Columbia Agar with 5% Sheep Blood- Composition, Principle, Preparation, Results, Uses

Columbia Agar with 5% Sheep Blood is a type of enriched and differential culture medium that is used for the isolation and identification of various bacteria from clinical specimens. It consists of a base medium (Columbia Agar) and an additive (Sheep Blood).

The base medium, Columbia Agar, contains the following ingredients:

• Pancreatic digest of casein and enzymatic digest of animal tissue: These are peptones that provide nitrogen, carbon, vitamins, and trace elements necessary for the growth of bacteria.
• Yeast extract: This is a source of vitamin B complex that enhances bacterial growth and metabolism.
• Corn starch: This is a carbohydrate that serves as an energy source for bacteria and also neutralizes toxic metabolites by absorbing them.
• Sodium chloride: This is a salt that maintains the osmotic balance and pH of the medium.
• Agar: This is a solidifying agent that gives the medium a gel-like consistency.

The additive, Sheep Blood, is added to the base medium after sterilization. It has the following functions:

• Hemolysis indicator: Sheep Blood contains red blood cells that can be lysed by some bacteria, producing different types of hemolysis (alpha, beta, or gamma) that can be observed on the medium. Hemolysis is a useful characteristic for identifying bacteria, especially streptococci and staphylococci.
• X factor supplier: Sheep Blood also provides the X factor (heme) that is required for the growth of some bacteria, such as Haemophilus species. However, it does not provide the V factor (nicotinamide adenine dinucleotide, NAD) that is also needed by some Haemophilus species.

The final composition of Columbia Agar with 5% Sheep Blood (per liter) is as follows:

The pH of the medium is adjusted to 7.3 ± 0.2 at 25°C before sterilization. The medium appears dark red in color after adding sheep blood.

Columbia Agar with 5% Sheep Blood is a highly nutritious medium that supports the growth of a wide range of bacteria, both fastidious and non-fastidious. The medium contains peptones that supply nitrogen, carbon, vitamins, and trace elements necessary for the growth of the bacterium. Yeast extract act as the supplier of the vitamin B complex. Corn starch serves as the energy source for organisms and also neutralizes toxic metabolites by absorbing toxic by-products contained in the specimen. Sodium chloride is a source of essential electrolytes and maintains osmotic equilibrium.

Sheep blood is added to demonstrate hemolytic reactions and supplies the X factor (heme) necessary for the growth of many pathogenic species. On this medium, colonies tend to be larger and growth is more luxuriant than on media containing other blood agar bases. Hemolysis is the breakdown of red blood cells by bacterial enzymes. There are three types of hemolysis: alpha, beta, and gamma. Alpha hemolysis is a partial or greenish hemolysis around the colony. Beta hemolysis is a complete or clear hemolysis around the colony. Gamma hemolysis is no hemolysis around the colony.

Columbia Agar with 5% Sheep Blood can be used to differentiate bacteria based on their hemolytic patterns. For example, Streptococcus pyogenes produces beta hemolysis, Streptococcus pneumoniae produces alpha hemolysis, and Enterococcus faecalis produces gamma hemolysis. Some bacteria may also show different types of hemolysis depending on the medium composition or incubation conditions. For example, Streptococcus agalactiae may produce alpha or beta hemolysis on Columbia Agar with 5% Sheep Blood.

Columbia Agar with 5% Sheep Blood is recommended as a frequently used primary isolation medium for isolation of non-fastidious and fastidious microorganisms from clinical specimens. It is a primary isolation medium on which most microorganisms, such as Enterobacteriaceae, Pseudomonas, and other non-fermenting Gram-negative rods, streptococci, enterococci, staphylococci, coryneforms, Candida species, and many others will grow.

Columbia Agar Base is a nutrient-rich medium that can support the growth of a wide range of bacteria. To prepare Columbia Agar Base, you will need the following ingredients:

• Peptone: 23 g
• Yeast extract: 5 g
• Corn starch: 1 g
• Sodium chloride: 5 g
• Agar: 15 g
• Distilled or deionized water: 1 L

The preparation steps are as follows:

1. Weigh out the required amounts of peptone, yeast extract, corn starch, sodium chloride and agar and add them to a large flask or beaker containing distilled or deionized water. Mix well to dissolve the ingredients.
2. Transfer the flask or beaker to a hot plate or a water bath and gently heat until boiling. Stir occasionally to prevent scorching or clumping of the agar.
3. Once the medium reaches boiling point, remove it from the heat source and cover it with a lid or foil. Sterilize the medium by autoclaving it for 15 minutes at 15 psi pressure and 121°C.
4. After autoclaving, let the medium cool down to 45°–50°C before adding sheep blood or pouring into plates or tubes.

Note: Do not overheat or overcook the medium as this may affect its performance and quality. Do not store the medium for long periods of time as this may lead to deterioration or contamination. Always use sterile equipment and aseptic techniques when handling the medium.

To prepare Columbia Agar with 5% Sheep Blood, you will need the following materials:

• Columbia Agar Base
• Sterile, defibrinated sheep blood
• Distilled or deionized water
• Autoclave
• Water bath
• Sterile Petri dishes or tubes
• Pipette or dispenser

The steps for preparing Columbia Agar with 5% Sheep Blood are as follows:

1. Prepare Columbia Agar Base according to the instructions given in the previous section. You will need 950 mL of the base for each liter of the final medium.
2. Cool the Columbia Agar Base to 45°C to 50°C in a water bath. Do not overcool or solidify the base.
3. Aseptically add 50 mL of sterile, defibrinated sheep blood to the cooled base. Use a pipette or a dispenser to measure and transfer the blood. Mix well by swirling or stirring gently.
4. Pour the medium into sterile Petri dishes or distribute into sterile tubes as desired. Avoid bubbles and spills. Label the plates or tubes with the name and date of preparation.
5. Allow the medium to solidify at room temperature. Do not refrigerate or incubate the medium before use as this may affect the hemolytic reactions.
6. Store the prepared medium at 2°C to 8°C in a dark place until use. Do not use the medium if it shows signs of contamination, discoloration, deterioration, or hemolysis.

Columbia Agar with 5% Sheep Blood can be used to observe the hemolytic reactions of different bacteria. Hemolysis is the breakdown of red blood cells by bacterial enzymes called hemolysins. There are three types of hemolysis: alpha, beta, and gamma.

• Alpha hemolysis is the partial or incomplete lysis of red blood cells, resulting in a greenish discoloration of the agar around the colonies. Alpha hemolysis is also called green hemolysis or viridans hemolysis. Some examples of alpha-hemolytic bacteria are Streptococcus pneumoniae, Streptococcus viridans, and Enterococcus faecalis.
• Beta hemolysis is the complete lysis of red blood cells, resulting in a clear zone of hemolysis around the colonies. Beta hemolysis is also called complete hemolysis or true hemolysis. Some examples of beta-hemolytic bacteria are Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes.
• Gamma hemolysis is the absence of hemolysis, resulting in no change in the appearance of the agar around the colonies. Gamma hemolysis is also called non-hemolysis or no hemolysis. Some examples of gamma-hemolytic bacteria are Escherichia coli, Salmonella typhi, and Proteus mirabilis.

The interpretation of hemolytic reactions on Columbia Agar with 5% Sheep Blood should be done in conjunction with other biochemical and serological tests to identify the bacteria. Some bacteria may exhibit different types of hemolysis depending on the source and quality of the blood used in the medium. For example, some beta-hemolytic streptococci may show alpha or weak hemolysis on this medium due to the high starch content that inhibits their hemolytic activity. Therefore, it is important to use a standardized and quality-controlled medium for accurate results.

Here is a table summarizing some common bacteria and their hemolytic reactions on Columbia Agar with 5% Sheep Blood:

Columbia Agar with 5% Sheep Blood is a general-purpose medium that supports the growth of a wide range of non-fastidious and fastidious microorganisms from clinical specimens. Some of the organisms that can grow on this medium are:

• Enterobacteriaceae: This is a large family of Gram-negative bacteria that includes many common pathogens such as Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter, Citrobacter, Serratia and others. They are usually facultative anaerobes that ferment glucose and other carbohydrates. They can be differentiated by their biochemical properties and antigenic structures. On Columbia Agar with 5% Sheep Blood, they typically produce large, moist, smooth colonies that may or may not show hemolysis.

• Pseudomonas and other non-fermenting Gram-negative rods: These are aerobic bacteria that do not ferment carbohydrates but oxidize them. They include Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and others. They are often opportunistic pathogens that cause infections in immunocompromised patients or those with chronic diseases. They can be distinguished by their pigment production, odor, motility and resistance to antibiotics. On Columbia Agar with 5% Sheep Blood, they usually produce small, dry, irregular colonies that are non-hemolytic.

• Streptococci: These are Gram-positive cocci that form chains or pairs. They are classified into different groups based on their hemolytic patterns and carbohydrate antigens. The most important groups are group A streptococci (Streptococcus pyogenes), group B streptococci (Streptococcus agalactiae), group D streptococci (Enterococcus faecalis and Enterococcus faecium) and viridans streptococci (Streptococcus pneumoniae and Streptococcus mutans). They are responsible for various infections such as pharyngitis, cellulitis, endocarditis, meningitis and dental caries. On Columbia Agar with 5% Sheep Blood, they usually produce small to medium-sized colonies that may show alpha (greenish), beta (clear) or gamma (no) hemolysis.

• Staphylococci: These are Gram-positive cocci that form clusters or grape-like structures. They are divided into two main groups: coagulase-positive staphylococci (Staphylococcus aureus) and coagulase-negative staphylococci (Staphylococcus epidermidis, Staphylococcus saprophyticus and others). They are common inhabitants of the skin and mucous membranes and can cause infections such as boils, abscesses, impetigo, mastitis and septicemia. They can be differentiated by their coagulase test, catalase test and antibiotic susceptibility. On Columbia Agar with 5% Sheep Blood, they usually produce large, round, opaque colonies that may show beta or gamma hemolysis.

• Coryneforms: These are Gram-positive rods that have an irregular or club-shaped appearance. They include Corynebacterium diphtheriae, Corynebacterium jeikeium, Corynebacterium urealyticum and others. They are part of the normal flora of the skin and mucous membranes and can cause infections such as diphtheria, endocarditis and urinary tract infections. They can be identified by their morphology, biochemical reactions and toxin production. On Columbia Agar with 5% Sheep Blood, they usually produce small to medium-sized colonies that are non-hemolytic.

• Candida species: These are yeast-like fungi that form oval or spherical cells. They include Candida albicans, Candida tropicalis, Candida glabrata and others. They are part of the normal flora of the mouth, vagina and gastrointestinal tract and can cause infections such as oral thrush, vaginitis and candidemia. They can be distinguished by their morphology on Gram stain, germ tube test and carbohydrate assimilation test. On Columbia Agar with 5% Sheep Blood, they usually produce white to cream-colored colonies that may show pseudohyphae or chlamydospores.

Different organisms may exhibit different colony characteristics on Columbia Agar with 5% Sheep Blood. Some of the common examples are:

• Staphylococcus aureus: This organism produces large, round, opaque, golden-yellow colonies with a smooth surface and entire edges. It also shows beta-hemolysis (clearing of the agar around the colonies) due to the production of hemolysins that lyse red blood cells.
• Streptococcus pyogenes: This organism produces small, round, translucent, gray-white colonies with a smooth surface and entire edges. It also shows beta-hemolysis (clearing of the agar around the colonies) due to the production of streptolysin O and S that lyse red blood cells.
• Streptococcus pneumoniae: This organism produces small, round, translucent, greenish colonies with a smooth surface and entire edges. It also shows alpha-hemolysis (greening of the agar around the colonies) due to the production of hydrogen peroxide that oxidizes hemoglobin to methemoglobin.
• Enterococcus faecalis: This organism produces small, round, translucent, gray-white colonies with a smooth surface and entire edges. It may show alpha-hemolysis (greening of the agar around the colonies), beta-hemolysis (clearing of the agar around the colonies), or no hemolysis (no change in the agar around the colonies) depending on the strain and the type of sheep blood used.
• Escherichia coli: This organism produces large, round, opaque, cream-colored colonies with a smooth surface and entire edges. It does not show any hemolysis (no change in the agar around the colonies) as it does not produce any hemolysins.
• Pseudomonas aeruginosa: This organism produces large, round, opaque, greenish-blue colonies with a smooth surface and entire edges. It does not show any hemolysis (no change in the agar around the colonies) as it does not produce any hemolysins. It may also produce a characteristic fruity odor and a fluorescent pigment that can be seen under ultraviolet light.

These are some of the common colony characteristics on Columbia Agar with 5% Sheep Blood. However, it is important to note that colony morphology may vary depending on the incubation conditions, inoculum size, and other factors. Therefore, it is recommended that additional tests be performed for confirmation and identification of the organisms. 

Columbia Agar with 5% Sheep Blood is a versatile and widely used medium for the isolation and cultivation of a variety of microorganisms from clinical specimens. Some of the main uses of this medium are:

• It is a primary isolation medium on which most microorganisms, such as Enterobacteriaceae, Pseudomonas, and other non-fermenting Gram-negative rods, streptococci, enterococci, staphylococci, coryneforms, Candida species, and many others will grow. It can also support the growth of some fastidious organisms, such as Streptococcus pneumoniae and Streptococcus pyogenes, by providing the X factor (heme) required for their growth.
• It is a differential medium that can demonstrate hemolytic reactions of various microorganisms. The presence of sheep blood in the medium allows the detection of alpha, beta, or gamma hemolysis by observing the changes in the color and transparency of the agar around the colonies. Alpha hemolysis is characterized by a greenish discoloration of the agar due to partial lysis of red blood cells. Beta hemolysis is characterized by a clear zone of complete lysis of red blood cells around the colonies. Gamma hemolysis is characterized by no change in the appearance of the agar around the colonies.
• It is a selective medium that can inhibit or enhance the growth of certain microorganisms by adding specific supplements or antibiotics to the medium. For example, adding colistin and nalidixic acid (CNA) to the medium can inhibit the growth of Gram-negative bacteria and select for Gram-positive bacteria. Adding bacitracin to the medium can inhibit the growth of most streptococci except Streptococcus pyogenes (group A streptococcus), which is bacitracin-sensitive. Adding esculin to the medium can differentiate enterococci from other streptococci by their ability to hydrolyze esculin and produce a black color in the agar.
• It is a base medium that can be modified or supplemented with various indicators or substrates to detect specific metabolic or enzymatic activities of microorganisms. For example, adding mannitol salt to the medium can differentiate staphylococci by their ability to ferment mannitol and produce an acid reaction that turns the medium yellow. Adding phenylethyl alcohol (PEA) to the medium can inhibit the growth of Gram-negative bacteria and enhance the growth of anaerobic bacteria. Adding chromogenic substrates to the medium can detect specific enzymes produced by microorganisms, such as beta-glucuronidase or beta-galactosidase, by producing a color change in the colonies.

Columbia Agar with 5% Sheep Blood is therefore a useful and versatile medium that can be employed for various purposes in microbiology laboratories. It can provide valuable information about the identity and characteristics of microorganisms isolated from clinical specimens.

Columbia Agar with 5% Sheep Blood is a versatile and widely used medium for the isolation and cultivation of various bacteria from clinical specimens. However, it also has some limitations that should be considered when using it for microbiological purposes. Some of these limitations are:

• It is recommended that biochemical, immunological, molecular and mass spectrometry testing be performed on colonies from pure culture for complete identification. Columbia Agar with 5% Sheep Blood does not provide enough information to differentiate between closely related species or strains of bacteria, especially those that have similar hemolytic patterns or colony morphology.
• The medium lacks V factor (nicotinamide adenine dinucleotide, NAD) since sheep blood contains NADase which destroys the NAD. Hence, Haemophilus influenzae which requires both the X and V factors, will not grow on this medium. Other bacteria that need V factor for growth include some species of Neisseria, Moraxella and Pasteurella.
• Neisseria gonorrhoeae does not grow well on this medium. This is because the organism is very sensitive to the fatty acids present in sheep blood, which inhibit its growth. Other bacteria that may be inhibited by sheep blood include some species of Campylobacter and Helicobacter.
• The medium is not suitable for the isolation and growth of Mycobacterium, Legionella, Bordetella and other organisms with highly specific nutritive requirements. These bacteria need special media that contain specific supplements or selective agents to support their growth and prevent contamination by other microorganisms.
• Due to the rather high carbohydrate (starch) content in the Columbia Agar Base, beta-hemolytic streptococci may exhibit alpha rather than beta hemolytic reactions or may exhibit weak hemolytic reactions on media based on this formulation. This may lead to misidentification or confusion of these bacteria with other alpha-hemolytic streptococci such as Streptococcus pneumoniae or viridans streptococci.

Therefore, Columbia Agar with 5% Sheep Blood should be used with caution and in conjunction with other media and tests for accurate identification and characterization of bacteria from clinical specimens. It is also important to follow the manufacturer`s instructions and quality control procedures when preparing and using this medium to ensure optimal performance and reliability.

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