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.

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

Ingredients	Gms / Litre
Peptone, special	23.000
Corn starch	1.000
Sodium chloride	5.000
Agar	15.000
Final pH at temperature 25°C	7.3±0.2
With addition of Sheep Blood	5.000

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 species	Add rehydrated contents of 1 vial of Brucella Selective Supplement to 500 ml sterile molten base
Campylobacter species	Add 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 species	Add rehydrated contents of 1 vial of G.Vaginalis Selective Supplement  to 500 ml sterile molten base.
For Cocci	Add rehydrated contents of 1 vial of Staph-Strepto Supplement or Strepto Supplement or Streptococcus Selective Supplement to 500 ml sterile molten base.

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

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.

Microorganisms	Growth	Type of haemolysis
Neisseria meningitidis	luxuriant	none
Staphylococcus aureus	luxuriant	beta / gamma
Staphylococcus epidermidis	luxuriant	gamma
Streptococcus pneumoniae	luxuriant	alpha
Streptococcus pyogenes	luxuriant	beta
Escherichia coli	luxuriant	beta
Clostridium perfringens	luxuriant	XX
Clostridium sporogenes	luxuriant	XX

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