Chocolate agar with Campylobacter and its related informations

Chocolate agar

Campylobacter on chocolate agar (CHOC)

Chocolate agar preparation and uses: 5% sheep blood agar heated to make chocolate agar. Campylobacter fetus inoculated into it  and the plate incubated for 2 days at 37°C in 5% CO2 incubator. After that we have as above shown image .

Morphology of Campylobacters

They are small delicate, spirally curved, motle gram negative bacteria. Faecal smear stained with  1% basic fuchsin, the organisms have been linked  to the wings of gulls and some forms appear S or comma shaped as shown below-

As the bacteria develop, they  become longer and look more like spirochaetes.

Key note- The distinctive appearance of Campylobacters in a basic fuchsin stained feacal smear  containing blood and pus  enables a presumptive diagnosis of Campylobacter enteritis to be made.

Use of selective culture media containing antimicrobial agents to isolate campylobacters are as follows-

  1. Skiirow’s blood agar: It contains lyzed horse blood  ( on blood agar base) with antimicrobial agents like vancomycin, polymyxin B, and trimethoprim.
  2. Blaser’s medium: It contains 10% shhep blood agar(  on Brucella agar base) with antibiotics like vancomycin, polymyxin B, trimethoprim, cephalothin and amphotericin B.
  3. Improved Preston blood free-medium: It clears from the title, there is no need of blood and it contains antibiotics for selective are cefoperazone and amphotericin B.

Extra informations

Introduction of Chocolate agar 

Chocolate Agar (CHOC) 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.

Tool and techniques for isolation of Haemophilus influenzae as shown below-

Principle of Chocolate Agar

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.

Requirements for Chocolate Agar Preparation

  • 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)

Preparation of Chocolate Agar

It can be prepared by following methods.

Simplest method

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.

Another Method

  1. Suspend 40.5 grams in 1000 ml 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 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 the quality control inoculate N. meningitidis, S. pneumoniae, and H. influenzae QC strains inoculate into prepared chocolate agar (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

N. meningitidis     luxuriant

S. pneumoniae      luxuriant

H. influenzae           luxuriant

Colony Characteristics in Chocolate Agar

  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 Chocolate Agar

  1. 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) .
  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 selective medium for screening H. influenzae from specimens e.g. sputum containing a mixed flora of microorganisms.
  6. Its modified forms uses given below.

Modification of Chocolate Agar (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 uses to improve the primary isolation of Haemophilus influenzae from specimens containing a mixed flora of microorganism.
  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.

Culture media the simplest way of identification | Blood |MacConkey | Chocolate |RCM| Nurient agar as shown below-

E. coli on MacConkey agar, blood agar and Chocolate agar as shown below-

Note: There is no need of using chocolate agar for cultivation E. coli because it can  grown even on ordinary medium like nutrient agar, but want to share one thing is that all the organisms growing on nutrient agar/ MacConkey agar/ blood agar can easily grow on chocolate agar but not vice versa.

Campylobacter on chocolate agar-

Satellitism test Positive of Haemophilus influenzae is shown below-

Haemophilus influenzae on Gram’s stained smear under the microscope showing gram negative cocobacilli , small to large rod and pleomorphic forms as shown below-

CSF processing for Haemophilus and penumococcus identification is shown below-

Variety of Haemophilus species identification on basis of X, V,XV factors, blood agar and Xylose test as shown below-

References

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  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://www.ncbi.nlm.nih.gov/pmc/articles/PMC271442/
  7. https://jcm.asm.org/content/jcm/20/4/822.full.pdf
  8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533390/
  9. https://anaerobesystems.com/products/plated-media/chocolate-agar-choc
  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.  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
  12.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042752/
  14. https://www.webmd.com/children/meningococcal-meningitis-symptoms-causes-treatments-and-vaccines
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