Culture Media

Blood Agar Definition, Preparation, Composition, Application, and limitation.

production of blood agar after the addition of liquid blood.

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This article writter by MN Editors on November 02, 2021

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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Blood Agar Definition, Preparation, Composition, Application, and limitation.
Blood Agar Definition, Preparation, Composition, Application, and limitation.

Blood Agar Definition

Blood Agar is an enriched medium provided with multiple nutrients that generally comes as a basal media for the preparation of blood agar by supplementation with blood.

  • Blood Agar comes as a base medium that is able to be used for its role as an all-purpose growth medium. The medium used for the base can be Columbia Agar or Tryptic Soy Agar.
  • The blood agar medium is a fantastic medium to cultivate extremely discerning bacteria that require specific nutrients, and do not grow rapidly on other media such as Agar for Nutrients. Agar.
  • Around 5% of blood of mammals that have been defibrinated (human sheep, horses) will be added autoclaved basal medium to make blood medium for agar.
  • It is an enhanced medium that aids in the development of fastidious bacteria. It also reduces the growth of bacteria, such as Neisseria as well as Haemophilus..
  • To promote the development of these bacteria, the inhibitors present in blood must be eliminated by heating blood Agar. The heated blood agar transforms into Chocolate Agar which supports Neisseria and Haemophilus.
  • One of the primary applications of blood agar is to study the hemolysis triggered by the growth of bacteria, that can later be used to determine the identity of the organism.
  • Blood agar is mainly used to cultivate pathogenic organisms capable of creating extracellular enzymes that can cause hemolysis in blood.
  • We can make the blood agar specific for specific pathogens through the addition of chemicals, antibiotics or dyes. Examples include crystal violet blood agar that is used to identify Streptococcus Pyogens from swabs of the throat and neomycin and kanamycin blood agar that is used to identify anaerobes in pus.
  • Within the US “blood agar” is generally prepared using Tryptic Soy Agar as well as Columbia Agar base that contains 5% sheep blood. 
  • Horse blood or rabbit blood can be used to develop NAD-dependent organisms like as Haemophilus species, however, the hemolytic patterns could be different from those in sheep blood. (Human blood is generally not recommended due to the possibility of being exposed to blood-borne pathogens , including HIV or Hepatitis.)

Blood Agar Composition 

Like many other nutritional media, Blood Agar includes at least one protein source salt, as well as beef extract to provide minerals and vitamins. In addition to these ingredients, 5percent defibrinated mammalian red blood is added to the medium. The blood agar base can be available for sale by various sellers and can be made in the laboratory when the ingredients readily available. Its exact formula for blood agar is described below:

IngredientsGram/liter
Peptone10.0
Tryptose10.0
Sodium chloride5.0
Agar15.0

In addition to the base medium, 5 % sterile mammalian blood is added after autoclaving prior to pouring on the plates.

Principle of Blood Agar

  • The blood agar is an enriching healthy medium that aids in the development of fastidious organisms through the addition of blood in general media that is not enriched with the blood.
  • The blood that is added to the base adds nutrients to the medium, by providing growth factors that are required by these species that are extremely meticulous.
  • It also assists in the visualization of hemolytic reactions of diverse bacteria. The hemolytic reactions depend on the kind of blood of animals that is used.
  • The majority of sheep blood is utilized for Group A Streptococci as it has the highest effectiveness however, it does not help the development of Haemophilus haemolyticus. This is due to the fact that sheep blood is not rich in pyridine nucleotides.
  • The hemolysis and growth from H. haemolyticus can be best when blood is from horses and the patterns of hemolysis could be similar to Streptococcus Pyogenes, a bacterium found in sheep blood.
  • In addition to the blood that is used in the medium peptone and tryptose also provide carbon amino acids, nitrogen as well as vitamins and minerals needed by the bacteria. Peptone and tryptose are both water-soluble and make it much easier for bacteria to absorb nutrients.
  • The addition of sodium chloride the medium to ensure the osmotic balance of the medium as well as prevent the alteration in pH of the medium throughout growth.
  • The distilled water provides an environment to dissolve nutrients and make it easier for bacteria to take them in.
  • Agar is the agent that solidifies that creates a solid substrate for the organism to develop on. This allows the examination of the morphology of colonies and the being able to count the organism.
  • In certain cases it is possible to add phenolphthaleinphosphate to the medium for determination of phosphate-producing Staphylococci and is accompanied by salt and agar to assess the degree of contamination on the surface.

Blood Agar Hemolysis/Greening reaction on blood agar

Hemolysis is the process of destroying red blood cells within the blood because of extracellular enzymes that are produced by specific bacteria. The extracellular enzymes made from these bacteria can be referred to as hemolysins, which radiate outwards from colonies, leading to complete and partial destruction of red blood cells. Different types of hemolysis are visible on blood agar, which can be distinguished from an area of hemolysis within the colonies that are growing.

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.

Blood Agar and Hemolysis
Blood Agar and Hemolysis

a. Alpha hemolysis on Blood Agar

  • Alpha hemolysis refers to the reduction of red blood cell hemoglobin into methemoglobin in the colony’s surrounding medium. 
  • The result is the appearance of a brown or green discoloration within the medium. This color could be associated to “bruising” the cells. 
  • The microscopic examination of alpha hemolyzed red blood cells reveals that the cell’s membrane remains intact, and therefore it’s not in fact real hemolysis. 
  • Textbook authors often use the term alpha to mean “partial hemolysis,” which can be confusing to students. It is crucial not to confuse with this “partial” or “incomplete” hemolysis with the “weak” or “subtle” hemolysis that occurs in Streptococcus Agalactiae, or Listeria monocytogenes, which is evident above. 
  • During a-hemolysis H2O2 produced by the bacteria triggers hemoglobin in the RBCs of the medium to be converted into methemoglobin.
  • Certain species of a-hemolytics are part of the normal human flora. However, certain species such as Streptococcus pneumonia can cause pneumonia as well as other serious infections.

b. Beta hemolysis on Blood Agar

  • Beta hemolysis (β) is defined as complete or true lysis of red blood cells. A clear zone, approaching the color and transparency of the base medium, surrounds the colony.
  • Many species of bacteria produce toxic by-products that are capable of destroying red blood cells.
  • The clear area appears as result of the complete destruction of the red blood cells in the medium. This causes hemoglobin’s denaturation to create colorless products.
  • B-hemolytic bacteria comprise streptococci of group A such as S. Pyogenes as well as group B streptococcus, such as S. Agalactiae, both of which can cause serious infections in humans.

c. Gamma hemolysis on Blood Agar

  • Gamma hemolysis is also known as non-hemolysis because there is no hemolysis of red blood cells is observed.
  • In the end, there is no color change or a hemolysis-like zone is seen under or around the colonies.
  • The species like Neisseria meningiditis are not hemolytic or Gamma-hemolytic.

d. Alpha prime or wide zone alpha hemolysis

  • Alpha prime hemolysis can be defined as small areas of erythrocytes in good condition close to the colony of bacterial cells and includes the complete lysis of RBCs that surrounds the area of intact erythrocytes.
  • It could be confused with B-hemolysis due the presence of a clear area within the colonies.
Blood Agar and Hemolysis
Image Source: https://paramedicsworld.com/microbiology-practicals/preparation-blood-agar-medium-using-trypticase-soy-agar-base/medical-paramedical-studynotes

Preparation of Blood Agar

  1. Approximately 40 grams of the media is added to 1000ml deionized or distilled water.
  2. The suspension is heated to a point of boiling, which dissolves the medium completely.
  3. Then it is sterilized by autoclaving at 15 lbs pressure and 121degC over 15 minutes.
  4. Then the medium is removed from the autoclave and then cooled down to 40-45degC.
  5. In addition to this, 5% v/v defibrinated sterile blood is added in a sterile manner and well mixed.
  6. It is later poured onto sterilized Petri plates in sterilized conditions.
  7. After the media has solidified and the plates are ready to be placed in the oven on a lower setting for a short time to dry out any moisture on the plates prior to using.

Storage and self-life of Blood Agar

  • The powdered media in its form must be stored between 10 and 30 degrees Celsius in a tightly-closed container. The prepared media should be kept between 20 and 30 degrees Celsius.
  • Following the opening process, the bottle should be properly stored once dry and sealed tightly around the bottle to avoid lump formation since the material is hygroscopic nature , and therefore retains moisture very quickly.
  • The container containing the liquid should be kept in a dry and ventilated space safe from extreme temperatures and ignition sources.
  • The product must be used up before the expiry date listed on the label.

Result Interpretation on Blood Agar

The media’s basal layer appears like a light amber color, which could appear transparent to appear slightly opalescent gel. After adding the 5%, v/v, defibrinated sterile blood, however the opaque cherry red gel develops on Petri plates. The following table illustrates the development of key medical bacteria along with their colony morphologies Blood Agar Medium:

OrganismGrowthColony MorphologyHemolysis
Neisseria meningiditisGood-luxuriantGrey and unpigmented colonies that appear round, smooth, moist, glistening, and convex, with a clearly defined edge.Non-hemolytic or γ-hemolytic.
Salmonella TyphiGood-luxuriantSmooth colorless colonies that are smooth, moist, and flat with a diameter range of 2-4 mm.Non-hemolytic or γ-hemolytic.
Staphylococcus aureusLuxuriantGolden yellow colored circular, convex and smooth colonies of the diameter range of 2-4 mm; opaque colonies with a zone of hemolysis.β-hemolytic.
Staphylococcus epidermidisLuxuriantCircular, colonies of the size 1-4 mm in diameter; grey to white-colored with low convex elevation; moist, glistening colonies.Non-hemolytic or γ-hemolytic.
Streptococcus pyogenesLuxuriantWhite-greyish-colored colonies with a diameter of > 0.5 mm; the colonies are surrounded by a zone of β-hemolysis that is often two to four times as large as the colony diameter.β-hemolytic.
Streptococcus pneumoniaLuxuriantsmall, grey, moist (sometimes mucoidal in encapsulated virulent strains), colonies with the characteristic zone of alpha-hemolysis (green); due to autolysis, often produces a dimple-like zone of hemolysis than the typical crater-like appearance.α-hemolytic.
Pseudomonas aeruginosa Good-luxuriantLarge colonies of the size 2-5mm in diameter; flat colonies that are grey to white-colored with an undulate margin with a zone of β-hemolysis.β-hemolytic.

Application of Blood Agar

  • The principal function of Blood Agar is to facilitate growing and separating extremely fastidious organisms such as Neisseria as well as Streptococcus.
  • It is also used to distinguish the type of hemolysis that is based on bacteria (a- hemolytic, b- or the g-hemolytic) they create on Agar.
  • Blood agar may be used to detect the phosphate-producing Staphylococci by adding phenolphthalein-phosphate to the medium.
  • It is frequently used to make Salmonella Typhi Antigens.
  • The base of blood agar is considered to be a common method to test food samples.

Limitations of Blood Agar

  • The development of Haemophilus hemolyticus is hindered on Blood Agar because of the presence of inhibitors that can be deactivated only by heating the medium following the addition of blood.
  • The patterns of hemolysis may vary depending on the blood type utilized.
  • The addition of horse or rabbit blood to the base medium aids in the development of H. hemolyticus. However, the growth is similar to that of the Streptococcus species. Therefore, it is recommended to be confirmed.
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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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