Usage, Advantages and Caveats
The gentamicin protection assay is commonly used in pathogen research. The contribution of specific genes or proteins to the bacteria's ability to invade cells can be easily assayed using this method. The gene in question can be knocked out, and the bacteria's invasiveness compared with that of normal, wild type bacteria. Environmental conditions, such as pH level and temperature, can also be assayed for their effect on invasiveness.
The gentamicin protection assay is very sensitive, as it can detect the internalization of even single bacteria. It has several drawbacks:
- Gentamicin can sometimes penetrate eukaryotic cells and kill the internalized bacteria. This may happen if the permeability of the cells somehow increased during the assay, sometimes due to poor handling of the cells.
- Internalized bacteria may sometimes not be entirely protected from the outside environment, such as when the phagosome (the vacuole surrounding the bacterium inside the cell) is defective in some way. Gentamicin may kill those bacteria.
- Gentamicin may fail to kill all the bacteria that remained outside the cells.
To help assess the accuracy of a particular assay, positive and negative controls should be performed. When performing the assay as described above, bacteria that are known to be entirely invasive (positive control) and bacteria that are known as non-invasive (negative control) should be included in the assay.
An alternative invasion assay is the differential immunostaining assay, based on the binding of antibodies to bacteria before and after invasion. The antibodies emit fluorescent, colored light, and the results of this assay are viewed under the microscope.
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