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 influenzaewhen incubated at 35-37°C in a 5% CO2  incubator.

Chocolate Agar: Introduction,Composition, Principle, Preparation Requirements,Testing Procedure, Colony Characteristics, Uses, Keynotes and Chocolate Agar Footages
Fig. Haemophilus influenzae growth on Chocolate Agar

Composition of Blood Agar Base

IngredientsGms / Litre
Casein enzymic hydrolysate14.0
Peptic digest of animal tissue4.5
Yeast extract4.5
Sodium chloride5.0
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. meningitidisS. 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 6303luxuriant
Haemophilus influenzae ATCC 19418luxuriant
Table: Control strains growth status

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

Freshly prepared chocolate agar without inoculation
Fig. Freshly prepared chocolate agar 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% COatmosphere.
  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.


  • 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

Campylobacter colony morphology on chocolate agar
Fig. Campylobacter colony morphology on chocolate agar (CHOC)

Haemophilus influenzae growth around bacitracin disk in CHOC of sputum culture

Haemophilus influenzae growth around bacitracin disk in chocolate agar of sputum culture
Fig. Haemophilus influenzae growth around bacitracin disk in chocolate agar of sputum culture

E. coli colony morphology on CHOC

E. coli colony morphology on chocolate agar
Fig. 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
  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

28 thoughts on “Chocolate Agar: Introduction, Composition, Principle, Preparation Requirements, Testing Procedure, Colony Characteristics, Uses, Keynotes, and Chocolate Agar Footages”

  1. Someone essentially lend a hand to make significantly posts I’d state. That is the first time I frequented your web page and to this point? I surprised with the analysis you made to make this actual submit incredible. Wonderful job!

  2. Admiring the commitment you put into your website and detailed information you offer. It’s good to come across a blog every once in a while that isn’t the same old rehashed material. Great read! I’ve bookmarked your site and I’m adding your RSS feeds to my Google account.

  3. Great write-up, I?m regular visitor of one?s website, maintain up the excellent operate, and It is going to be a regular visitor for a lengthy time.

  4. You really make it seem so easy with your presentation but I find this matter to be
    actually something which I think I would never understand.
    It seems too complicated and extremely broad for me.

    I’m looking forward for your next post, I will try to get the hang of it!

  5. I do love the manner in which you have framed this particular issue plus it does indeed give me personally some fodder for consideration. Nonetheless, through what precisely I have observed, I only trust as other responses stack on that folks stay on point and don’t start upon a soap box regarding some other news du jour. Still, thank you for this excellent piece and whilst I can not go along with this in totality, I respect your point of view.

  6. What an insightful and well-researched article! The author’s thoroughness and ability to present intricate ideas in a understandable manner is truly admirable. I’m thoroughly enthralled by the breadth of knowledge showcased in this piece. Thank you, author, for sharing your expertise with us. This article has been a true revelation!

  7. Together with every thing that appears to be developing throughout this subject matter, all your viewpoints tend to be somewhat radical. Nonetheless, I beg your pardon, but I can not subscribe to your whole plan, all be it exhilarating none the less. It appears to me that your comments are generally not completely validated and in actuality you are generally yourself not even totally confident of your assertion. In any case I did take pleasure in looking at it.

  8. certainly like your web site but you have to test the spelling on quite a few of your posts. Many of them are rife with spelling issues and I find it very bothersome to inform the truth however I?ll definitely come back again.

  9. I like what you guys are up also. Such intelligent work and reporting! Carry on the superb works guys I?ve incorporated you guys to my blogroll. I think it will improve the value of my website 🙂

  10. I would also love to add when you do not currently have an insurance policy otherwise you do not form part of any group insurance, you might well take advantage of seeking assistance from a health insurance professional. Self-employed or people having medical conditions normally seek the help of a health insurance brokerage service. Thanks for your text.

  11. I would like to thank you for the efforts you’ve put in writing this website. I am hoping the same high-grade web site post from you in the upcoming as well. Actually your creative writing abilities has inspired me to get my own website now. Really the blogging is spreading its wings rapidly. Your write up is a good example of it.

  12. Appreciating the time and effort you put into your site and detailed information you provide. It’s good to come across a blog every once in a while that isn’t the same unwanted rehashed material. Wonderful read! I’ve saved your site and I’m including your RSS feeds to my Google account.

  13. I?ve been exploring for a little for any high quality articles or blog posts on this sort of area . Exploring in Yahoo I at last stumbled upon this site. Reading this information So i am happy to convey that I have an incredibly good uncanny feeling I discovered exactly what I needed. I most certainly will make certain to do not forget this website and give it a look on a constant basis.

  14. Wow! This can be one particular of the most helpful blogs We’ve ever arrive across on this subject. Actually Fantastic. I’m also an expert in this topic therefore I can understand your effort.


Leave a Comment