Blood Agar Base / Infusion Agar

Intended Use

Blood Agar Base / Infusion Agar (DM040) is used with blood for the isolation and cultivation of a wide variety of microorganisms.

Product Summary and Explanation

Blood Agar Bases are typically supplemented with 5-10% sheep, rabbit, or horse blood, and used for isolating, cultivating, and determining hemolytic reactions of fastidious pathogenic microorganisms. Without enrichment, this blood agar base can be used as a general purpose medium. Blood Agar Base is a nutritious base, and a variety of supplements, including blood, are often added to enhance this medium. Blood Agar Base is formulated to achieve good growth and improve hemolytic reactions of important pathogenic bacteria.

Principles of the Procedure

Blood Agar Base is prepared using specially selected raw materials to support good growth of a wide variety of fastidious microorganisms. Blood Agar Base contains Brain heart infusion and Tryptose, which provide nitrogen, carbon, and essential vitamins to stimulate organism growth. Along with essential nutritive properties, the peptones improve and enhance hemolysin production. Sodium Chloride maintains the osmotic balance of the medium. Agar is the solidifying agent. In general, blood agar bases are relatively free of reducing sugars, which have been reported to adversely influence the hemolytic reactions of α-hemolytic streptococci.(3) Supplementation with blood (5 – 10%) provides additional growth factors for fastidious microorganisms, and aids in determining hemolytic reactions. Hemolytic patterns may vary with the source of animal blood and the type of basal medium used.(4)

Formula / Liter

 Precautions

1. For Laboratory Use only.
2. IRRITANT. Irritating to eyes, respiratory system, and skin.

Directions

1. Suspend 40 g of the medium in one liter of distilled water.
2. Heat to boiling to completely dissolve the medium.
3. Autoclave at 121°C, 15 psi pressure, for 15 minutes.
4. Prepare 5% blood agar by aseptically adding the appropriate volume of sterile defibrinated blood to melted sterile
molten agar base, cooled to 45 – 50°C.

Quality Control Specifications

Expected Cultural Response:

The organisms listed are the minimum that should be used for quality control testing.

Test Procedure

1. Inoculate directly onto the surface of the medium. Streak for isolation with an inoculating loop, and stab the agar
several times to deposit beta-hemolytic streptococci beneath the agar surface. Subsurface growth will display the
most reliable hemolytic reactions.(4)
2. Incubate plates aerobically, anaerobically, or under conditions of increased CO2 (5 – 10%).

Results

Examine the medium for growth and hemolytic reactions after 18 – 24 and 48 hours incubation. There are four types of
hemolysis on blood agar media described as:
(4)
1. Alpha hemolysis (α) is the reduction of hemoglobin to methemoglobin in the medium surrounding the colony. This
produces a green discoloration of the medium.
2. Beta hemolysis (β) is the lysis of red blood cells, producing a clear zone surrounding the colony.
3. Gamma hemolysis (γ) indicates no hemolysis.
4. Alpha-prime-hemolysis (Ü) is a small zone of complete hemolysis, surrounded by an area of partial lysis.

Storage

Store the sealed bottle containing the dehydrated medium at 10 – 30°C. Once opened and recapped, place container i n a
low humidity environment at the same storage temperature. Protect from moisture and light.

Expiration

Refer to the expiration date stamped on the container. The dehydrated medium should be discarded if not free flowi ng,
or if the appearance has changed from the original color. Expiry applies to medium in its intact container when stored as
directed.

Limitations of the Procedure

1. The incubation environment can influence hemolytic reactions of beta-hemolytic streptococci.(4)
2. Hemolytic reactions of some strains of group D streptococci have been shown to be affected by differences in animal
blood. (4)