Chocolate Agar- Composition, Principle, Preparation, Results, Uses
Chocolate agar is a type of culture medium that is used to grow bacteria that are difficult to cultivate with ordinary media. It is especially useful for isolating and identifying fastidious pathogens, such as Haemophilus influenza and Neisseria meningitides, which cause respiratory infections and meningitis.
Chocolate agar is a variant of blood agar, which contains red blood cells that provide nutrients for bacterial growth. However, unlike blood agar, chocolate agar is prepared by heating the blood agar to 80°C, which causes the red blood cells to lyse (break down) and release their contents into the medium. This process gives the medium a chocolate-brown color and also makes it enriched with hemoglobin, hemin (X factor), and nicotinamide adenine dinucleotide (NAD or V factor), which are essential for the growth of some bacteria.
Chocolate agar is a non-selective medium, meaning that it can support the growth of many types of bacteria. However, it can be modified by adding antibiotics or other supplements to make it selective for certain bacteria. For example, chocolate agar with bacitracin can select for Haemophilus species, while chocolate agar with vancomycin, nystatin, and colistin can select for Neisseria species.
Chocolate agar is widely used in clinical microbiology laboratories for the isolation and identification of bacteria that cause serious infections in humans. It is also used for quality control and susceptibility testing of these bacteria. Chocolate agar is easy to prepare and has a long shelf life compared to other media.
In this article, we will discuss the composition, principle, preparation, results, uses, and limitations of chocolate agar in detail. We will also explore some of the modifications of chocolate agar that are available for different purposes.
Chocolate agar is a type of blood agar that has been heated to lyse the red blood cells and release their contents into the medium. The name comes from the dark brown color of the medium after heating. Chocolate agar is enriched with nutrients that support the growth of fastidious bacteria, especially Haemophilus and Neisseria species.
The composition of chocolate agar may vary depending on the manufacturer or the preparation method, but generally, it contains the following ingredients:
- Casein/animal tissue digest: This provides nitrogen, amino acids, and other essential elements for bacterial growth.
- Cornstarch: This helps to neutralize toxic substances such as fatty acids that may inhibit the growth of Neisseria species.
- Potassium phosphate: This acts as a buffer to maintain a stable pH during bacterial growth.
- Sodium chloride: This maintains the osmotic balance of the medium and prevents cell lysis.
- Agar: This is a solidifying agent that gives the medium its gel-like consistency.
- Hemoglobin solution: This is a 2% solution of hemoglobin that provides hemin (X factor) for Haemophilus species and other bacteria that require it for growth. Hemin is a component of hemoglobin that contains iron and porphyrin.
- Isovitox enrichment: This is a 10% solution of isovitalex that provides nicotinamide adenine dinucleotide (NAD or V factor) for Haemophilus species and other bacteria that require it for growth. NAD is a coenzyme that participates in many metabolic reactions.
The final pH of chocolate agar is 7.2 ± 0.2 at 25°C.
Chocolate agar is a type of enriched medium that supports the growth of fastidious bacteria, especially Haemophilus and Neisseria species. These bacteria require certain factors, such as hemin (X factor) and nicotinamide adenine dinucleotide (NAD or V factor), that are not present in ordinary media. Chocolate agar provides these factors by lysing red blood cells (RBCs) during the preparation of the medium. The lysis of RBCs releases hemoglobin, which contains hemin, and NAD, which is a coenzyme for many metabolic reactions. The heat also inactivates enzymes that could degrade NAD in the medium.
Chocolate agar also contains other ingredients that enhance the growth of fastidious bacteria. Casein and animal tissue digest provide nitrogenous nutrients, amino acids, and other essential elements for bacterial metabolism. Cornstarch helps neutralize fatty acids and other toxic metabolites that may inhibit the growth of Neisseria species. Potassium phosphate buffers the pH of the medium and prevents acidification by bacterial fermentation. Sodium chloride maintains the osmotic balance and cell integrity of the bacteria. Agar acts as a solidifying agent and allows the formation of discrete colonies.
Chocolate agar can be modified by adding different supplements to make it more selective or differential for certain bacteria. For example, Thayer-Martin media is a chocolate agar supplemented with antibiotics (vancomycin, nystatin, and colistin) to inhibit the normal flora and selectively isolate Neisseria gonorrhoeae and Neisseria meningitidis. Chocolate agar supplemented with bacitracin is used to selectively isolate Haemophilus influenzae from mixed specimens. Chocolate agar supplemented with GC base and growth supplement is used to enhance the growth of fastidious organisms that require hemin and NAD, such as Haemophilus influenza when incubated in a 5% CO2 atmosphere.
The principle of chocolate agar is based on providing the necessary factors and nutrients for the growth of fastidious bacteria by lysing RBCs and adding supplements to the medium. Chocolate agar is a useful medium for isolating and identifying these bacteria from clinical specimens.
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Chocolate agar is prepared by heating blood agar, which in turn ruptures the red blood cells (RBCs) and releases nutrients that aid in the growth of fastidious bacteria. The lysis of RBCs gives the medium a chocolate-brown color. The following steps are involved in the preparation of chocolate agar:
Preparation of the hemoglobin solution
- Add hemoglobin to distilled water and bring volume to 500 ml.
- Mix thoroughly and autoclave for 15 min at 15lbs pressure at 121°C.
- Cool at 45°C -50°C.
Preparation of medium
- Add components except for hemoglobin solution to distilled water and bring volume to 500ml.
- Mix thoroughly and gently heat until boiling.
- Autoclave for 15 min at 15lbs pressure at 121°C.
- Cool at 45°C -50°C.
- Add 500ml of sterile hemoglobin solution and mix thoroughly.
- Pour into sterile Petri dishes or distribute into sterile tubes.
Alternatively, you can use a commercially available chocolate agar base and follow the manufacturer`s instructions. You will need to add 5-7% v/v of defibrinated blood (horse or sheep blood) and place the media in a water bath of 75 -80°C, and keep swirling gently until the color changes to dark brown. Then pour into sterile Petri plates under aseptic conditions after the media has cooled to 50-55°C.
Some modifications can also be made for the growth of specific organisms. For example, you can add supplements such as isovitalex, vancomycin, nystatin, colistin, or bacitracin to enhance or inhibit the growth of certain bacteria.
The appearance of bacterial colonies on chocolate agar depends on the type and species of the organism. Some common examples are:
- Neisseria gonorrhoeae produces small, grey-to-white mucoid colonies, smooth consistency, and defined margins.
- Neisseria meningitides produces larger bluish-grey, mucoid, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
- Haemophilus influenzae produces small, colorless, moist colonies with a characteristic seminal odor.
- Streptococcus pneumoniae produces small, greyish-white colonies with alpha-hemolysis (greenish zone around the colonies) on blood agar.
- Streptococcus pyogenes produces small, white to cream-colored colonies with beta-hemolysis (clear zone around the colonies) on blood agar.
- Corynebacterium diphtheriae produces greyish-black colonies with brown halos on Tinsdale agar or Mueller-Hinton tellurite blood agar.
- Legionella pneumophila produces small, white to greyish-blue colonies on buffered charcoal yeast extract (BCYE) agar.
Gram staining of the isolated colonies can further confirm the identity of the organism by showing its morphology and Gram reaction. For example:
- Neisseria species are Gram-negative diplococci that resemble coffee beans.
- Haemophilus species are Gram-negative coccobacillus that may require special stains such as methylene blue or gentian violet.
- Streptococcus species are Gram-positive cocci in chains or pairs.
- Corynebacterium species are Gram-positive bacilli that may show club-shaped ends and form palisades or Chinese letters.
- Legionella species are Gram-negative bacilli that are faintly staining and may require silver stain.
Some additional biochemical tests may be required to differentiate between closely related species or strains. For example:
- Coagulase test can distinguish between coagulase-positive Staphylococcus aureus and coagulase-negative Staphylococcus epidermidis.
- Catalase test can differentiate between catalase-positive Staphylococcus species and catalase-negative Streptococcus species.
- X and V factor tests can identify Haemophilus species based on their requirement for hemin (X factor) and NAD (V factor) for growth.
Chocolate agar is a non-selective, enriched medium that supports the growth of fastidious microorganisms, particularly Haemophilus and Neisseria species. These bacteria require certain growth factors, such as hemin (X factor) and nicotinamide adenine dinucleotide (NAD or V factor), that are released from the lysed red blood cells in chocolate agar.
Chocolate agar is used for the isolation of Neisseria gonorrhoeae from chronic and acute cases of gonococcal infections. N. gonorrhea is the causative agent of gonorrhea, a sexually transmitted disease that can affect the urethra, cervix, rectum, throat, and eyes. Chocolate agar can also be used to isolate Neisseria meningitides, the causative agent of meningococcal meningitis, a serious infection of the meninges that can lead to septicemia and death. N. meningitidis can be found in the nasopharynx of asymptomatic carriers and can be transmitted through respiratory droplets.
Chocolate agar is also used for the isolation of Haemophilus influenzae, a gram-negative coccobacillus that can cause respiratory infections, such as otitis media, sinusitis, bronchitis, and pneumonia. H. influenza can also cause invasive infections, such as meningitis, epiglottitis, septic arthritis, and cellulitis. H. influenzae requires both X and V factors for growth and can be differentiated from other Haemophilus species by its requirement for the V factor.
Chocolate agar can be modified by adding various supplements or antibiotics to enhance the growth or selectivity of certain organisms. For example:
- Chocolate agar with bacitracin acts as a selective medium for screening H. influenzae from specimens containing a mixed flora of microorganisms.
- Thayer-Martin agar is a chocolate agar supplemented with vancomycin, nystatin, and colistin to inhibit the normal flora, including nonpathogenic Neisseria, for the selective isolation of N. gonorrhoeae and N. meningitidis.
- Chocolate agar supplemented with GC base and growth supplement is another modification that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO2 atmosphere.
Chocolate agar is a useful medium for the diagnosis of various diseases caused by fastidious bacteria that require hemin and NAD for growth.
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- Chocolate Agar is an enriched medium that can support the growth of many bacteria and fungi, not only the fastidious ones. Therefore, it is not a selective medium and may allow the overgrowth of normal flora or contaminants that can mask or inhibit the growth of the target organisms. For example, if the specimen contains nonpathogenic Neisseria species, they may grow on Chocolate Agar and interfere with the isolation of N. gonorrhoeae or N. meningitidis. To overcome this limitation, selective media such as Thayer-Martin Agar or Martin-Lewis Agar can be used in parallel with Chocolate Agar to inhibit the unwanted microorganisms and enhance the recovery of pathogenic Neisseria species.
- Chocolate Agar is also not a differential medium and does not provide any distinctive characteristics to distinguish between different types of bacteria or fungi. Therefore, it is not sufficient to identify the isolated colonies based on their appearance on Chocolate Agar alone. Additional biochemical and/or serological tests are required to confirm the identity of the microorganisms and their susceptibility to antibiotics. For example, oxidase test, catalase test, carbohydrate fermentation test, coagulase test, Gram stain, and agglutination test can be used to differentiate between various Haemophilus and Neisseria species and other bacteria that may grow on Chocolate Agar.
- Chocolate Agar may contain some precipitated hemoglobin that appears as dark spots on or in the media. This does not affect the performance of the media or the growth of the microorganisms, but it may cause some confusion or misinterpretation of the results. For example, some bacteria, such as Streptococcus pneumoniae or Staphylococcus aureus, may produce alpha-hemolysis or beta-hemolysis on blood agar plates, which are indicated by a greenish or clear zone around the colonies. However, on Chocolate Agar plates, these zones may not be visible due to the presence of precipitated hemoglobin. Therefore, it is important to use blood agar plates along with Chocolate Agar plates to observe the hemolytic reactions of the bacteria.
- Chocolate Agar does not guarantee the detection of all pathogenic microorganisms that may be present in a specimen. Some microorganisms may require special media or conditions for their growth and survival, such as anaerobic bacteria or Mycobacterium tuberculosis. Some microorganisms may be present in very low numbers or in a dormant state and may not grow on Chocolate Agar within the incubation period. Some microorganisms may be inhibited by substances present in the specimen or in the media, such as antibiotics or fatty acids. Therefore, it is recommended to use other methods, such as microscopy, culture-independent techniques, or molecular methods, to complement the culture results and ensure a comprehensive diagnosis of infections.
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