Columbia Agar with 5% Sheep Blood- Composition, Principle, Preparation, Results, Uses

Columbia Agar with 5% Sheep blood is a highly nutritious general-purpose medium for the isolation and cultivation of the non-fastidious and fastidious microorganisms from various clinical specimens and non-clinical specimens of public health importance.  Columbia Blood Agar was first described in 1966 by Ellner et al who incorporated animal-derived peptone, enzymatic digests of casein, and enriched medium by addition of the defibrinated sheep blood into one medium. It was found to be an improved blood agar, promoting both luxuriant and rapid growth, improved pigment production, typical colony morphology, and sharply defined hemolytic reactions.

Columbia Agar with 5% Sheep Blood

Composition of Columbia Agar with 5% Sheep Blood

Pancreatic Digest of Casein12.0 g
Peptic Digest of Animal Tissue5.0 g
Yeast Extract3.0 g
Beef Extract3.0 g
Corn Starch1.0 g
Sodium Chloride5.0 g
Agar13.5 g
Sheep Blood, Defibrinated5 % (50ml)

pH 7.3 ± 0.2 at 25°C

Principle of Columbia Agar with 5% Sheep Blood

The medium contains peptones that supply nitrogen, carbon, vitamins, and trace elements necessary for the growth of the bacterium. Yeast extract act as the supplier of the vitamin B complex. Corn starch serves as the energy source for organisms and also neutralizes toxic metabolites by absorbing toxic by-products contained in the specimen. Sodium chloride is a source of essential electrolytes and maintains osmotic equilibrium. Sheep blood is added to demonstrate hemolytic reactions and supplies the X factor (heme) necessary for the growth of many pathogenic species. On this medium, colonies tend to be larger and growth is more luxuriant than on media containing other blood agar bases.

Preparation of Columbia Agar with 5% Sheep Blood

Preparation of Columbia Agar Base

  1. Add components to distilled/ deionized water and bring volume to 1.0L.
  2. Mix thoroughly.
  3. Gently heat until boiling.
  4. Autoclave for 15 min at 15 psi pressure–121°C.
  5. Cool to 45°–50°C.

Preparation of Columbia Agar with 5% Sheep Blood

  1. To 950.0mL of cooled, sterile Columbia agar base, aseptically add 50.0mL of sterile, defibrinated sheep blood.
  2. Mix thoroughly.
  3. Pour into sterile Petri dishes or distribute into sterile tubes.

Results Interpretation of Columbia Agar with 5% Sheep Blood

Organisms Colony characteristics
Streptococci (non-group D)Small, white to grayish.

Beta or alpha hemolysis

Enterococci (Group D)Small, but larger than group A streptococci, grayish.

 Alpha (rarely beta) hemolysis

StaphylococciLarge, white to gray or cream to yellow, with or without hemolysis
CorynebacteriaSmall to large, white to gray or yellow, with or without hemolysis
Listeria monocytogenesSmall to medium-sized, grayish, with weak beta hemolysis
EnterobacteriaceaeMedium-sized to large, grey colonies, with or without hemolysis
Candida speciesSmall, white colonies

Uses of Columbia Agar with 5% Sheep Blood

  • Columbia Blood Agar is recommended as a frequently used primary isolation medium for isolation of non-fastidious and fastidious microorganisms from clinical specimens.
  • It is a primary isolation medium on which most microorganisms, such as Enterobacteriaceae, Pseudomonas, and other non-fermenting Gram-negative rods, streptococci, enterococci, staphylococci, coryneforms, Candida species, and many others will grow.

Limitations of Columbia Agar with 5% Sheep Blood

  • It is recommended that biochemical, immunological, molecular and mass spectrometry testing be performed on colonies from pure culture for complete identification.
  • The medium lacks V factor (nicotinamide adenine dinucleotide, NAD) since sheep blood contains NADase which destroys the NAD. Hence, Haemophilus influenzae which requires both the X and V factors, will not grow on this medium.
  • Neisseria gonorrhoeae does not grow well on this medium.
  • The medium is not suitable for the isolation and growth of Mycobacterium, Legionella, Bordetella and other organisms with highly specific nutritive requirements.
  • Due to the rather high carbohydrate (starch) content in the Columbia Agar Base, beta-hemolytic streptococci may exhibit alpha rather than beta hemolytic reactions or may exhibit weak hemolytic reactions on media based on this formulation.


  1. Ronald M. Atlas and James W. Snyder (2014). Handbook of media for clinical and public health microbiology. CRC Press. Taylor & Francis Group, LLC. Page no.148-149.
  2. Becton Dickinson
  3. Himedia
  4. Hardy Diagnostics
  5. Thermo Fisher

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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