of defibrinated mammalian blood (usually sheep or horse) is added after sterilization. The blood provides extra nutrients, such as iron, growth factors, and hemin, that are essential for some bacteria. The blood also serves as an indicator of hemolysis, which is a characteristic reaction of some bacteria that produce enzymes called hemolysins. Hemolysins can cause partial or complete lysis of red blood cells, resulting in different patterns of color change on the agar surface.

There are four types of hemolysis that can be observed on blood agar: alpha (α), beta (β), gamma (γ), and alpha prime (α`). Alpha hemolysis is a partial lysis of red blood cells that causes a greenish discoloration around the bacterial colonies. Beta hemolysis is a complete lysis of red blood cells that causes a clear zone around the bacterial colonies. Gamma hemolysis is no lysis of red blood cells and no change in color on the agar. Alpha prime hemolysis is a double zone of hemolysis, with a narrow zone of intact red blood cells surrounded by a wider zone of complete lysis.

The type of hemolysis produced by a bacterium can be used as a clue for its identification. For example, Streptococcus pneumoniae produces alpha hemolysis, Streptococcus pyogenes produces beta hemolysis, and Staphylococcus epidermidis produces gamma hemolysis. However, hemolysis alone is not sufficient for identification, as different bacteria can produce the same type of hemolysis or different types of hemolysis depending on the source of blood used. Therefore, other tests and characteristics are also needed to confirm the identity of a bacterium.

Blood agar is an important clinical medium that can help isolate and identify fastidious and pathogenic bacteria from various specimens. Blood agar can also be modified by adding other ingredients or heating the blood to create specialized media for specific purposes. For example, chocolate agar is a heated blood agar that supports the growth of Neisseria and Haemophilus species. Phenolphthalein phosphate can be added to blood agar to detect phosphate-producing Staphylococci.

In this article, we will discuss the composition, principle, preparation, uses and limitations of blood agar in detail. We will also explain how to interpret the results on blood agar based on the growth and hemolysis patterns of different bacteria.

Blood agar is a type of enriched medium that contains 5% defibrinated mammalian blood (usually sheep or horse) added to a basal medium such as Columbia agar or tryptic soy agar. Blood agar is used to grow fastidious organisms that require additional nutrients and to differentiate bacteria based on their hemolytic properties. Hemolysis is the lysis of red blood cells by bacterial enzymes called hemolysins, which can produce different patterns of color change on the agar surface.

Columbia agar is a type of enriched medium that contains animal-derived peptones, yeast extract, corn starch, and hemin. Columbia agar can support the growth of a wide range of bacteria, including anaerobes, and can also be supplemented with blood or other additives to enhance its performance. Columbia agar is often used as a base for blood agar or chocolate agar (heated blood agar).

Tryptic soy agar is a type of general purpose medium that contains soybean meal and casein digest as sources of organic nitrogen, glucose as a source of energy, sodium chloride as an osmotic stabilizer, and dipotassium phosphate as a buffer. Tryptic soy agar can support the growth of many bacteria, both gram-positive and gram-negative, and can also be enriched with blood or other supplements to increase its versatility. Tryptic soy agar is commonly used as a base for blood agar or other selective or differential media.

Nutrient agar is a type of general purpose medium that contains beef extract and peptone as sources of nutrients, sodium chloride as an osmotic stabilizer, and agar as a solidifying agent. Nutrient agar can support the growth of many bacteria that are not fastidious, but it may not be sufficient for some bacteria that require specific growth factors or complex nutrients. Nutrient agar can also be modified with various additives to create selective or differential media.

The main differences between blood agar and the other three media are:

• Blood agar contains blood, which provides additional nutrients and allows for hemolysis detection.
• Blood agar is more enriched than nutrient agar, which may not support the growth of some fastidious bacteria.
• Blood agar can be prepared from either Columbia agar or tryptic soy agar as a base, which have different compositions and properties.
• Blood agar may show different hemolysis patterns depending on the type of blood used (sheep, horse, rabbit, etc.).

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:

• Agar: Agar, derived from seaweed, serves as the solidifying agent that transforms the liquid medium into a gel-like consistency.
• Peptone: Peptone is a mixture of peptides and amino acids obtained by the partial hydrolysis of proteins. It provides carbon, nitrogen, vitamins and minerals for the bacteria.
• Beef extract or yeast extract: These are sources of organic nitrogen, carbon and vitamin B complex. They also enhance the growth of fastidious bacteria by providing additional growth factors.
• Sodium chloride: Sodium chloride maintains the osmotic balance and pH of the medium.
• Sheep blood: Sheep blood is the most commonly used blood for blood agar preparation. It provides iron, hemin (X factor) and other growth factors for the bacteria. It also allows the detection of hemolytic reactions by different bacteria.

The exact composition of blood agar may vary depending on the manufacturer or the laboratory preparation. However, a typical formulation of blood agar base is as follows :

The pH of the medium should be adjusted to 7.2-7.6 at 25°C before sterilization. The medium is then autoclaved at 121°C for 15 minutes and cooled to 45-50°C. To this, 5% (v/v) sterile defibrinated sheep blood is added aseptically and mixed gently but well . The medium is then poured into sterile Petri plates and allowed to solidify.

Blood agar is an enriched medium that supports the growth of fastidious organisms by providing them with additional nutrients and growth factors that are present in blood. Fastidious organisms are those that have complex nutritional requirements and do not grow well on ordinary media. Some examples of fastidious organisms are streptococci, pneumococci, haemophilus, and neisseria .

The blood added to the base medium provides proteins, carbohydrates, lipids, vitamins, minerals, and other essential factors that these organisms need for their growth and metabolism . The blood also serves as an indicator of the hemolytic activity of the bacteria. Hemolysis is the lysis or breakdown of red blood cells by bacterial enzymes called hemolysins .

Hemolysis can be observed on blood agar plates by looking at the zone of clearing or discoloration around the bacterial colonies. There are three types of hemolysis that can be distinguished on blood agar :

• Alpha-hemolysis is the partial lysis of red blood cells, resulting in a greenish or brownish discoloration around the colonies. This is due to the conversion of hemoglobin to methemoglobin by hydrogen peroxide produced by the bacteria . Some alpha-hemolytic bacteria are normal flora of the human respiratory tract, such as Streptococcus pneumoniae and Streptococcus viridans, but they can also cause infections like pneumonia and endocarditis .
• Beta-hemolysis is the complete lysis of red blood cells, resulting in a clear zone of hemolysis around the colonies. This is due to the production of hemolysins that destroy both the red blood cells and the hemoglobin . Some beta-hemolytic bacteria are pathogenic and cause diseases like strep throat, scarlet fever, rheumatic fever, and necrotizing fasciitis. Examples of beta-hemolytic bacteria are Streptococcus pyogenes (group A streptococcus), Streptococcus agalactiae (group B streptococcus), and Staphylococcus aureus .
• Gamma-hemolysis is the absence of hemolysis, resulting in no change in the appearance of the medium around the colonies. This means that the bacteria do not produce hemolysins or have a very weak hemolytic activity . Some gamma-hemolytic bacteria are non-pathogenic or opportunistic pathogens, such as Staphylococcus epidermidis, Enterococcus faecalis, and Neisseria meningitidis .

By observing the type of hemolysis on blood agar plates, one can differentiate and identify some bacterial species based on their hemolytic patterns. However, hemolysis alone is not sufficient for identification, and other tests and criteria are needed to confirm the identity of the bacteria . Furthermore, some factors can affect the hemolytic reaction on blood agar plates, such as the type and concentration of blood used, the incubation temperature and time, and the presence of inhibitors or activators in the blood . Therefore, blood agar plates should be used with caution and proper controls when performing hemolysis tests.

References:

: Blood Agar Plates and Hemolysis Protocols - American Society for Microbiology : 22.4: Blood Agar Plates (BAP) - Biology LibreTexts : Blood Agar - Its Composition, Principle and uses : Blood Agar- Composition, Principle, Preparation, Uses and Hemolysis : Blood Agar (BA): Introduction, Composition, Preparation, Principle and Interpretation

Hemolysis is the lysis or breakdown of red blood cells (RBCs) due to the action of extracellular enzymes called hemolysins produced by some bacteria. Hemolysis can be observed on blood agar, which is a medium enriched with 5% sheep blood. Blood agar allows the growth of fastidious bacteria and also shows the type of hemolysis they produce. Hemolysis can be used as a criterion for the identification and differentiation of bacteria, especially streptococci.

There are four types of hemolysis that can be seen on blood agar:

Alpha hemolysis (α)

Alpha hemolysis is characterized by a greenish or brownish discoloration around the bacterial colonies, due to the partial lysis of RBCs and oxidation of hemoglobin to methemoglobin. Alpha hemolysis is also called green hemolysis or incomplete hemolysis. Some examples of alpha-hemolytic bacteria are Streptococcus pneumoniae, Streptococcus viridans, and Enterococcus faecalis.

Beta hemolysis (β)

Beta hemolysis is characterized by a clear zone of complete lysis of RBCs around the bacterial colonies, due to the production of potent hemolysins that destroy both the RBCs and the hemoglobin. Beta hemolysis is also called complete hemolysis or true hemolysis. Some examples of beta-hemolytic bacteria are Streptococcus pyogenes (group A streptococcus), Streptococcus agalactiae (group B streptococcus), Staphylococcus aureus, and Listeria monocytogenes.

Gamma hemolysis (γ)

Gamma hemolysis is actually the absence of hemolysis, as no lysis of RBCs occurs around the bacterial colonies. Gamma hemolysis is also called non-hemolysis. Some examples of gamma-hemolytic bacteria are Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis.

Alpha prime hemolysis (α`)

Alpha prime hemolysis is characterized by a small zone of intact RBCs adjacent to the bacterial colonies, surrounded by a larger zone of complete lysis of RBCs. Alpha prime hemolysis is also called wide zone alpha hemolysis or double zone hemolysis. It may be confused with beta hemolysis due to the presence of a clear zone, but it can be distinguished by holding the plate against transmitted light and observing the inner zone of intact RBCs. Some examples of alpha prime-hemolytic bacteria are Streptococcus intermedius and Clostridium perfringens.

The type of hemolysis produced by bacteria may vary depending on the source and concentration of blood used in the medium, the incubation time and temperature, and the presence of oxygen and carbon dioxide. Therefore, it is important to use standardized methods and conditions for observing and interpreting hemolysis on blood agar.

Blood agar is prepared by adding 5% sterile defibrinated blood to a basal medium that has been autoclaved and cooled to about 45°C. The basal medium can be either Columbia Agar or Tryptic Soy Agar, depending on the preference of the manufacturer or the user. The blood can be obtained from different sources, such as sheep, horse, or human, but sheep blood is the most commonly used. The blood should be fresh and free of any contaminants or inhibitors that might affect the growth or hemolysis of the bacteria. The following are the steps for preparing blood agar:

• Suspend the basal medium powder in distilled water and heat it to dissolve completely.
• Sterilize the medium by autoclaving at 121°C for 15 minutes.
• Transfer the medium to a water bath at 45°C and let it cool slightly.
• Add 5% v/v sterile defibrinated blood to the medium and mix gently but thoroughly. Avoid creating air bubbles or froth.
• Pour the medium into sterile Petri dishes under aseptic conditions.
• Allow the medium to solidify and dry slightly in a laminar flow hood or an incubator.
• Store the plates at 2-8°C until use or invert them and incubate them at 35-37°C for 24 hours to check for sterility.

Some variations of blood agar can be made by adding different supplements or modifying the base medium. For example, chocolate agar is made by heating the blood agar after adding the blood, which causes the release of growth factors from the lysed red blood cells. Chocolate agar is used for growing fastidious bacteria such as Neisseria and Haemophilus. Another example is phenolphthalein phosphate blood agar, which is used for detecting phosphate-producing Staphylococci. This medium contains phenolphthalein phosphate as a substrate and sodium chloride and agar as selective agents. The phosphate-producing bacteria produce alkaline phosphatase, which hydrolyzes the substrate and produces a pink color on the medium.

• The media in the powder form should be stored between 10 to 30°C in a tightly closed container, and the prepared medium should be stored at 2-8°C .
• After opening, the product should be properly stored when dry, after tightly capping the bottle in order to prevent lump formation as the medium is hygroscopic in nature and thus, absorbs moisture relatively quickly.
• The container with the medium should be stored in a dry ventilated area protected from extremes of temperature and sources of ignition.
• The product should be used before the expiry date on the label.
• The prepared plates should be preferably stored in sealed plastic bags to prevent loss of moisture .
• The shelf life of thus prepared blood agar plates is up to four weeks , but some sources suggest that they can last longer if properly stored.
• The plates should be checked for any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or changes in pH before use .
• The plates should also be warmed to room temperature before inoculation.

The basal medium appears light amber colored which might look clear to slightly opalescent gel. After the addition of 5%, v/v sterile defibrinated blood; however, the cherry red-colored opaque gel is formed on the Petri plates .

The growth and appearance of colonies on blood agar can provide useful information for the identification and differentiation of various bacteria. The most important feature to observe is the type of hemolysis (lysis of red blood cells) produced by the bacteria, which can be classified into four types: alpha, beta, gamma and alpha prime .

• Alpha hemolysis is defined by a greenish-grey or brownish discoloration around the colony as a result of the partial lysis of the red blood cells. During alpha-hemolysis, H2O2 produced by the bacteria causes hemoglobin present in the RBC of the medium to be converted into methemoglobin . Some examples of alpha-hemolytic bacteria are Streptococcus pneumoniae, Streptococcus viridans and Enterococcus faecalis.
• Beta hemolysis is defined by a clear zone of hemolysis under and around the colonies when grown on blood agar. The clear zone appears as a result of the complete lysis of the red blood cells present in the medium, causing denaturation of hemoglobin to form colorless products . Some examples of beta-hemolytic bacteria are Streptococcus pyogenes (group A streptococcus), Streptococcus agalactiae (group B streptococcus), Staphylococcus aureus and Listeria monocytogenes.
• Gamma hemolysis is also called non-hemolysis as no lysis of red blood cells occurs. As a result, no change of coloration or no zone of hemolysis is observed under or around the colonies . Some examples of gamma-hemolytic bacteria are Neisseria meningitidis, Neisseria gonorrhoeae and Moraxella catarrhalis.
• Alpha prime hemolysis is defined by a small zone of intact erythrocytes adjacent to the bacterial colony, with a zone of complete lysis of RBCs surrounding the zone of intact erythrocytes. This might be confused with beta-hemolysis due to the appearance of a clear zone around the colonies . Some examples of alpha prime-hemolytic bacteria are Clostridium perfringens and Staphylococcus lugdunensis.

Besides hemolysis, other characteristics such as growth, colony morphology, color, odor and pigmentation can also be observed on blood agar. For example, some bacteria produce pigments that can change the color of the medium or the colonies. Some examples are Serratia marcescens (red pigment), Pseudomonas aeruginosa (green pigment) and Chromobacterium violaceum (purple pigment). Some bacteria also produce distinctive odors that can be detected on blood agar. For example, Pseudomonas aeruginosa produces a fruity odor, Proteus mirabilis produces a fishy odor and Clostridium difficile produces a horse manure odor.

However, blood agar alone is not sufficient for the definitive identification of bacteria. Other tests such as Gram stain, biochemical tests, molecular tests and serological tests are also required to confirm the identity and characteristics of the bacteria isolated on blood agar.

Blood agar is a versatile medium that has many applications in microbiology. Some of the common uses of blood agar are:

• Cultivation and isolation of fastidious organisms. Blood agar is an enriched medium that provides additional nutrients and growth factors for the growth of fastidious organisms that do not grow well on general media. Some examples of fastidious organisms that can be cultivated and isolated on blood agar are Streptococcus, Neisseria, Haemophilus, Corynebacterium, and Listeria.
• Differentiation of bacteria based on hemolysis. Blood agar is also a differential medium that allows the distinction of bacteria based on their ability to produce hemolysins that lyse the red blood cells in the medium. The type of hemolysis (alpha, beta, gamma, or alpha prime) can be observed by the color change or zone of clearing around the colonies. Hemolysis is an important characteristic for the identification and classification of some bacteria, especially Streptococcus species.
• Detection of phosphate-producing Staphylococci. Blood agar can be modified by adding phenolphthalein phosphate to the medium to detect phosphate-producing Staphylococci. Phenolphthalein phosphate is a substrate that is hydrolyzed by phosphatases produced by some Staphylococci species. The hydrolysis results in the release of phenolphthalein, which turns pink in alkaline pH. Therefore, phosphate-producing Staphylococci colonies appear pink on blood agar with phenolphthalein phosphate.
• Preparation of Salmonella Typhi antigens. Blood agar can be used for the preparation of Salmonella Typhi antigens for serological tests such as Widal test. Salmonella Typhi is a pathogenic bacterium that causes typhoid fever in humans. The antigens are prepared by growing Salmonella Typhi on blood agar and then harvesting the bacterial cells and extracting their surface proteins.
• Analysis of food samples. Blood agar base is considered a standard method for the analysis of food samples for the presence of microorganisms. The blood agar base can be supplemented with different selective agents or indicators to enhance the growth or detection of specific microorganisms in food samples. For example, blood agar base with sodium azide can be used for the isolation of Streptococcus thermophilus from yogurt samples.

These are some of the common uses of blood agar in microbiology. However, blood agar is not suitable for all types of bacteria and has some limitations that should be considered before using it.

Blood agar is a useful medium for the cultivation and differentiation of many bacteria, but it also has some limitations that should be considered.

• Blood agar is not suitable for the growth of some bacteria that are inhibited by the inhibitors present in blood, such as Haemophilus hemolyticus. To grow these bacteria, the blood must be heated to deactivate the inhibitors, resulting in chocolate agar.
• Blood agar may not provide complete information for the identification of bacterial isolates, as additional tests and media may be required for confirmation.
• Blood agar may show different patterns of hemolysis depending on the type and source of blood used. For example, sheep blood is preferred for group A streptococci, but it may not support the growth or hemolysis of H. hemolyticus. Horse blood may support the growth of H. hemolyticus, but it may mimic the hemolysis of Streptococcus pyogenes on sheep blood. Human blood is not recommended for blood agar due to safety risks and poor bacterial isolation rates.
• Blood agar may be affected by environmental factors such as temperature, humidity, and oxygen exposure, which may alter the quality and performance of the medium. The medium must be prepared, dispensed, and packaged under oxygen-free conditions to prevent oxidation. The medium must also be stored properly and used before the expiry date to ensure optimal results.

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