Chocolate Agar: Principle, Composition, Preparation,Uses And Results Interpretation

Introduction

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.

Principle

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
Autoclave
Weighing balance
Water bath
Hot air oven ( optional)

Procedure

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).

Alternatively

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.

Quality Control

Organism	Growth
N. meningitidis	luxuriant
S. pneumoniae	luxuriant
H. influenzae	luxuriant

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% CO₂atmosphere.
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.

Further References

Power, D.A. (ed.), and P.J. McCuen. 1988. Manual of BBL products and laboratory procedures, 6th ed. Becton Dickinson Microbiology Systems, Cockeys ville, Md.
Martin, J.E., T.E. Billings, J.F. Hackney, and J.D. Thayer. 1967. Primary isolation of N.
gonorrhoeae with a new commercial medium. Public Health Rep. 82:361-363.
Vastine, D.W., C.R. Dawson, I. Hoshiwara, C. Yonega, T. Daghfous, and M. Messadi. 1974.
Comparison of media for the isolation of Haemophilus species from cases of seasonal
conjunctivitis associated with severe endemic trachoma. Appl. Microbiol. 28:688-690.
Ruoff, L.R. 2003. Aerococcus, Abiotrophia, and other infrequently isolated aerobic catalasenegative, gram-positive cocci. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8thed. American Society for
Microbiology, Washington, D.C.
Anonymous. 1998. DIFCO Manual, 11th edition. DIFCO Laboratories, Division of Becton
Dickinson and Co. Sparks, MD, USA.
Ruoff, K.L. 2003. Aerococcus, Abiotrophia, and other infrequently isolated aerobic catalasenegative, gram-positive cocci. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller,and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.
S. Mishra, G.B. Nair, R.K. Bhadra, S.N. Sikder, S.C. Pal, Comparison of selective media for primary isolation of Aeromonas speciesfrom human and animal faeces, J. Clin. Microbiol., 25, 2040 (1987).
J.E. Sondag, R.K. Morgens, J.E. Hoppe, J.J. Marr, Detection of pneumococci in respiratory secretions: clinical evaluation of gentamicin blood agar, J. Clin. Microbiol., 5, 397 (1977).
National Committee for Clinical Laboratory Standards. Quality Assurance Standard for Commercially Prepared Microbiological Culture Media; Approved Standard. NCCLS publication M22-A3, Vol. 24 No.19 Table 2, 2004.
Gunn B.A (1984). “Chocolate agar: A differential medium for gram positive cocci”. Journal of Clinical Microbiology. 20 (4): 822–823.