Blood Agar Base (Infusion Agar)

Intended Use

Blood Agar Base (Infusion Agar), with the addition of sterile blood, is used for the isolation, cultivation and detection of hemolytic activity of streptococci and other fastidious microorganisms.

Summary and Explanation

Infusion Agar is an all-purpose medium which has been used for many years as a base for the preparation of blood agars. In a study of viability of streptococci, Snavely and Brahier performed comparative studies of horse, rabbit and sheep blood with Blood Agar Base, and found that sheep blood gave the clearest and most reliable colony and hemolysis characteristics at both 24 and 48 hours.¹ In the course of the investigation, about 1,300 isolations of streptococci were made with Blood Agar Base containing 5% sheep blood.
Blood Agar Base media are specified in standard methods for food testing.^2-4 Infusion Agar has been largely replaced as a blood agar base by the Tryptic/Trypticase™ Soy Agar formulations, which contain milk and plant peptones in place of the variable infusion component.

Principles of the Procedure

Infusion from heart muscle, casein peptone and yeast extract provide nitrogen, carbon, amino acids and vitamins in Blood Agar Base. Medium contains sodium chloride to maintain osmotic equilibrium and agar is the solidifying agent.
Supplementation with blood (5-10%) provides additional growth factors for fastidious microorganisms, and is the basis for determining hemolytic reactions. Hemolytic patterns may vary with the source of animal blood or type of base medium used.⁵

User Quality Control

Identity Specifications
Blood Agar Base (Infusion Agar)
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       4.0% solution, soluble in purified water upon
                                   boiling. Solution is medium, yellow to tan, clear
                                   to slightly hazy. 
Prepared Appearance:    Plain – Medium, yellow to tan, clear to slightly hazy.
                                   With 5% sheep blood – Cherry red, opaque.
Reaction of 4.0%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response
Blood Agar Base (Infusion Agar)
Prepare the medium per label directions without (plain) and with 5% defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2°C for 18-24 hours (incubate streptococci with 3-5% CO2).

Formula

Blood Agar Base (Infusion Agar)
Approximate Formula* Per Liter
Heart Muscle, Infusion from (solids).............................. 2.0 g
Pancreatic Digest of Casein......................................... 13.0 g
Yeast Extract............................................................... 5.0 g
Sodium Chloride.......................................................... 5.0 g
Agar......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 40 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. For preparation of blood agar, cool the base to 45-50°C and aseptically add 5% sterile, defibrinated blood. Mix well.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. After streaking, stab the agar several times to deposit beta-hemolytic streptococci beneath the agar surface. Subsurface growth will display the most reliable hemolytic reactions owing to the activity of both oxygen-stable and oxygen-labile streptolysins.⁵
Since many pathogens require carbon dioxide on primary isolation, plates may be incubated in an atmosphere containing approximately 3-10% CO₂. Incubate plates at 35 ± 2°C for 18-24 hours.

Expected Results

Colonial morphology on blood agar containing 5% sheep blood is as follows:
1. Hemolytic streptococci may appear as translucent or opaque, grayish, small (1 mm), or large matte or mucoid (2-4 mm) colonies, encircled by a zone of hemolysis. Gram stains should be made and examined to check the macroscopic findings. (Other organisms which may cause hemolysis include Listeria, various corynebacteria, hemolytic staphylococci, Escherichia coli and Pseudomonas.) Approximate quantitation of the number of colonies of hemolytic streptococci may be helpful to the clinician.
2. Pneumococci usually appear as very flat, smooth, translucent, grayish and sometimes mucoid colonies surrounded by a narrow zone of “green” (alpha) hemolysis.
3. Staphylococci appear as opaque, white to gold-yellow colonies with or without zones of beta hemolysis.
4. Listeria may be distinguished by their rod shape in stains, and by motility at room temperature. Small zones of beta hemolysis are produced.
5. Other organisms representing minimal flora and clinically significant isolates can also be expected to grow on this nonselective formulation.

Limitations of the Procedure

Colonies of Haemophilus haemolyticus are beta-hemolytic on horse and rabbit blood agar and must be distinguished from colonies of beta-hemolytic streptococci using other criteria.⁶ The use of sheep blood has been suggested to obviate this problem since sheep blood is deficient in pyridine nucleotides and does not support growth of H. haemolyticus.⁵

*Store at 2-8°C.

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