How to prepare Blood and Chocolate agar using Columbia Agar base

Columbia Agar Base is used as an efficient base for the preparation of culture media like blood and chocolate agar .It is also used for the preparation of various selective and differential media and for the isolation of most fastidious organisms from clinical and non clinical samples (Blood and chocolate agar).This medium contains special peptones that promote fast and luxurious growth of fastidious and non- fast organisms and also promotes typical colonial morphology; improved pigment production and haemolytic reactions more clearly defined.An addition of bacitracin makes the enriched Columbia Agar Medium selective for the isolation of Haemophilus species from clinical specimens, particularly from the upper respiratory tract.

Principle of Columbia Agar

  • Corn starch serves as an energy source and also neutralizes toxic metabolites.
  • Addition of 5% sheep blood permits the detection of haemolysis and also provides heme (X factor) which is required for the growth of many bacteria. However it is devoid of V factor (Nicotinamide adenine dinucleotide) and hence Haemophilus influenzae which needs both the X and V factors, will not grow on this medium.Some other microbiology agar plates like brain heart infusion agar (bhi agar),nutrient agar,tryptic soy agar and tryptone soya agar can be used to make chocolate agar or blood agar

Some Modification of Columbia Agar

  • Columbia Agar Base with added sterile serum (some laboratories uses fetal bovine serum or horse serum) provides an efficient medium for Corynebacterium diphtheriae virulence test medium. After following the established technique for Corynebacterium diphtheriae, lines of toxin-antitoxin precipitation are clearly visible in 48 hours. Many pathogens require carbon dioxide; therefore, plates may be incubated in an atmosphere containing approximately 3-10% CO2.
Also read  How to prepare tryptic Soy Agar(TSA) : Principle,Composition and Colony response

Precaution for handling Highly infectious organism

  • Brucella cultures are highly infective and must be handled carefully; incubate in 5-10% CO2 .
  • Campylobacter species are best grown at 42°C in a microaerophilic atmosphere.
  • Plates with Gardnerella supplements plates should be incubated at 35°C for 48 hours containing 7% CO2

Composition of Columbia agar

The following are ingredient used in the composition of Columbia blood agar base

IngredientsGms / Litre
Peptone, special23.000
Corn starch1.000
Sodium chloride5.000
Agar15.000
Final pH at temperature 25°C7.3±0.2
With addition of Sheep Blood5.000

How to Prepare Columbia agar

Note that standard petri dish sizes can hold a volume of 2 ml when media is poured

  1. Start by suspending 44.0 grams of in 1000 ml distilled water.
  2. Heat to boiling in order to completely dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. Cool to 45-50°C before adding heat sensitive compounds.

For Blood Agar: Add 5% v/v sterile defibrinated sheep blood to sterile cool base.

For Chocolate Agar: Add 10% v/v sterile defibrinated sheep blood to sterile cool base.

  1. Heat to 80°C for 10 minutes with constant agitation.

Modification of Columbia Agar for increase selectivity

The medium can be made selective by adding different antimicrobials to sterile base.

Brucella speciesAdd rehydrated contents of 1 vial of Brucella Selective Supplement to 500 ml sterile molten base
Campylobacter speciesAdd rehydrated contents of 1 vial of Campylobacter Supplement- I or Campylobacter Supplement- II or Campylobacter Supplement- III or Campylobacter Selective Supplement or Campylobacter Supplement- VI to 500 ml sterile molten base along with rehydrated contents of 1 vial of Campylobacter Growth Supplement and 5-7% v/v horse or sheep blood.
For Gardnerella speciesAdd rehydrated contents of 1 vial of G.Vaginalis Selective Supplement  to 500 ml sterile molten base.
For CocciAdd rehydrated contents of 1 vial of Staph-Strepto Supplement or Strepto Supplement or Streptococcus Selective Supplement to 500 ml sterile molten base.
Also read  Albert staining of metachromatic granules ideal for Corynebacterium diphtheriae

Appearance of Medium after preparation

  • The basal medium appears light amber coloured clear to slightly opalescent gel.
  • After addition of 5%w/v sterile defibrinated blood it change to cherry red coloured opaque gel forms in petri dishes
Uninoculated Columbia agar base
Uninoculated Columbia agar base
Columbia chocolate agar
Columbia chocolate agar
Columbia blood agar base
Columbia blood agar base

Type of specimens

Clinical specimen such as

  • Blood
  • respiratory exudates

How to Inoculate on Columbia Agar

Either streak, inoculate or surface spread the test inoculum aseptically on the plate.

  • For Brucella cultures incubate in 5-10% CO2 .
  • Campylobacter species are best grown at 42°C in a microaerophilic atmosphere.
  • Plates with Gardnerella supplements plates should be incubated at 35°C for 48 hours containing 7% CO2

Plate reading and Colonies morphology

The following cultural characteristics  are observed with added 5% w/v sterile defibrinated blood, after an incubation at  a temperature of 35-37°C for 24-48 hours.

MicroorganismsGrowthType of haemolysis
Neisseria meningitidisluxuriantnone
Staphylococcus aureusluxuriantbeta / gamma
Staphylococcus epidermidisluxuriantgamma
Streptococcus pneumoniaeluxuriantalpha
Streptococcus pyogenesluxuriantbeta
Escherichia coliluxuriantbeta
Clostridium perfringensluxuriantXX
Clostridium sporogenesluxuriantXX
Streptococcus pyogenes on Columbia Blood Agar

Streptococcus pyogenes on Columbia Blood Agar
Neisseria subflava on Columbia chocolate agar

Neisseria subflava on Columbia chocolate agar
Streptococcus pneumoniae on Columbia blood agar.jpg

Streptococcus pneumoniae on Columbia blood agar.jpg
Nonhemolytic colonies on columbia blood agar

Nonhemolytic colonies on columbia blood agar

Some Limitation of Columbia Agar

  • Certain fastidious organisms like Haemophilus influenzae may not grow on the medium, blood supplementation may be required.
  • As this medium have a relatively high carbohydrate content, beta-haemolytic Streptococci may exhibit a greenish haemolytic reaction which may be mistaken for the alpha haemolysis.
  • Carry out confirmatory tests of all the colonies

Reference

  • Ellner P. P., Stoessel C. J., Drakeford E. and Vasi F., 1966, Am. J. Clin. Pathol., 45:502.
  • Fildes P., 1920, Br. J. Exp. Pathol., 1:129. 3.Fildes P., 1921, Br. J. Exp. Pathol., 2:16.
  • Chapin K. C. and Doern G. V., 1983, J. Clin. Microbiol., 17:1163.
  • Bailey R. K., Voss J. L. and Smith R. F., 1979, J. Clin. Microbiol., 9 ; 65-71

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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