page_title: Gentamicin protection assay
section_title: Procedure
hierarchical_section_title: Gentamicin protection assay / Procedure
context_page_description: The gentamicin protection assay or survival assay or invasion assay is a method used in microbiology. It is used to quantify the ability of pathogenic bacteria to invade eukaryotic cells.
  The assay is based on several observations made in the 1970s, in which the ability of internalized bacteria to avoid killing by antibiotics was reported. The assay started to be used in biological research in the early 1980s.
context_section_description: HeLa cells are commonly used as eukaryotic cells in the gentamicin protection assay, but other cells can be used as well. As for bacteria, only species susceptible to gentamicin can be assayed.
  The assay is performed in plastic microtiter plates, which are commonly used in laboratories for culturing eukaryotic cells. The cells are allowed to grow in the wells overnight, creating a flat layer. Bacteria are separately grown overnight. On the next day the eukaryotic cells are inoculated with the bacteria and are incubated together for an hour. Centrifuging the plates for a few minutes may help bring cells and bacteria in contact and initiate infection.
  After infection gentamicin is added to the plates, and they are incubated for an hour, allowing the antibiotic to kill all bacteria that were not able to penetrate the cells and remained outside. The plates are then washed well to remove the dead bacteria. Next the eukaryotic cells are lysed using a detergent, most commonly Triton X-100.
  The bacteria that penetrated the cells and remained alive are now released, and they are plated on solid medium plates. Counting the colonies formed on the plates on the next day, and knowing how many bacteria were used in the beginning of the assay, enables the researcher to calculate the percentage of bacteria that were able to invade the eukaryotic cells.
caption: The chemical structure of gentamicin