Blood Agar

Blood agar is an enriched medium prepared in microbiology laboratory to culture fastidious organisms i.e. supply specific bacteria with hemin (X factor), nicotinamide adenine dinucleotide (NAD, or V factor), amino acids, and iron.

Use of Blood in Blood Agar

Blood agar is prepared using mammalian blood including that of sheep, humans or horse. The used blood must be defibrinated so that clotting does not occur and the red blood cells are available to use for bacteria.

Common bacteria grown in hospitals in blood agar include:

• Streptococcus species

• Neisseria gonorrhoea

• Haemophilus

Hemolysis in Blood Agar

Use of blood agar becomes important in an microbiology laboratory when it comes to classifying bacteria by identifying their hemolytic patterns.

• Alpha-hemolysis: The red blood cells are only partially hemolyzed. After proper incubation, clear zone of hemolysis can be seen in the blood agar plates.

• Beta-hemolysis: The red blood cells are completely hemolyzed. After proper incubation, greenish discoloration is observed around the colonies.

• Gamma-hemolysis: The red blood cells are not hemolyzed. Once properly incubated, the integrity of remains as original i.e. there is no change in the color of the culture media.

Type Result on Blood Agar Common Bacteria

Alpha (α) hemolysis Partial hemolysis of red blood cells, a Streptococcus pneumoniae, Viridans streptococci greenish or brownish discoloration around colonies

Beta (β) hemolysis Complete hemolysis of red blood cells, Streptococcus pyogenes, Staphylococcus aureus a clear transparent zone around colonies

Gamma (γ) hemolysis No hemolysis; no change in the agar Enterococcus faecalis, Staphylococcus epidermidis surrounding colonies

Composition of Blood Agar

• Peptone Broth

• Tryptose

• Sodium chloride

• Agar

Extra ingredient needed: Sterile defibrinate blood

Component Function / Purpose

Peptone Broth Provides nitrogen, amino acids, peptides, and nutrients necessary for bacterial growth

Tryptose Supplies additional protein nutrients and growth factors that support fastidious organisms

Sodium Chloride Maintains osmotic balance and proper cell environment (NaCl)

Agar Acts as a solidifying agent to provide a surface for bacterial colony growth; generally not used as a nutrient by bacteria

Blood Enriches the medium and allows detection of hemolysis patterns (alpha, beta, gamma)

Steps to Prepare Blood Agar

1. Introduce 40 grams of the blood agar powder in 1000 ml of distilled water (standard for HiMedia Blood Agar Base).

2. Let the mix boil, make sure the agar dissolves completely.

3. For sterilization, autoclave the media. Standard: maintain 15 lbs pressure for 15 minutes at pressure 15 lbs.

4. After removing from the autoclave, let the agar come to a temperature of 45-50°C and maintain aseptic conditions, add 5% v/v sterile defibrinated blood.

5. Give a proper and slow shake to the conical flask you have prepared on.

6. Now, it’s time to plate the agar. Under strict aseptic conditions, pour into sterile petriplates. Maintain a depth of 3 to 5 mm.

7. Let the plate dry and it’s ready to use. If you have excess plates or tend to use it later on, store properly in a refrigerator. Keep in mind the blood agar media is nutrients rich and not being able to maintain aseptic conditions can favor growth of contaminants.

Uses of Blood Agar Media

1. It is used for the growth of bacteria that do not grow under normal growing agar like nutrient agar.

2. As it is an enriched medium, it is used to prevent delay of growth of important clinical isolates.

3. It is important in the classification of organisms such as Streptococci and Staphylococci on the basis of hemolytic reactions.

4. It is an important medium in hospitals to grow all the pathogenic bacteria, commonly used for routine bacteriological culture and diagnostic purposes.

Variation of Blood Agar Media

• Chocolate agar media: Chocolate agar is a variant of blood agar media, in the preparation step mentioned above, you just need to heat the blood to around 80°C. The purpose of applying heat is to lyse red blood cells. This releases the nutrients present inside the RBCs, making them easily accessible to the fastidious bacteria. On the other hand, it also destroys enzymes that can destroy essential nutrients in the culture media. Some of the fastidious organisms that grow well in chocolate agar are Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae.

Hence, the name chocolate agar does not mean the use of chocolate in the media. Rather, the name is based on the darkish brown color the media gets once the blood is heated.

References

1. Parija SC. Textbook of Microbiology and Immunology. 3rd ed. New Delhi: Elsevier India; 2016.

2. Textbook of Medical Microbiology. New Delhi: Jaypee Brothers Medical Publishers.

3. Textbook of Microbiology. Hyderabad: Universities Press