Chocolate agar (CHOC) or chocolate blood agar (CBA), is a nonselective, enriched growth medium used for isolation of pathogenic bacteria.It is a variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80°C.The name is itself derived from the fact that red blood cell (RBC) lysis gives the medium a chocolate-brown color. Chocolate agar is used for growing fastidious respiratory bacteria, such as Haemophilus influenzae and Neisseria meningitidis.The composition of chocolate agar and the Blood Agar is the same and the only difference is while preparing chocolate agar, the red blood cells are lysed.
Medium contains Proteose peptone is a nitrogen source required for the growth of wide variety of
organisms. Dextrose acts a carbon energy source. Disodium phosphate buffers the medium whereas sodium chloride maintains the osmotic equilibrium. Agar is the solidifying agent. Heated sheep blood is added to give the medium its “chocolate” appearance. This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.
The composition of chocolate agar is same as the blood agar and the only difference is while preparing Chocolate agar, the red blood cells are lysed changing the medium color chocolate brown.The lysis of RBC during the heating process releases intracellular coenzyme nicotinamide adenine dinucleotide (Factor V or NAD) into the agar for utilization by fastidious bacteria (the heating process also inactivates growth inhibitors). Hemin (factor X) is available from non-hemolyzed as well as hemolyzed blood cells.The most common species that require this enriched medium for growth include Neisseria meningitidis and Haemophilus spp. H. influenzae is not able to grow on sheep blood agar.
Preparation Of Chocolate Agar
- Prepared blood agar
- Incubator for simplest method
- But for other method are
- Blood agar base
- Sheep blood
- Distilled water
- Measuring cylinder
- Weighing balance
- Water bath
- Hot air oven ( optional)
- Suspend 40.5 grams in 1000 ml distilled water or deionized water.
- Heat to boiling to dissolve the medium completely.
- Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
- Heat-lyse a volume of sheep blood that is 5% of the total volume of media being prepared very slowly to 56°C in a water bath.
- Dispense 20 ml into 15×100 mm Petri dishes. Allow the media to solidify and condensation to dry.
- Place the plates in sterile plastic bags and store at 4ºC until use.
- As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO2 (or in a candle-jar).
- Take already prepared blood agar plates (5% sheep blood agar) and put those plates into hot air oven for 2 hours at 55°C.
- Take out those plates and you will get chocolate agar.
- Place the plates in sterile plastic bags and store at 4°C until use.
- As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO2.
Result Interpretation/Colony Characteristics of Chocolate Agar
- Neisseria meningitidis: Growth on chocolate agar is grayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
- Neisseria gonorrhoeae produces small, grey to white, mucoid colonies, smooth consistency, and defined margins.
- Haemophilus influenzae: Non hemolytic, opaque cream to gray colonies.
Modification of ChocolateAgar (CHOC
- Thayer-Martin agar/medium uses for the selective isolation of N. gonorrhoeae and N. meningitidis. This Media is a chocolate agar supplemented with vancomycin, colistin and nystatin (VCN) to inhibit the normal flora, including non-pathogenic Neisseria from the clinical specimens.
- Chocolate Agar with bacitracin: CHOC with bacitracin is a selective medium uses to improve the primary isolation of Haemophilus influenzae from specimens containing a mixed flora of microorganism.
- 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.
- 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%CO2atmosphere.
Uses of Chocolate Agar
- It is very useful medium to isolate fastidious organisms in Microbiology Laboratory from various clinical specimens like sputum ( H. influenzae), urethral discharge ( N. gonorrhoeae), CSF/blood (N. menigitidis) .
- And thus, Chocolate agar uses to isolate and cultivate fastidious microorganisms such as Haemophilus species and Neisseria species.
- It is also useful in isolating N. gonorrheae from both acute and chronic cases of gonococcal infections.
- It is also useful in isolating N. meningitidis from bacterial meningitis.
- Chocolate agar with bacitracin acts as selective medium for screening H. influenzae from specimens e.g. sputum containing a mixed flora of microorganisms.
Limitations of Chocolate Agar
- It is recommended that additional biochemical and/or serological tests should be performed on isolated colonies from the pure culture in order to complete identification.
- Chocolate Agar is an enriched medium, thus non-pathogenic organisms may overgrow pathogenic bacteria. If isolation of N. gonorrhoeae is desired, a selective medium such as Thayer Martin Agar, Modified should be used in parallel with this non-selective formula.
- Precipitated hemoglobin may appear as dark spots on or in the media and does not affect the performance of the media.
- The presence or absence of N. gonorrhoeae in a specimen does not rule out the possible presence of other pathogenic organisms.
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