Determination of hemolysis type is a key classification system in medical diagnostics and microbiology, and is especially useful in classifying streptococcal species. Serious infection diseases such as meningitis, bacterial pneumonia, endocarditis are caused by streptococci.
Blood agar plates composed of 5-10% defibrinated animal blood (for example, bovine, sheep) are widely used to detect hemolytic activities. Streptococcal colonies which grow on blood agar plates have the ability to disrupt red blood cells either partially, completely or not at all. The hemolytic pattern on blood agar plates can help differentiate such specific abilities (Figure 1).
Figure 1. Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= non-hemolytic, S. salivarius)
A greenish halo around the colony represents the alpha hemolysis. Partial hemolysis of the red blood cells generates the halo. The reason behind this is the production of hydrogen peroxide by the bacterium, which causes oxidation of erythrocyte hemoglobin to green methemoglobin.
A clear halo around the colony represents beta hemolysis, which is the result of complete hemolysis of red blood cells. The bacterium secretes an enzyme called endotoxin Streptolysin, which completely lyses erythrocytes. Streptolysin’s interaction with cholesterol in the membrane of eukaryotic cells eventually leads to the breakup of the cells. Gamma hemolysis is shown in Figure 1 as no hemolysis or discoloration of the blood.
High quality blood agar plates are required to reliably determine hemolysis type. They are typically light red in color (Figure 2), which is a characteristic feature of intact erythrocytes. The brown color of agar plates consisting of lysed blood cells causes difficulty in reading the hemolysis pattern.
The temperature of the agar must be kept low to prevent spontaneous lysis of erythrocytes. Besides minimizing cell lysis, the time the blood is retained in the hot agar must be as short as possible. For most laboratories, satisfying these requirements is a challenging task.
Figure 2. Brownish color of an agar plate if erythrocytes are lysed (left). Typical light red color of high quality blood agar plates (right).
Success Factors for High-Quality Blood Agar
Using the INTEGRA media preparation system
This section of the article discusses an automated protocol that shortens the exposure time of blood to hot agar medium, thereby efficiently reducing thermally induced lysis of erythrocytes.
The dispensing temperature can be fixed and maintained at low temperature by using an INTEGRA MEDIACLAVE media preparator for blood agar preparation. Moreover, red blood cell lysis can be significantly reduced by adding the blood to the agar just before pouring the plate, using the combination of the MEDIAJET Petri Dish filler, a DOSE-IT peristaltic pump, and special silicone blood agar tubing.
Figure 3. MEDIAJET Petri dish filler in combination with the peristaltic pump DOSE IT and the blood agar tubing set.
The Right Dispensing Temperature of the Agar Medium
The temperature of the agar needs to be appropriate during dispensing to achieve a reduction in thermal stress to erythrocytes. Lysis of red blood cells will occur if the temperature is too high, causing the lysis of erythrocytes which can be observed from the color change of agar from light red to brown.
In contrast, the mixing performance with the blood deteriorates when the temperature is very low, causing inhomogeneous blood distribution on the plates. This can be observed from the formation of streaks on the plates. These streaks, generally, do not affect the bacterial growth but complicate the reading of hemolysis pattern. Hence, the dispensing temperature must be set low, ideally between 45 to 47 °C.
Blood at Room Temperature
For homogeneous mixing with the agar, it is vital that the blood has to be maintained at room temperature. Sterile blood is generally stored at low temperatures between 4 to 8 °C. The refrigerated blood needs to be tempered to room temperature before it is added to the agar, since cold blood below room temperature can result in partial gelling of the agar inside the silicone tubing, and the subsequent generation of streaks on the plates.
Precise Synchronization of MEDIAJET and DOSE IT
The DOSE IT peristaltic pump and MEDIAJET can be precisely synchronized if the flow rate of the DOSE IT is appropriate. Accurate synchronization is important to maintain the desired mixing performance and to avoid the blood volume variation between the dispensing steps. The flow rate of the peristaltic pump must be set to a point, where the dispensing time of the DOSE IT is equal or slightly shorter than that of the MEDIAJET.
Agitation of Blood During Dispensing
Erythrocytes sink below the plasma due to their high specific density, when the blood is allowed to stand for a longer period without agitation. It is enough to shake the small blood containers (capacity of 0.5 L) manually immediately before starting the automated addition to the agar. Agitation might be required if larger blood containers are used in order to prevent sinking of the red blood cells.
Tested Instrument Settings
The following instrument settings for blood agar plate production were widely tested by INTEGRA Biosciences and blood agar producers.
MEDIACLAVE 10/30 Settings
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Sterilization temperature
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121 °C
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Sterilization time
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15 minutes
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Stirrer speed at sterilization
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150 rpm
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Dispensing temperature
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47 °C
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Stirrer speed at sterilization
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100 rpm
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MEDIAJET Settings
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Dosing volume
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16 ml
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Pump speed
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60%
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Pause time
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0.0 s
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Rotor movement
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spread
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DOSE IT Settings
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Tubing ID
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2.0 mm
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Volume
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0.8 ml
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Flow rate
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25.2 ml/minute
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Conclusion
The production of high quality blood agar is facilitated by the INTEGRA Biosciences media preparation system. The agar medium can be cooled down to the optimal dispensing temperature using the media sterilizer MEDIACLAVE, reducing blood cell lysis.
Moreover, the exposure time of red blood cells to high temperature is reduced using the combination of the MEDIAJET, DOSE IT, and a special blood agar tubing set. High quality light red blood agar plates can be achieved across the whole batch if:
- The agar medium’s dispensing temperature is set as low as possible
- The blood is tempered to room temperature
- Precise synchronization of the DOSE IT pump with the MEDIAJET filler
- Large blood containers are agitated during dispensing
About INTEGRA
INTEGRA provides innovative solutions for Liquid Handling and Media Preparation applications which serve the needs of their customers in research, diagnostics and quality control laboratories.
Their instruments and plastic consumables are developed and manufactured in Zizers, Switzerland and Hudson, NH USA. In order to remain close to their customers, they maintain a direct sales and support organization in North America, the UK, France and Germany, as well as a network of over 100 highly trained distribution partners worldwide.
In recent years they have focused on developing a new and technologically advanced range of handheld electronic pipettes which are simple to use and meet the ergonomic needs of their customers.
Today they are proud to offer the widest range of electronic pipettes in the market spanning a range from single channel pipettes up to 384 channel bench-top instruments.
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