This fluorescence microscopy experiment assesses the viability of individual bacteria associated with host cells and determines bacterial viability in different subcellular locations. First, to identify external bacteria, expose the infected cells to a fluorescent reagent that is specific to the bacteria permease, the infected host in the presence of fluorescent dyes that discriminate viable from non-viable bacteria based on bacterial membrane integrity. Then to indicate where bacteria are found inside host cells incubate the infected cells with a fluorescently coupled antibody to a marker of interest.
Resulting immunofluorescent images can determine the percent of viable bacteria inside and external to host cells and the subcellular localization of viable versus non-viable intracellular bacteria. Unlike colony count assays, gentamycin protection assays, and electron microscopy, this method allows direct assessment of the viability of individual bacteria. It also can reveal if bacteria in different subcellular compartments have differences in their viability.
Demonstrating this procedure will be Brittany Johnson, a graduate student from my laboratory, Atory to cells cultured on circular glass cover slips in 24, well plates add bacteria of interest and incubate for the desired time. Rinse the infected cells once gently. Then add Alexa Fluor 6 4 7 coupled antibody, or a bacterial specific lectin to detect external bacteria and incubate for 10 minutes in the dark at room temperature.
After two rinses, aspirate the media and add 0.5 milliliters of live dead staining solution containing cyto nine and propidium iodine. Incubate the cells for 15 minutes at room temperature in the dark, then rinse twice in mops magnesium chloride. Invert the cover slips face down onto glass slides.
Seal with clear nail polish. Acquire the images within 30 minutes using a fluorescent microscope with filter sets detailed in the text protocol to label the bacteria. Add 10 micrograms per milliliter of DPI in mors defined medium and incubate for 20 minutes at room temperature in the dark.
Now infect the adherent cells with DPI labeled bacteria. Do not fix the cells with aldehydes or organic solvents. Rinse the cells once.
Then add Alexa Fluor 6 4, 7 coupled antibody or lectin and incubate for 10 minutes at room temperature in the dark. After two rinses with mops buffer, add an Alexa Fluor 5 5, 5 coupled antibody against the subcellular marker of interest and incubate for 20 minutes After two, RINs with mops buffer, wash the cells once with mops magnesium chloride. Then aspirate the media and add 0.4 micromolar cyt green incubate cells for five minutes at room temperature in the dark, rin the cells twice in mops, magnesium chloride.
Then wash cells. One more time in mops magnesium chloride for five minutes. Invert the cover slips face down onto glass slides seal with clear nail polish, acquire images of the slides within 30 minutes on the fluorescence microscope.
In this experiment, human neutrophils were infected with gonorrhea. The Alexa Fluor 6 4 7 coupled soybean lectin detects extracellular gonorrhea. Then the green fluorescent viability die, and the red fluorescent propidium iodide were added in the presence of saponin, which sequesters cholesterol to preferentially perme the host cell plasma membranes, but does not permeate the gonorrhea membrane in infected cells, the eukaryotic cell nucleus stains with both cyto nine and propidium iodide.
These images of gonorrhea infected neutrophils were generated using the viability dyes cyt talk screen and dpi. The propidium iodide dye was not used as it fluoresces in both red and ultraviolet channels on the fluorescence microscope. All gonorrhea stain with dpi, but only bacteria with compromised membranes stain with ox green.
The nonviable intracellular bacteria has a ring of staining for CD 63 surrounding the bacteria. This ring indicates primary granules are enriched at this phagosome. The viable intracellular bacterium lacks staining for CD 63 indicating primary granules are not enriched at its phagosome.Taken.
Together, the data shows a correlation between the viability of gonorrhea and residents in a primary granule negative phagosome. After watching this video, you should have a good understanding of how to evaluate the viability of bacteria in host cells. Additionally, you'll be able to determine the viability of bacteria localized to different compartments in host cells by using antibodies directed against these subcellular compartments.
