Chocolate Agar: Introduction, Composition, Principle, Preparation Requirements, Testing Procedure, Colony Characteristics, Uses, Keynotes, and Chocolate Agar Footages

Introduction of Chocolate agar

Chocolate Agar short form is (CHOC). It is a non-selective, enriched growth medium that is the lysed blood agar. The agar is named for its color when the red blood cells (RBCs) lysis gives the medium a chocolate-brown color without having chocolate products. It is used for the isolation of fastidious bacteria, such as Haemophilus influenzae, when incubated at 35-37°C in a 5% CO₂  incubator.

Composition of Blood Agar Base

Ingredients	Gms / Litre
Casein enzymic hydrolysate	14.0
Peptic digest of animal tissue	4.5
Yeast extract	4.5
Sodium chloride	5.0
Agar	12.5
Final pH (at 25°C)	7.3±0.2

Principle of Chocolate Agar

The composition of chocolate agar is the same as the blood agar and the only difference is while preparing Chocolate agar, the red blood cells are lysed changing the medium color to 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.

Requirements for CHOC Preparation

1. Prepared blood agar
2. Incubator for the simplest method
3. But other methods are
4. Blood agar base
5. Sheep blood
6. Distilled water
7. Measuring cylinder
8. Autoclave
9. Weighing balance
10. Water bath
11. Hot air oven (optional)

Preparation of CHOC

It can be prepared by the following methods.

Simplest method

• Take already prepared blood agar plates (5% sheep blood agar) and put those plates into a 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 them at 4°C until use.
• As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO₂.

Another Method

1. Suspend 40.5 grams in 1000 ml of distilled water or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
4. 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.
5. Dispense 20 ml into 15×100 mm Petri dishes. Allow the media to solidify and condensation to dry.
6. Place the plates in sterile plastic bags and store them at 4ºC until use.
7. As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO₂ (or in a candle jar).

Quality Control

For quality control inoculate N. meningitidis, S. pneumoniae, and H. influenzae QC strains inoculate into prepared CHOC for 18-24 hours at 35-37°C with 5% CO₂ (or in a candle jar but it can only provide up to 3% CO₂).

Organisms	growth
Neisseria meningitidis ATCC 13090	luxuriant
Streptococcus pneumoniae ATCC 6303	luxuriant
Haemophilus influenzae ATCC 19418	luxuriant

Storage and Shelf life of CHOC

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Freshly prepared CHOC without inoculation

Test Requirements

• Test specimens (Blood, vaginal samples, sputum, or growth of bacteria)
• Inoculating loop
• Bunsen burner
• Incubator
• Chocolate agar
• Control strains

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC for 18-24 hours with 5% CO₂.
6. Examine colonial characteristics.

Result -Interpretation

• Control strains i.e. Neisseria meningitidis ATCC 13090 and Haemophilus influenzae ATCC 19418: Good-luxuriant
• Test Organisms: Colony morphology depends on the nature of the organisms.

   

Colony Characteristics in CHOC

1. Haemophilus influenzae: Non-hemolytic, opaque cream to gray colonies.
2. Neisseria meningitidis: Growth on chocolate agar is grayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
3. Neisseria gonorrhoeae: Colonies on CHOC are pinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter.

Uses of CHOC

1. It is a 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. meningitidis).
2. And thus, Chocolate agar uses to isolate and cultivate fastidious microorganisms such as Haemophilus species and Neisseria species.
3. It is also useful in isolating N. gonorrheae from both acute and chronic cases of gonococcal infections.
4. It is also useful in isolating N. meningitidis from bacterial meningitis.
5. Chocolate agar with bacitracin acts as a selective medium for screening H. influenzae from specimens e.g. sputum containing a mixed flora of microorganisms.
6. Its modified forms use given below.

Modification of CHOC

1. 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
2. Chocolate Agar with bacitracin: CHOC with bacitracin is a selective medium used to improve the primary isolation of Haemophilus influenzae from specimens containing a mixed flora of microorganisms.
3. 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.
4. 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.

Keynotes

• Chocolate agar is a recommended medium for the isolation of Neisseria gonorrhoeae from chronic and acute cases of gonococcal infections as well as H. influenzae from the sputum of upper respiratory tract infection.
• It is the medium of choice for the cultivation of fastidious bacteria.
• The organisms which grow on MacConkey medium and blood agar also grow on chocolate agar but not vice versa.
• Thayer-Martin agar, chocolate agar with bacitracin, chocolate agar with GC base and growth supplement, and chocolate agar with TSA and growth supplements are modified forms of chocolate agar.

CHOC Footages

Campylobacter colony morphology on CHOC

Haemophilus influenzae growth around bacitracin disk in CHOC of sputum culture

E. coli colony morphology on CHOC

Further Readings

1. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
4. https://www.thermofisher. com/order/catalog/product/R01293#/R01293
5. https://www.sciencedirect.com/topics/immunology-and-microbiology/chocolate-agar
6. https://jcm.asm.org/content/jcm/20/4/822.full.pdf
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533390/
8. https://anaerobesystems.com/products/plated-media/chocolate-agar-choc
9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC271442/
10. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
11. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
12. Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042752/
14. https://www.webmd.com/children/meningococcal-meningitis-symptoms-causes-treatments-and-vaccines