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| Haemophilus influenzae on chocolate agar |
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What is the Principle of Chocolate agar
CHOC agar is used for growing fastidious respiratory bacteria like Haemophilus influenzae and Neisseria meningitidis and gonorrhoeae which Neither of these species is able to grow on Sheep Blood Agar.In addition, some of these bacteria, most notably H. influenzae, need specific growth factors such as NAD (factor V) and hemin (factor X), which are inside red blood cells; thus, a prerequisite to growth for these bacteria is lysis of the red blood cells. The heat also inactivates enzymes which could otherwise degrade NAD. The agar is named for the color and contains no actual chocolate.
Note that The composition of Chocolate agar and the Blood Agar is the same but the only difference is while preparing Chocolate agar, the red blood cells are lysed by gently heating.Better explaination are provided in preparation.
What are those Microorganism that can be culture on Chocolate agar
- Haemophilus influenzae
- Neisseria gonorrheae
- Neisseria meningitidis
- streptococcus pneumoniae.
What is the Composition of Chocolate Agar?
The composition of CHOC is the same as blood agar. that is
Ingredients Gms / Litre
Proteose peptone 20.000
Dextrose 0.500
Sodium chloride 5.000
Disodium phosphate 5.000
Agar 15.000
Final pH ( at 25°C) 7.3±0.2
How is Chocolate agar Prepared ?
- Heat-lyse a volume of horse or sheep blood that is 5% of the total volume of media being prepared very slowly to 56°C in a water bath.(Some people uses human blood and still yield the same result)
- Dispense 20 ml into 15x100 mm Petri dishes. Allow the media to solidify and condensation to dry.
- Place the plates in sterile plastic bags and Store below 30°C in tightly closed container and prepared medium at 2-8°C. until use.
- As a sterility test or quality control, incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
Another method is : Blood agar is put in a water bath for about 1 hour until its color changes from red to chocolate brown . Heating converts Hamoglobin into hematin.
What is the color of Chocolate Agar : Opaque Chocolate color
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| uninoculate chocolate agar (Opaque chocolate color |
What are the Colony characteristics in chocolate agar
- Neisseria meningitidis: Growth on chocolate agar is grayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
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| Haemophilus Influenzae in chocolate agar |
- Neisseria gonorrhoeae: Colonies on Chocolate agar are pinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter.
- Haemophilus influenzae: Non hemolytic, opaque cream-to-gray colonies (accompanying Sheep blood agar shows no growth)
What are other Modification of Chocolate Agar:
Thayer-Martin agar: It is used for the selective isolation of N. gonorrhoeae and N. meningitidis. Thayer-Martin Media is a chocolate agar supplemented with vancomycin (3.0mg/L), colistin (7.5mg/L), and nystatin (1250units/L).to inhibit the normal flora, including nonpathogenic Neisseria.
Chocolate Agar with bacitracin: CAP with bacitracin is a selective medium used to improve the primary isolation of H. influenzae from specimens containing a mixed flora of bacteria and/or fungi.
Chocolate agar with GC base and growth supplement: It is a medium 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 it is composed of 2% hemoglobin, and coenzyme enrichments.
Chocolate agar with TSA and growth supplements: It is a medium 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.
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