Chocolate Agar, also known as Hektoen Enteric Agar (HE) is a type of culture medium used for the isolation and cultivation of gram-negative enteric bacteria, particularly Salmonella and Shigella species. The medium is made by adding blood, typically sheep or horse blood, to a base agar medium, which provides a source of nutrients for the bacteria. The medium also contains a pH indicator, typically phenol red, which helps to differentiate between different types of bacteria based on their ability to produce characteristic colonies. Chocolate agar is typically incubated at 35-37°C under a microaerophilic environment. It is commonly used to isolate and identify enteric bacteria from clinical specimens such as feces, urine and blood. It can also be used for antimicrobial susceptibility testing.
Difference between blood agar and chocolate agar:
Blood agar and chocolate agar are both types of culture media used for the growth of bacteria. However, there are some differences between them:
- Composition: Blood agar contains nutrients such as peptones, yeast extract, and agar, along with sheep or horse blood. The blood provides the necessary growth factors and helps in the differentiation of bacteria based on their ability to hemolyze red blood cells. On the other hand, chocolate agar is made by heating blood agar to lyse the red blood cells, which releases more nutrients and growth factors for the bacteria.
- Hemolysis: Blood agar is used to differentiate bacteria based on their hemolytic activity. Some bacteria can lyse red blood cells and produce a clear zone around their colonies on blood agar, which is called beta-hemolysis. Others can partially lyse red blood cells, producing a greenish discoloration around their colonies, which is called alpha-hemolysis. Some bacteria do not have any hemolytic activity, and their colonies appear unchanged. In contrast, chocolate agar is not used for hemolysis differentiation.
- Nutrient content: Chocolate agar is richer in nutrients than blood agar because the heating process releases more nutrients from the blood. This makes it a preferred medium for the growth of fastidious bacteria that require specific nutrients for growth.
- Applications: Blood agar is commonly used to isolate and identify Streptococcus and Staphylococcus species, while chocolate agar is often used for the growth of Haemophilus influenzae and Neisseria meningitidis.
In summary, blood agar is a general-purpose medium that is useful for the cultivation of many bacteria, whereas chocolate agar is a nutrient-rich medium that is particularly useful for the growth of fastidious bacteria. Blood agar is used for hemolysis differentiation, while chocolate agar is not.
Composition of chocolate agar?
- Casein/Animal Tissue Digest: This is a source of amino acids and peptides derived from the digestion of casein or animal tissues. It provides nutrients for bacterial growth.
- Cornstarch: Cornstarch may be used as a carbohydrate source in the medium, providing energy for bacterial metabolism.
- Sodium Chloride: Sodium chloride (salt) is added to maintain the osmotic balance of the medium.
- Dipotassium Phosphate and Monopotassium Phosphate: These are phosphate salts that help buffer the pH of the medium. The specific combination of dipotassium and monopotassium phosphate can be adjusted to achieve the desired pH for bacterial growth.
- Hemoglobin Solution: Hemoglobin is a source of iron and other nutrients required by many bacteria for growth. The hemoglobin solution likely contains dissolved hemoglobin, which provides essential nutrients to support bacterial growth.
- Coenzyme Enrichment: Coenzymes are organic molecules that assist in enzymatic reactions. Coenzyme enrichment likely contains various coenzymes necessary for specific metabolic pathways of certain bacteria.
- Agar: Agar is a solidifying agent derived from seaweed. It is used to solidify the medium, allowing for the growth of bacteria on agar plates.
| Ingredient | Quantity (Grams per liter) | Purpose |
|---|---|---|
| Casein/Animal Tissue Digest | 15.0 Grams | Source of amino acids and peptides for bacterial growth. |
| Cornstarch | 1.0 Grams | Provides carbohydrates as an energy source for bacteria. |
| Sodium Chloride | 5.0 Grams | Maintains osmotic balance in the medium. |
| Dipotassium Phosphate | 4.0 Grams | Acts as a pH buffer to maintain the desired pH for bacterial growth. |
| Monopotassium Phosphate | 1.0 Grams | Acts as a pH buffer to maintain the desired pH for bacterial growth. |
| Hemoglobin Solution 2% | 500 ml | Provides iron and other nutrients necessary for bacterial growth. |
| Coenzyme Enrichment | 10 ml | Contains coenzymes needed for specific metabolic pathways of bacteria. |
| Agar | 10 Grams | Solidifying agent that allows the medium to form a solid surface for bacterial growth. |
The exact concentrations of these ingredients can vary slightly depending on the specific formulation used in different laboratories or by different manufacturers.
How to prepare a chocolate agar?
- A volume of blood either from a horse or sheep is heat-lyse very slowly in a water bath at a 56 degrees Celsius. The blood must only be 5% of the total media volume.
- Place about 20 ml into the Petri dish and let it solidify and condensation to dry.
- Put the plate in a sterile plastic and store it at a temperature of 4 degrees Celsius until use. Before using the medium, it should be warmed to room temperature at a 25 degrees Celsius.
- To test for sterility, the uninoculated plate should be incubated for 48 hours at a temperature of 35 degrees Celsius.
Medium preparation
- Add all the components except for the hemoglobin solution to the distilled water. The total volume should be 500 ml and should be thoroughly mixed. Bring to boil and autoclave for about 15 minutes at 12 pounds pressure at a 21 degrees Celsius. Allow to cool at 45 degrees Celsius. About 500 mL of the hemoglobin solution should be added. Gently mix and pour into the sterile Petri dish.
Principle of chocolate agar
If a supplement is added on a chocolate agar base, it would be a perfect growth place for fastidious organisms. For the organism to grow, various nutrients are added such as casein and animal tissue, which serves as amino acids, nitrogenous nutrients, and other vital elements for the organism’s growth.
Modifications can be made to grow other organisms. Some of the modifications include:
- Thayer-Martin media – It is a chocolate agar supplemented with nystatin, vancomycin, and colistin. The purpose is to inhibit the normal flora and non-pathogenic Neisseria for the isolation of N. meningitides and N. gonorrhoeae.
- Chocolate agar with bacitracin – It is a selective medium for the improvement of primary isolation of H. influenza from a specimen that has a mixed flora of bacteria and fungi.
- Chocolate agar with GC base and growth supplement – It supports the growth requirements needed to isolate fastidious organisms.
Purpose of Chocolate agar?
Chocolate agar is a nutrient-rich growth medium used for the cultivation of fastidious bacteria, which are bacteria that have complex nutritional requirements and are difficult to culture on standard media. The purpose of using chocolate agar is to provide a rich source of nutrients and growth factors that are not present in other media, allowing the growth of a wider range of bacterial species.
The blood used in the preparation of chocolate agar provides essential nutrients such as hemin, NAD, and other growth factors that are required for the growth of many fastidious bacteria. The heating process used in the preparation of chocolate agar further releases additional nutrients and growth factors, making it an ideal medium for the growth of these bacteria.
Some examples of bacteria that grow well on chocolate agar include Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Chocolate agar is also used in the diagnosis of bacterial infections such as meningitis and pneumonia.
In summary, the purpose of chocolate agar is to provide a nutrient-rich medium that supports the growth of fastidious bacteria, allowing their isolation, identification, and study.
List of Bacterias Growth on Chocolate Agar :
Chocolate agar is a nutrient-rich medium that contains lysed red blood cells, which provides essential nutrients for the growth of a wide range of fastidious bacteria. Some of the bacteria that can grow on chocolate agar include:
- Haemophilus influenzae
- Neisseria gonorrhoeae
- Neisseria meningitidis
- Streptococcus pneumoniae
- Moraxella catarrhalis
- Legionella pneumophila
- Bordetella pertussis
- Brucella species
- Francisella tularensis
- Actinobacillus actinomycetemcomitans
- Pasteurella multocida
- Campylobacter species
- Helicobacter pylori
- Gardnerella vaginalis
- Capnocytophaga species
These bacteria have different nutritional requirements and may require the addition of specific growth factors such as hemin and NAD (V factor) to the medium, which are present in chocolate agar.
Colony characteristics in chocolate agar:
The colony characteristics of bacteria grown on chocolate agar can vary depending on the species of bacteria being cultured. However, there are some general characteristics that are commonly observed:
- Appearance: The colonies on chocolate agar are typically smooth and moist with a glossy appearance. They are often described as having a “chocolate-like” color due to the presence of lysed red blood cells.
- Size: The size of the colonies can vary depending on the species of bacteria. Some bacteria form small, pinpoint colonies, while others form larger, more diffuse colonies.
- Hemolysis: Unlike blood agar, chocolate agar is not used for hemolysis differentiation. Therefore, the colonies on chocolate agar do not show any hemolytic activity.
- Shape: The shape of the colonies can vary depending on the species of bacteria. Some bacteria form circular colonies, while others may be irregular or have a filamentous appearance.
- Odor: Some bacteria may produce a characteristic odor when grown on chocolate agar. For example, Streptococcus pneumoniae can produce a sweet, fruity odor.
In summary, the colony characteristics of bacteria grown on chocolate agar are generally smooth, moist, and glossy with a chocolate-brown color. The size, shape, and odor of the colonies can vary depending on the species of bacteria. Unlike blood agar, chocolate agar is not used for hemolysis differentiation.
Neisseria meningitides
Neisseria meningitidis is a fastidious gram-negative bacterium that can cause meningitis and septicemia. When grown on chocolate agar, it typically exhibits the following colony characteristics:
- Appearance: The colonies are small, round, and translucent, with a smooth and slightly convex surface. They may appear grayish-white to translucent in color.
- Size: The colonies are usually small, ranging from 1-2 mm in diameter.
- Texture: The colonies are smooth and slightly moist.
- Hemolysis: N. meningitidis is a non-hemolytic organism and does not produce any hemolytic activity on chocolate agar.
- Other features: N. meningitidis is an oxidase-positive bacterium, meaning that it produces an enzyme called cytochrome oxidase. This can be detected by adding a reagent called oxidase to the colony, which will produce a color change if the enzyme is present. N. meningitidis is also non-motile and non-spore-forming.
In summary, N. meningitidis colonies on chocolate agar are small, round, and translucent with a smooth and slightly convex surface. They are non-hemolytic and have a smooth and slightly moist texture. Additionally, they are oxidase-positive, non-motile, and non-spore-forming.
S. pneumonia
Streptococcus pneumoniae is a fastidious gram-positive bacterium that can cause various infections such as pneumonia, meningitis, and sepsis. When grown on chocolate agar, it typically exhibits the following colony characteristics:
- Appearance: The colonies are usually small, smooth, and round with a slightly raised or flat surface. They appear opaque, and the color may range from grayish-white to greenish-gray.
- Size: The colonies are small, ranging from 1-2 mm in diameter.
- Texture: The colonies are smooth, glistening, and moist.
- Hemolysis: S. pneumoniae is an alpha-hemolytic organism that produces a greenish discoloration around the colony due to the partial breakdown of red blood cells.
- Other features: S. pneumoniae is catalase-negative, meaning that it does not produce the enzyme catalase. It is also optochin-sensitive, meaning that it is susceptible to a chemical called optochin, which can be used to differentiate it from other alpha-hemolytic streptococci.
In summary, S. pneumoniae colonies on chocolate agar are small, smooth, and round with a slightly raised or flat surface. They are alpha-hemolytic and produce a greenish discoloration around the colony. They are catalase-negative and optochin-sensitive.
Neisseria gonorrhoeae
Neisseria gonorrhoeae is a fastidious gram-negative bacterium that is the causative agent of the sexually transmitted infection gonorrhea. When grown on chocolate agar, it typically exhibits the following colony characteristics:
- Appearance: The colonies are small, round, and translucent with a smooth, slightly convex surface. They appear grayish-white to translucent in color.
- Size: The colonies are usually small, ranging from 0.5-1 mm in diameter.
- Texture: The colonies are smooth and slightly moist.
- Hemolysis: N. gonorrhoeae is a non-hemolytic organism and does not produce any hemolytic activity on chocolate agar.
- Other features: N. gonorrhoeae is an oxidase-positive bacterium, meaning that it produces an enzyme called cytochrome oxidase. This can be detected by adding a reagent called oxidase to the colony, which will produce a color change if the enzyme is present. N. gonorrhoeae is also non-motile and non-spore-forming.
In summary, N. gonorrhoeae colonies on chocolate agar are small, round, and translucent with a smooth, slightly convex surface. They are non-hemolytic and have a smooth and slightly moist texture. Additionally, they are oxidase-positive, non-motile, and non-spore-forming. It is important to note that chocolate agar is not a selective medium and other non-pathogenic Neisseria species may also grow on it. Therefore, additional tests are required to confirm the identification of N. gonorrhoeae.
Haemophilus influenza
Haemophilus influenzae is a fastidious gram-negative bacterium that can cause respiratory tract infections such as pneumonia, otitis media, and sinusitis. When grown on chocolate agar, it typically exhibits the following colony characteristics:
- Appearance: The colonies are small, round, and translucent with a smooth, slightly convex surface. They appear grayish-white to translucent in color.
- Size: The colonies are usually small, ranging from 1-2 mm in diameter.
- Texture: The colonies are smooth and slightly moist.
- Hemolysis: H. influenzae is a non-hemolytic organism and does not produce any hemolytic activity on chocolate agar.
- Other features: H. influenzae is a facultative anaerobe and requires X and V factors for growth. X factor, which is hemin, is present in blood, and V factor, which is nicotinamide adenine dinucleotide (NAD), is produced by some bacteria. Chocolate agar contains both X and V factors, which is why it is an ideal medium for H. influenzae growth. H. influenzae is also oxidase-positive, meaning that it produces an enzyme called cytochrome oxidase.
In summary, H. influenzae colonies on chocolate agar are small, round, and translucent with a smooth, slightly convex surface. They are non-hemolytic and have a smooth and slightly moist texture. Additionally, they require X and V factors for growth, which are present in chocolate agar, and are oxidase-positive.
Limitations:
Although chocolate agar is a useful medium for the cultivation of fastidious microorganisms, there are several limitations to its use, including:
- Contamination: Chocolate agar can be contaminated easily during preparation or storage, which can affect the growth of microorganisms and lead to false results.
- Nutrient depletion: Over time, the nutrients in chocolate agar can become depleted, leading to reduced growth of microorganisms or the growth of contaminating bacteria.
- Selectivity: Chocolate agar is not a selective medium, which means that it can support the growth of a wide range of bacteria, including non-pathogenic organisms. This can make it difficult to isolate and identify specific pathogens.
- Cost: The production of chocolate agar is more complex and expensive than the production of other types of agar, which can make it more expensive to use.
- Storage: Chocolate agar is a perishable medium and must be stored properly to maintain its integrity. Improper storage can result in a loss of nutrients and a decline in the quality of the medium.
In summary, while chocolate agar is a useful medium for the cultivation of fastidious microorganisms, it has some limitations, including contamination, nutrient depletion, lack of selectivity, cost, and storage requirements. These limitations must be taken into consideration when using chocolate agar for microbiological studies.
FAQs:
What is chocolate agar used for?
Chocolate agar is used for the cultivation of fastidious bacteria, including Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae.
What is the difference between blood agar and chocolate agar?
Blood agar contains whole red blood cells and can support the growth of a wider range of microorganisms compared to chocolate agar, which contains lysed red blood cells and is used for the cultivation of fastidious bacteria.
What is the composition of chocolate agar?
Chocolate agar is made from sheep blood, peptone, and agar.
What bacteria can grow on chocolate agar?
Bacteria that can grow on chocolate agar include Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Moraxella catarrhalis, Legionella pneumophila, and Bordetella pertussis, among others.
What is the color of chocolate agar?
Chocolate agar is a brownish-red color due to the lysed red blood cells in the medium.
What is the difference between chocolate agar and MacConkey agar?
Chocolate agar is used for the cultivation of fastidious bacteria, while MacConkey agar is a selective and differential medium used for the isolation and differentiation of Gram-negative bacteria.
How do you prepare chocolate agar?
Chocolate agar is prepared by adding lysed sheep blood to a base medium consisting of peptone and agar.
What is the temperature for incubating chocolate agar?
Chocolate agar should be incubated at 35-37°C in a CO2-enriched atmosphere.
Can you make chocolate agar at home?
It is not recommended to make chocolate agar at home due to the risk of contamination and the specialized equipment required for proper sterilization.
What is the pH of chocolate agar?
The pH of chocolate agar is typically around 7.4.
What is the shelf life of chocolate agar?
The shelf life of chocolate agar depends on the manufacturer and storage conditions, but it typically ranges from 4-8 weeks.
What is the difference between TSA and chocolate agar?
TSA (tryptic soy agar) is a general-purpose medium used for the cultivation of a wide range of microorganisms, while chocolate agar is used for the cultivation of fastidious bacteria.
Can you freeze chocolate agar?
Chocolate agar can be frozen, but the thawed medium may have reduced quality and nutrient content.
What is the difference between chocolate agar and Columbia agar?
Columbia agar is a general-purpose medium that can be used for the cultivation of a wide range of microorganisms, while chocolate agar is used for the cultivation of fastidious bacteria.
What is the difference between Mueller Hinton agar and chocolate agar?
Mueller Hinton agar is a medium used for antibiotic susceptibility testing, while chocolate agar is used for the cultivation of fastidious bacteria.
What is the difference between Sabouraud agar and chocolate agar?
Sabouraud agar is a medium used for the cultivation of fungi, while chocolate agar is used for the cultivation of fastidious bacteria.
Can you use chocolate agar for anaerobic bacteria?
Chocolate agar is not suitable for the cultivation of anaerobic bacteria, which require specialized anaerobic culture techniques.
What is the difference between chocolate agar and blood agar plates?
Blood agar plates contain whole red blood cells and can support the growth of a wider range of microorganisms compared to chocolate agar, which contains lysed red blood cells and is used for the cultivation of fastidious bacteria
Conclusion:
In conclusion, chocolate agar is a type of bacterial growth medium used for the cultivation of fastidious bacteria that have complex nutritional requirements. Chocolate agar is made by lysing sheep red blood cells and adding them to a base medium consisting of peptone and agar. The medium is typically incubated at 35-37°C in a CO2-enriched atmosphere. Bacteria that can grow on chocolate agar include Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Moraxella catarrhalis, Legionella pneumophila, and Bordetella pertussis, among others. Chocolate agar is a useful tool for diagnosing bacterial infections and studying bacterial physiology, but it has some limitations such as the requirement for specialized equipment and the risk of contamination.
