Introduction
Blood agar is actually a couple of related media, all of which are rich formulations containing peptones, yeast extracts, liver or heart extracts (depending on the medium), and blood. The blood is usually sheep’s red blood cells (RBC), though horse and other species may be used. Blood agar is used to grow fastidious organisms (species that do not grow easily) requiring a rich media providing many nutrients and growth factors that are largely supplied by blood. It is also a differential media in allowing the detection of hemolysis (destroying the RBC) by cytolytic toxins secreted by some bacteria, such as certain strains of Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Aerococcus.
In blood agar process, blood agar plates,are routinely used, they allow for the growth of fastidious organisms and the differentiation of cells according to three hemolytic activities:
- A clear zone around bacterial growth -RBC hemolyzed completely (Beta-hemolysis and pathogenic).
- A greenish zone around growth -RBC partially hemolyzed (Alpha-hemolysis).
- No change around growth -blood is not hemolyzed (Gamma-hemolysis or no hemolysis).
Purpose
“Blood Agar” is not a consistently defined medium. The term “blood agar”
generally refers to an enriched base medium to which defibrinated
mammalian blood has been added.It is often used to grow
fastidious organisms and to differentiate bacteria based on their
hemolytic properties.
Principle Of Blood Agar
Haemolysins are exotoxins produced by bacteria that lyse red blood cells. The haemolytic reaction can be visualized on blood agar plates observing through the bright transmitted light. On blood agar plates colonies of haemolytic bacteria may be surrounded by clear, colorless zone where the red blood cells have been lysed and the haemoglobin destroyed to a colorless compound and which is beta haemolysis. Other types of bacteria can reduce haemoglobin to methaemoglobin which produces a greenish zone around the colonies and is called alpha haemolysis . Gamma haemolysis is lacking haemolysis where no change in the medium is observed. Sheep blood agar base with added sheep blood was developed to allow maximum recovery of organisms without interfering with their haemolytic reactions. Sheep blood agar base was formulated to be compatible with sheep blood and give improved haemolytic reactions of organisms. Casein enzymic hydrolysate and yeast extract provide nitrogen, carbon, amino acids and vitamins. Peptic digest of animal tissue (PDAT) is the nitrogen source. Sodium chloride (NaCl) maintains the osmotic balance. Sheep blood agar base showed considerable improvement and the expected beta haemolytic reactions with S. pyogenes in comparison to other blood agar bases supplemented with blood.
Blood Agar And Hemolysis
Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify certain microorganisms. This is particularly useful in classifying streptococcal species. A substance that causes hemolysis is a hemolysin. Three different types of hemolysis are observed on blood agar which can each be identified by a zone of hemolysis present around the growing colonies.To read the hemolytic reaction on a blood agar plate, the plate must be held up to a light source and observed with the light coming from
behind (transmitted light).
Alpha
When alpha hemolysis (α-hemolysis) is present, the agar under the colony is dark and greenish. Streptococcus pneumoniae and a group of oral streptococci (Streptococcus viridans or viridans streptococci) display alpha hemolysis. This is sometimes called green hemolysis because of the color change in the agar. Alpha hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin producing the green oxidized derivative methemoglobin.
Beta
Beta hemolysis (β-hemolysis), sometimes called complete hemolysis, is a complete lysis of red cells in the media around and under the colonies: the area appears lightened (yellow) and transparent. Streptolysin, an exotoxin, is the enzyme produced by the bacteria which causes the complete lysis of red blood cells.
Beta hemolysis will never include the brown or green discoloration of the cells in the surrounding medium. On prolonged incubation, many alpha hemolytic organisms will begin to appear more
clear, but if the surrounding medium contains any shades of brown or green the “hemolysis” is still considered “alpha.”
Gamma
If an organism does not induce hemolysis, the agar under and around the colony is unchanged, and the organism is called non-hemolytic or said to display gamma hemolysis (γ-hemolysis).
Preparation Of Blood Agar
Composition Of Blood Agar
| COMPONENTS | QUANTITY (in grams) |
|---|---|
| Pancreatic digest of casein | 17.0 |
| Papaic digest of soybean meal | 3.0 |
| Dextrose (Glucose) | 2.5 |
| Dipotassium hydrogen phosphate | 2.5 |
| Sodium Chloride | 5.0 |
| Agar-Agar | 15.0 |
Procedure
- Suspend 40.5 grams in 1000 ml distilled water or deionized water.
- Heat to boiling to dissolve the medium completely.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- Cool to 45-50°C and aseptically add 7% sterile sheep blood.
- Mix well and pour into sterile Petri plates. Avoid formation of air bubbles. You must have warmed the blood to room temperature at the time of dispensing to molten agar base.
- Dispense 15 ml amounts to sterile Petri plates aseptically
- Label the medium with the date of preparation and give it a batch number (if necessary).
Quality Control of Blood Agar
- The pH of the blood agar range from 7.2 to 7.6 at room temperature.
- Inoculate the plates with 5-hour broth cultures of Streptococcus pyogenes and S. pneumoniae. Inoculate also a plate with H. influenzae and streak with S. aureus (i.e. Satellitism Test).
- Incubate the plates in a carbon dioxide enriched atmosphere at 35-37°C overnight.
- Check for the growth characteristics of each species. (a)
Storage and Shelf Life
Store below 30°C in tightly closed container and the prepared medium at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture. The shelf life of thus prepared blood agar is up to four weeks. Use before expiry date on the label.
Result Interpretation on Blood Agar
| Organism | Growth | Colony Morphology | Hemolysis |
| Neisseria meningiditis | Good-luxuriant | Grey and unpigmented colonies that appear round, smooth, moist, glistening, and convex, with a clearly defined edge. | Non-hemolytic or γ-hemolytic. |
| Salmonella Typhi | Good-luxuriant | Smooth colorless colonies that are smooth, moist, and flat with a diameter range of 2-4 mm. | Non-hemolytic or γ-hemolytic. |
| Staphylococcus aureus | Luxuriant | Golden yellow colored circular, convex and smooth colonies of the diameter range of 2-4 mm; opaque colonies with a zone of hemolysis. | β-hemolytic. |
| Staphylococcus epidermidis | Luxuriant | Circular, colonies of the size 1-4 mm in diameter; grey to white-colored with low convex elevation; moist, glistening colonies. | Non-hemolytic or γ-hemolytic. |
| Streptococcus pyogenes | Luxuriant | White-greyish-colored colonies with a diameter of > 0.5 mm; the colonies are surrounded by a zone of β-hemolysis that is often two to four times as large as the colony diameter. | β-hemolytic. |
| Streptococcus pneumonia | Luxuriant | small, grey, moist (sometimes mucoidal in encapsulated virulent strains), colonies with the characteristic zone of alpha-hemolysis (green); due to autolysis, often produces a dimple-like zone of hemolysis than the typical crater-like appearance. | α-hemolytic. |
| Pseudomonas aeruginosa | Good-luxuriant | Large colonies of the size 2-5mm in diameter; flat colonies that are grey to white-colored with an undulate margin with a zone of β-hemolysis. | β-hemolytic. |
Uses Of Blood Agar
- Determine the type of hemolysis, whether alpha, beta or gamma.
- For culture of streptococci as well as antimicrobial susceptibility testing (AST).
- Use of optochin disc for presumptive identification of Streptococcus pneumoniae.
- Similarly use of bacitracin disc (0.04U) for presumptive identification of Streptococcus pyogenes.
- To perform CAMP test for Streptococcus agalactiae.
- To perform satellitism test for Haemophilus.
- It is also used for the isolation and cultivation of other than streptococci like Neisseria and other fastidious microorganisms.
- It is also useful for the preparation of Salmonella Typhi antigens.
Further References
- Difco Laboratories. 1998. Difco Manual, 11th ed. Difco Laboratories, Sparks, MD, USA.
- Harley J.P. and Prescott L.M. 2002. Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.
- Pickett M.J. 1989. Methods for identification of flavobacteria. J. Clin. microbiol. 27:2309.
- Ruoff, K.L. 1995. Streptococcus, p. 299. In P.R. Murray, E.J. Baron, M.A Pfaller, F.C. Tenover, and R.H. Yoken (eds.). Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
- U.S. Food and Drug Administration, 1995, Bacteriological Analytical Manual, 8th Ed., AOAC International, Gaithersburg, Md.
- Muir, Robert and James Ritchie. 1903. Manual of Bacteriology. The MacMillan Company, London. (p. 226-229)
- Bulloch, William. 1938. The History of Bacteriology. Oxford University Press, London. (p. 42).