Columbia Blood Agar Base w/ Hemin – Composition, Preparation, Principle, Result, Uses

What is Columbia Blood Agar Base?

• Columbia Blood Agar Base is an enriched base used for the preparation of various types of agar media, including chocolate agar, blood agar, and selective or identification media. It is highly nutritious and supports the growth of both nonfastidious and fastidious microorganisms obtained from clinical specimens.
• The development of Columbia Agar was reported by Ellner et al. in 1966. This formulation incorporates two peptones and yeast extract, which provide essential nutrients and B complex vitamins, respectively. The addition of corn starch in the medium helps absorb toxic by-products present in the specimen, while also serving as an energy source for organisms that produce alpha-amylases. The inclusion of 5% sheep blood in Columbia Agar enables the detection of hemolytic reactions and supplies the necessary X factor (heme) required for the growth of many pathogenic species.
• Columbia Blood Agar is known for promoting larger colony size and more luxuriant growth compared to other blood agar bases. It is widely recommended as a primary isolation medium in various diagnostic manuals and standards, such as the MiQ standards. In many European countries, Columbia Blood Agar has become the preferred medium for primary isolation of clinical specimens.
• Overall, Columbia Blood Agar Base provides a highly supportive environment for the growth and cultivation of a wide range of microorganisms, making it a valuable tool in the laboratory for diagnostic purposes and microbial research.

Principle of Columbia Blood Agar Base

The principle of Columbia Blood Agar Base lies in its composition and functionality in supporting the growth and differentiation of various bacterial species. Here are the key principles of Columbia Blood Agar Base:

1. Base for Blood and Selective Media: Columbia Agar Base serves as the foundation for media formulations containing blood and selective agents. Different combinations of antimicrobial agents can be added to create selective media.
2. Hemolytic Reactions and X-Factor Supply: The inclusion of sheep blood in Columbia Agar Base allows for the detection of hemolytic reactions, aiding in the identification and differentiation of bacterial species. Sheep blood also provides the X-factor (heme) necessary for the growth of many bacterial species.
3. Lack of V-Factor: Sheep blood lacks the V-factor (Nicotinamide Adenine Dinucleotide), as it contains NADase, an enzyme that destroys NAD. Therefore, Haemophilus influenzae, which requires both the X and V factors, will not grow on this medium.
4. Selectivity for Haemophilus Species: The addition of bacitracin to enriched Columbia Agar Medium makes it selective for the isolation of Haemophilus species from clinical specimens, particularly those obtained from the upper respiratory tract.
5. Growth-Supporting Properties: Columbia Blood Agar Base, supplemented with peptones prepared from tryptone, peptone, and HM peptone B, provides superior growth-supporting properties. Cornstarch present in the medium serves as an energy source and neutralizes toxic metabolites.
6. Detection of Hemolysis: Sheep blood enables the detection of hemolysis, which is indicative of the ability of bacteria to lyse red blood cells. This observation aids in the identification and classification of bacterial species.
7. Carbon Dioxide Requirement: Some pathogens require carbon dioxide for optimal growth. Therefore, incubation of Columbia Blood Agar plates can be performed in an atmosphere containing approximately 3-10% CO2.
8. Precautions for Specific Pathogens: Certain pathogens, such as Brucella species and Campylobacter species, have specific incubation requirements, such as incubation in a CO2-enriched or microaerophilic atmosphere, respectively. It is important to follow the recommended precautions and conditions for the growth of these pathogens.

Composition of Columbia Blood Agar Base

Preparation of Columbia Blood Agar Base

To prepare Columbia Blood Agar Base, follow the instructions below:

1. Suspend 44.01 grams of the medium in 1000 ml of purified or distilled water.
2. Heat the suspension to boiling to ensure complete dissolution of the medium.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Allow the sterilized medium to cool down to 45-50°C before adding any heat-sensitive compounds.
5. For Blood Agar: Add 5% v/v sterile defibrinated sheep blood to the sterile and cooled base.
6. For Chocolate Agar: Add 10% v/v sterile defibrinated sheep blood to the sterile and cooled base. Heat the mixture to 80°C for 10 minutes with constant agitation.
7. To make the medium selective for specific organisms, you can add different antimicrobials to the sterile base as per the desired formulation.
   • For Brucella species: Add the rehydrated contents of 1 vial of NPBCVN Selective Supplement (FD005) to 500 ml of sterile molten base.
   • For Campylobacter species: Add the rehydrated contents of 1 vial of Blaser-Wang Selective Supplement (FD006), Butzler Selective Supplement (FD007), Skirrow Selective Supplement (FD008), VTCA Selective Supplement (FD090), or Butzler VI Selective Supplement (FD106) to 500 ml of sterile molten base. Also, add the rehydrated contents of 1 vial of Minerals Growth Supplement (FD009).
   • For Gardnerella species: Add the rehydrated contents of 1 vial of GNA Selective Supplement (FD056) to 500 ml of sterile molten base.
   • For Cocci: Add the rehydrated contents of 1 vial of NC Selective Supplement (FD030), NNP Selective Supplement (FD031), or CO Selective Supplement (FD119) to 500 ml of sterile molten base.

By following these steps, you can prepare Columbia Blood Agar Base and customize it for various selective or differential media formulations to meet specific laboratory requirements.

Results Interpretation of Columbia Agar

When interpreting the results obtained from Columbia Agar, it is crucial to examine the colony characteristics of the organisms grown on the medium. Here are the typical colony characteristics associated with different microorganisms:

1. Streptococci (non-group D):
   • Colony Characteristics: Small, white to grayish colonies.
   • Hemolysis: Beta or alpha hemolysis.
2. Enterococci (Group D):
   • Colony Characteristics: Small colonies, but larger than group A streptococci, grayish in color.
   • Hemolysis: Alpha (rarely beta) hemolysis.
3. Staphylococci:
   • Colony Characteristics: Large colonies, ranging from white to gray or cream to yellow. Hemolysis may or may not be present.
4. Corynebacteria:
   • Colony Characteristics: Colonies can vary in size from small to large and appear white to gray or yellow. Hemolysis may or may not be present.
5. Listeria monocytogenes:
   • Colony Characteristics: Small to medium-sized colonies with a grayish color.
   • Hemolysis: Weak beta hemolysis.
6. Enterobacteriaceae:
   • Colony Characteristics: Colonies are medium-sized to large and appear grey. Hemolysis may or may not be present.
7. Candida species:
   • Colony Characteristics: Small, white colonies.

These characteristics provide valuable information for the identification and differentiation of various microorganisms. It is important to note that additional confirmatory tests or techniques may be required to accurately identify the species within each microbial group.

By examining the colony morphology, color, and hemolytic patterns, healthcare professionals and microbiologists can make preliminary identifications and further investigate the isolates. These results help guide the diagnosis and treatment of infections, as well as contribute to the understanding of the epidemiology and pathogenicity of different microorganisms.

Uses of Columbia Blood Agar Base

• Columbia Blood Agar Base is a versatile medium widely used in microbiology laboratories for various purposes. It serves as a primary isolation medium, facilitating the growth and identification of both non-fastidious and fastidious microorganisms obtained from clinical specimens. This agar base provides a favorable environment for the cultivation of a broad range of microorganisms, making it an essential tool in microbiological research and diagnostics.
• One of the primary applications of Columbia Blood Agar Base is the isolation of non-fastidious microorganisms. The medium supports the growth of various bacterial species, including Enterobacteriaceae, Pseudomonas, and other non-fermenting Gram-negative rods. These organisms are commonly encountered in clinical samples and play a significant role in infectious diseases. Columbia Blood Agar Base promotes their growth, enabling researchers and healthcare professionals to isolate and identify these pathogens accurately.
• In addition to non-fastidious bacteria, Columbia Blood Agar Base also supports the growth of fastidious microorganisms. This characteristic makes it particularly useful in the isolation and identification of streptococci, enterococci, staphylococci, coryneforms, Candida species, and various other microorganisms. Fastidious bacteria are often more delicate and require specific growth conditions to thrive. The balanced composition of Columbia Blood Agar Base provides the necessary nutrients and growth factors to support their growth, allowing for their isolation and subsequent characterization.
• Columbia Blood Agar Base contains a combination of peptones, meat extract, and blood to create an enriched medium that encourages the growth of a wide range of microorganisms. The peptones and meat extract provide a source of essential nutrients, while the addition of blood promotes the growth of fastidious bacteria by providing necessary growth factors such as vitamins, amino acids, and other important compounds.
• Moreover, Columbia Blood Agar Base can be supplemented with additional selective agents or antimicrobial agents to enhance its functionality for specific purposes. For example, selective supplements like antibiotics or dyes can be added to inhibit the growth of certain bacteria while allowing the growth of others, aiding in the isolation of specific pathogens or differentiating between bacterial species.
• Overall, Columbia Blood Agar Base is an indispensable tool in microbiology laboratories for primary isolation and cultivation of a wide array of microorganisms. Its ability to support the growth of both non-fastidious and fastidious bacteria makes it a versatile medium in clinical diagnostics, research, and epidemiological studies. By using this agar base, scientists and healthcare professionals can successfully isolate, identify, and study various pathogens, contributing to our understanding and management of infectious diseases.

Limitations of Columbia Blood Agar Base

Columbia Blood Agar Base has certain limitations that should be considered. Here are the key limitations:

• Identification Challenges: While Columbia Blood Agar Base can support the growth and differentiation of various microorganisms, it is recommended to perform additional tests such as biochemical, immunological, molecular, and mass spectrometry testing on colonies from pure culture for complete identification and confirmation of species.
• Lack of V Factor: The medium lacks the V factor (nicotinamide adenine dinucleotide, NAD) due to the presence of NADase in sheep blood, which destroys NAD. As a result, Haemophilus influenzae, which requires both the X and V factors for growth, will not grow on this medium.
• Poor Growth of Neisseria gonorrhoeae: Neisseria gonorrhoeae does not grow well on Columbia Blood Agar Base. Other specialized media or techniques are preferred for the isolation and cultivation of this specific organism.
• Unsuitability for Certain Organisms: Columbia Blood Agar Base is not suitable for the isolation and growth of organisms with highly specific nutritive requirements, such as Mycobacterium, Legionella, Bordetella, and others.
• Hemolysis Reactions: The relatively high carbohydrate (starch) content in Columbia Agar Base can lead to certain limitations in hemolysis reactions. Beta-hemolytic streptococci may exhibit alpha hemolytic reactions or weak hemolytic reactions on media based on this formulation.
• Limited Shelf Life: Prepared plates of supplemented media should be used within 18 hours of preparation to ensure optimum selectivity and performance.
• Confirmation of Results: Further biochemical and serological tests must be performed for confirmation of the results obtained on Columbia Blood Agar Base.

These limitations should be considered when using Columbia Blood Agar Base, and alternative media or techniques may be required for specific organisms or precise identification purposes.

References

References

• https://exodocientifica.com.br/_technical-data/M144.pdf
• https://www.bd.com/resource.aspx?IDX=8968
• https://www.himedialabs.com/in/mp144-columbia-5-sheep-blood-agar-plate.html
• http://www.biomerieux-culturemedia.com/product/30-columbia-agar-+-5-percent-sheep-blood
• https://www.bio-rad.com/en-in/sku/63784-columbia-agar-5-sheep-blood?ID=63784