Columbia CNA Agar – Composition, Preparation, Uses, and Results

What is Columbia CNA Agar?

• Columbia CNA Agar, also known as Columbia agar with colistin and nalidixic acid, is a selective and differential medium used for the isolation and differentiation of pathogenic gram-positive cocci from various clinical and non-clinical samples.
• The medium incorporates the antimicrobial agents colistin (C) and nalidixic acid (NA), which serve as selective agents by inhibiting the growth of gram-negative organisms. Colistin, belonging to the polymyxin group of antibiotics, disrupts the cell membranes of gram-negative bacteria. Nalidixic acid, a first-generation quinolone, blocks DNA replication in susceptible gram-negative bacteria.
• The base of Columbia CNA Agar is a nutritionally rich formula known as Columbia agar base. It contains three peptone sources and 5% defibrinated sheep blood. The sheep blood provides essential nutrients and acts as a diagnostic indicator for differentiating bacterial colonies based on their hemolytic reactions. The presence of sheep blood allows for the detection of hemolysis, which is the breakdown of red blood cells by certain bacterial species.
• Columbia CNA Agar selectively inhibits the growth of gram-negative bacteria, such as members of the Enterobacteriaceae family and Pseudomonas species. This selective property allows for the preferential growth of gram-positive organisms, including staphylococci, streptococci, enterococci, and yeast.
• By observing the growth and hemolytic reactions of colonies on Columbia CNA Agar, microbiologists can differentiate various gram-positive cocci based on their specific characteristics. This differentiation is important for the identification and classification of pathogenic bacteria in clinical and research settings.
• Columbia CNA Agar serves as a valuable tool in microbiology laboratories for the isolation and characterization of gram-positive cocci. Its selective and differential properties enable the identification of specific organisms and contribute to the diagnosis and treatment of infectious diseases.

Columbia CNA Agar with 5% Sheep Blood

• The selective medium Columbia CNA Agar with 5% Sheep Blood is used to isolate gram-positive bacteria (particularly staphylococci and streptococcus) from clinical specimens.
• Ellner et al. announced in 1966 the invention of a blood agar formulation, now known as Columbia Agar.
• This medium, which produces larger colonies and lusher growth than equivalent blood agar bases, is used for media containing blood and for specific formulations.
• Ellner et al. discovered that a medium containing 10 mg of colistin and 15 mg of nalidixic acid per litre in a Columbia agar base enriched with 5% sheep blood promotes the growth of staphylococci, hemolytic streptococci, and enterococci while inhibiting the growth of Proteus, Klebsiella, and Pseudomonas species.
• Columbia Agar provides a nutrient-rich medium basis. Colistin and nalidixic acid make the medium selective for gram-positive bacteria, particularly streptococci and staphylococci.
• The concentration of nalidixic acid in BD Columbia CNA Agar with 5% Sheep Blood has been decreased to 10 mg/l in order to increase the recovery of gram-positive cocci from clinical specimens.
• Sheep blood permits the detection of hemolytic responses, which are crucial in the presumptive diagnosis of streptococci.

Composition of Columbia CNA Agar per Liter of Medium

The composition of Columbia CNA Agar can be summarized as follows, considering the information provided:

Ingredients:

• Casein-Meat Peptone: 10 g/L
• Casein-Yeast Peptone: 10 g/L
• Heart Peptone: 3 g/L
• Sodium chloride: 5 g/L
• Corn starch: 1 g/L
• Agar: 13.5 g/L
• Defibrinated sheep blood: 50 mL/L
• Colistin sulfate: 10 mg/L
• Nalidixic Acid: 15 mg/L
• pH: 7.3 +/- 0.2

The base of Columbia CNA Agar is prepared using a nutritionally rich medium that includes different peptones to support the growth of microorganisms. This includes casein-meat peptone, casein-yeast peptone, and heart peptone. These peptones provide essential nutrients for the growth of bacteria.

Sodium chloride is added to provide proper osmotic balance in the medium. Corn starch acts as a neutralizer source, helping to counteract any toxic metabolites produced by microorganisms.

Agar is included as a solidifying agent, allowing the medium to solidify into agar plates or slants.

The significant component of Columbia CNA Agar is defibrinated sheep blood, which is added at a concentration of 50 mL/L. The sheep blood provides both the X and V factors required for the growth of fastidious gram-positive bacteria. The X factor is heme, which is necessary for the growth of certain bacteria, while the V factor is nicotinamide adenine dinucleotide (NAD), which supports the growth of others. Additionally, the presence of sheep blood aids in differentiating bacterial colonies based on their hemolytic reactions.

Colistin sulfate and nalidixic acid are added as selective agents to inhibit the growth of gram-negative bacteria, allowing for the preferential growth of gram-positive organisms.

It’s important to note that the composition of Columbia CNA Agar may vary slightly depending on the manufacturer, as stated in the information provided.

Ingredients	Amount
Casein-Meat Peptone	10.0 g
Casein-Yeast Peptone	10.0 g
Heart Peptone	3.0 g
Sodium Chloride	5.0 g
Corn Starch	1.0 g
Agar	13.5 g
Defibrinated Sheep Blood	50.0 mL
Colistin Sulfate	10 mg
Nalidixic Acid	15 mg

Preparation of Media

To prepare the media, follow the provided instructions:

1. Suspend 44.02 grams of the medium in 1000 ml of purified or distilled water.
2. Heat the mixture to boiling to ensure complete dissolution of the medium. Be cautious not to overheat the medium.
3. Sterilize the medium by autoclaving at 15 lbs pressure at 121°C for 15 minutes.
4. Allow the medium to cool down to a temperature of 45-50°C.
5. Aseptically add 5% v/v (volume/volume) sterile defibrinated blood to the cooled medium. It is important to maintain sterility during this step.
6. Mix the medium and the blood thoroughly to ensure even distribution.
7. Pour the prepared medium into sterile Petri plates. Each plate should contain approximately 20-25 ml of the medium for a 90 mm Petri plate.
8. Allow the plates to solidify before further use or storage.

Following these steps will result in the preparation of the Columbia CNA Agar medium in Petri plates, ready for use in the isolation and cultivation of gram-positive cocci from various samples.

Inoculation of the Medium 

To inoculate the Columbia CNA Agar medium, the following steps should be followed:

1. Temperature Equilibration: Allow the medium to reach room temperature (approximately 20-25°C) before inoculation. This ensures that the agar has solidified and is suitable for streaking or stabbing.
2. Direct Inoculum: Using a direct sample from the specimen, perform a four-quadrant streak on the surface of the agar. This technique helps to obtain well-isolated colonies. Alternatively, if desired, stabs can be made into the medium using an inoculating loop or needle. Stabs can facilitate the observation of hemolytic reactions within the agar.
3. Incubation Conditions: Depending on the specific organism of interest, Columbia CNA Agar plates can be incubated under different atmospheric conditions. For aerobic organisms, incubation in ambient air is suitable. For anaerobic organisms, plates can be incubated in an anaerobic environment. However, for optimal results, particularly for the observation of hemolytic reactions in streptococci, it is recommended to incubate the plates in a CO2-enriched environment containing 5% CO2. This can be achieved using a CO2 incubator.
4. Incubation Period: After inoculation, incubate the Columbia CNA Agar plates at a temperature of 35°C. It is important to examine the plates at regular intervals. Typically, initial observations are made after 24 hours of incubation, and final observations are made after 48 hours. This allows sufficient time for the growth of organisms and the development of characteristic hemolytic reactions.

By following these guidelines for inoculation, incubation conditions, and observation, microbiologists can maximize the effectiveness of Columbia CNA Agar in the isolation and differentiation of gram-positive cocci. The appropriate incubation conditions and incubation periods help ensure accurate and reliable results.

Interpretation of Results

To interpret the results of Columbia CNA Agar, the following guidelines can be followed:

1. Growth: Examine the plates for the presence of bacterial growth. The growth may appear as colonies of different sizes, shapes, and colors on the agar surface.
2. Hemolysis: To observe hemolysis, view the culture plates with a bright light source transmitting behind the plate. Hemolysis refers to the breakdown or lysis of red blood cells surrounding bacterial colonies. There are four types of hemolysis that can be observed on Columbia CNA Agar:

• Alpha-hemolysis: This type of hemolysis results in partial lysis of red blood cells, leading to a greenish discoloration around the bacterial colony. The agar surrounding the colony may appear slightly discolored.
• Beta-hemolysis: Beta-hemolysis indicates complete lysis of red blood cells, resulting in a clear zone around the bacterial colonies. The area surrounding the colonies appears transparent and devoid of blood.
• Gamma-hemolysis: Gamma-hemolysis indicates no hemolysis or absence of red blood cell lysis. In this case, there is no change in the appearance of the medium surrounding the colonies.
• Alpha-prime-hemolysis: This type of hemolysis is characterized by a small zone of complete hydrolysis, indicated by a clear zone, surrounded by an area of partial hemolysis. This zone can be seen as a combination of transparent and greenish discoloration around the colonies.

By carefully examining the growth and hemolysis patterns on Columbia CNA Agar, microbiologists can interpret and classify the bacterial isolates based on their hemolytic characteristics. This interpretation can provide valuable information for the identification and differentiation of different species and strains of bacteria.

Quality Control  

To ensure the quality and performance of the completed Columbia CNA Agar medium, various quality control measures can be conducted. These include checking pH, color, depth, and sterility. Additionally, specific organisms are used to assess the growth performance of the medium. Here is the information regarding quality control:

1. pH, Color, Depth, and Sterility: Prior to use, the pH of the prepared medium should be checked to ensure it falls within the specified range. The color and depth of the medium should also be consistent with the expected appearance. Furthermore, the medium should be sterile, free from any contamination or growth.
2. Growth Performance Organisms:

• Streptococcus pyogenes: When inoculated onto Columbia CNA Agar, Streptococcus pyogenes is expected to exhibit growth and display beta-hemolysis. Beta-hemolysis refers to the complete lysis of red blood cells surrounding the bacterial colonies, resulting in a clear zone around the colonies.
• Streptococcus pneumoniae: Inoculation of Streptococcus pneumoniae onto the medium is expected to result in growth and display alpha-hemolysis. Alpha-hemolysis refers to partial lysis of red blood cells, leading to a greenish discoloration around the colonies.
• Staphylococcus aureus: When Staphylococcus aureus is inoculated onto Columbia CNA Agar, it is expected to show growth and display either beta-hemolysis or no-hemolysis. The presence of a clear zone or the absence of hemolysis indicates the expected results.
• Proteus mirabilis: Inoculation of Proteus mirabilis onto the medium should result in partial inhibition of growth. This organism is expected to show some level of growth inhibition on Columbia CNA Agar.

By testing these organisms on Columbia CNA Agar, the laboratory can verify the growth performance and hemolytic reactions of the medium, ensuring its quality and reliability.

Uses 

Columbia CNA Agar has several important uses in microbiology, primarily related to the isolation and differentiation of gram-positive cocci, particularly Streptococcus species and Staphylococcus species, when they are present in mixed cultures with gram-negative bacilli. Here are the key uses of Columbia CNA Agar:

1. Isolation of Gram-Positive Cocci: Columbia CNA Agar is used to selectively isolate gram-positive cocci from mixed cultures. In conventional blood agar, the growth of gram-negative bacilli can often overshadow the growth of gram-positive cocci. However, the selective agents present in Columbia CNA Agar, such as colistin and nalidixic acid, inhibit the growth of gram-negative bacteria, allowing for the preferential growth of gram-positive cocci.
2. Differentiation of Streptococcus Species: The addition of sheep blood to Columbia CNA Agar provides important diagnostic information for the differentiation of Streptococcus species. The hemolytic reactions observed on the medium can help identify different species of Streptococcus based on their hemolytic patterns. For example, some Streptococcus species exhibit alpha-hemolysis (partial lysis of red blood cells), while others display beta-hemolysis (complete lysis of red blood cells), aiding in the identification and classification of these organisms.
3. Identification of Staphylococcus Species: Columbia CNA Agar can also be used for the isolation and preliminary identification of Staphylococcus species. The medium allows for the growth of Staphylococcus organisms while inhibiting the growth of competing bacteria. Subsequent biochemical tests and additional confirmatory tests are required for the accurate identification of specific Staphylococcus species.

In summary, Columbia CNA Agar is a valuable tool in the microbiology laboratory for the isolation and differentiation of gram-positive cocci, particularly Streptococcus and Staphylococcus species, in mixed cultures. Its selective properties and the addition of sheep blood aid in the detection and differentiation of these organisms, providing important diagnostic information for the identification and classification of bacterial isolates.

Storage and Shelf Life 

To ensure the longevity and quality of Columbia CNA Agar, proper storage conditions should be followed. Here is the information regarding storage and shelf life:

1. Storage Temperature: Columbia CNA Agar should be stored at a temperature of 4°C to 8°C (39°F to 46°F). This range helps maintain the stability and integrity of the medium.
2. Protection from Light: It is important to protect Columbia CNA Agar from direct light exposure. Storing the medium in a dark or opaque container, or in a covered storage area, helps prevent light-induced degradation.
3. Medium Orientation: When storing the plates or containers of Columbia CNA Agar, ensure that the medium side is placed uppermost. This positioning helps prevent excessive moisture accumulation on the agar surface, which could affect the performance of the medium.
4. Shelf Life: Under proper storage conditions, Columbia CNA Agar has a shelf life of 8 weeks from the date of manufacture. It is important to note the manufacturing date and adhere to the recommended expiration date.

By following these storage guidelines, laboratories can maintain the quality and efficacy of Columbia CNA Agar throughout its shelf life. It is essential to use the medium within the specified time frame to ensure accurate and reliable results in microbiological testing.

FAQ

References

• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology), 7th edition
• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition