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Anaerobic Blood Agar
- Anaerobic Blood Agar is a solid media recommended for use in qualitative procedures for primary isolation and cultivation of anaerobic organisms, including fastidious strains.
- This medium was formulated by V.R. Dowell and T.M. Hawkins at the Centers for Disease Control and Prevention in Atlanta, Georgia.
- Anaerobic Blood Agar supports good growth and typical pigmentation of fastidious and slow-growing anaerobes, as well as other anaerobes of significant clinical importance.
Composition of Anaerobic Blood Agar
|Casein enzymic hydrolysate||15.000|
|Papaic digest of soybean meal||5.000|
Final pH: 7.4±0.2
Principle of Anaerobic Blood Agar
- Anaerobic Blood Agar base serves as a nutritious, nonselective medium allowing the cultivation of not only fastidious anaerobes but also of aerobic and microaerophilic microorganisms.
- It contains peptones which supply nitrogenous substances and amino acids necessary for the growth of anaerobic bacteria.
- Yeast extract provides B-complex vitamins and serves as a growth enhancer.
- Hemin, vitamin K, and sheep blood stimulate the growth of anaerobes like Bacteroides species and gram-positive spore bearers like Clostridium species.
- Sodium chloride is a source of essential electrolytes and maintains osmotic equilibrium.
- Addition of blood provides nutrients and helps to differentiate hemolytic organisms.
Preparation and Method of Use of Anaerobic Blood Agar
- Suspend 44.0 grams in 1000 ml distilled water.
- Heat to boiling to dissolve the medium completely.
- Add the rehydrated contents of 1 vial of Vitamin K1 solution.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- Cool to 45-50°C.
- Aseptically add 5% v/v sterile defibrinated sheep blood.
- Mix well and pour into sterile Petri plates.
- Prior to use, reduce the plates for a minimum of 24 hours by placing them in an anaerobic environment at room temperature.
- Inoculate specimens for anaerobic culture on both selective and non-selective media as soon as possible after receipt in the laboratory; streak plates for isolation.
- Incubate anaerobically at 33-37°C for 48-72 hours.
- Confirm anaerobic growth by subculture to anaerobic blood agar plate.
Result Interpretation of Anaerobic Blood Agar
|Bacteroides fragilis||Luxuriant Growth|
|Bacteroides melaninogenicus||Luxuriant Growth|
|Peptostreptococcus anaerobius||Luxuriant Growth|
|Clostridium perfringens||Growth, double zone β-hemolysis|
|Prevotella melaninogenica||Growth; pigment (tan to brown)|
Uses of Anaerobic Blood Agar
- Anaerobic Blood Agar Base is recommended for the cultivation of anaerobic microorganisms, including very fastidious organisms from clinical specimens.
- It promotes both typical pigment formation in Bacteroides melaningenicus and displays double hemolytic reaction in Clostridium perfringens with added blood to the medium base.
- The media supports typical pigment production by pigmented Prevotella and Porphyromonas
Limitations of Anaerobic Blood Agar
- The media must be prepared, dispensed, and packaged under oxygen-free conditions to prevent the formation of oxidized products prior to use.
- Anaerobic Blood Agar will not provide complete information for identification of bacterial isolates. Additional test procedures and media are required for complete identification.
- It is recommended that a selective media such as Anaerobic Brucella Laked Blood Agar with Kanamycin and Vancomycin and/or Anaerobic Brucella Blood Agar with Phenylethyl Alcohol also be inoculated with clinical specimens to assure growth of all species present.
- Dowell, V.R., Jr. and T.M. Hawkins. 1974. Laboratory Methods in Anaerobic Bacteriology, CDC Laboratory Manual. U.S. Dept. of H.H.S. and CDC, Atlanta, GA.