The overall goal of this procedure is to perform capsular s stereotyping of streptococcus pneumoniae or the pneumococcus using the quelling reaction. This is accomplished by first inoculating heart infusion broth with a fresh, overnight pure culture of pneumococci. In the second step, a drop of inoculum is transferred to a glass microscope slide.
Next antis serum is added in equal volume onto the slide. In the final step, both drops are mixed and a cover slip is placed over the suspension. Ultimately, the suspension can be viewed under phase contrast to observe the positive or negative quell lung reaction.
This method demonstrates the gold standard method of pneumococcal syp. The method is simple, easy to perform, and easy to interpret. Visual demonstration of this method is important as individuals new to this technique may struggle to recognize what is a suitable cell density for performing the qualm test.
Begin by using a sterile disposable one microliter loop to obtain a small sweep of a fresh overnight pure culture of pneumococci grown on a solid, non-selective horse blood agar plate inoculate the pneumococci in 100 microliters of heart infusion broth. The suspension should appear to be just visibly turbid and gently agitate the tube to emulsify the cell solution. Use a new one microliter loop to transfer a drop of the inoculum onto a glass microscope.
Slide next, place a small circular cover slip over the suspension and check the density of the suspension under phase contrast at times 400 magnification. If the inoculum is too heavy, dilute the cell suspension with more broth and gently mix the cells again if the inoculum is too light. For example, if there are less than 50 cells visible, add more bacteria to the cell suspension and gently mix again.
With experience. The density of the cell suspension can be judged by shaking and holding the suspension up to the light where it should appear to be just visibly turbid. When viewed under the microscope, the cell solution should be about this dense.
Once the optimal bacterial cell dilution has been achieved, transfer about one microliter of the inoculum onto the glass microscope. Slide next. Using a new loop, add an equal volume of room temperature.
Antiserum onto the slide and to mix both drops well. With the loop, immediately place a small cover slip onto the suspension, taking care not to touch the drop with the forceps. Then view the suspension under phase contrast as just demonstrated.
A negative quell lung reaction is observed when little to no swelling of the pneumococcal cells is observed. Similar to the negative control, a pneumococcal cell suspension with no antisera added. A positive quell lung reaction occurs when type specific antibody binds to the capsule of the pneumococcus leading to a change in its refractive index.
When viewed under phase contrast, the bacteria appear swollen and more visible. Don't forget that when you're doing this procedure that you're actually working with live microorganisms. For that reason, you need to be careful of to follow good lab practices, wear protective clothing, the appropriate lab equipment, and dispose of all of the equipment and reagents at the end of the process safely.
Once mastered, this technique can be done in approximately 10 minutes if it is performed properly. After viewing this procedure, you should have a good idea of how to serotype the pneumococcus using the qua lung reaction. This technique is important for understanding pneumococcal epidemiology, both in nasopharyngeal carriage and an invasive disease.
