This method provides a rapid, high-throughput detection of bacteria adherence to host cells. Compared to the conventional plating methods, it reduce hands-on time required for the quantification of the adherent bacteria. This technique is automated, time-saving and very reproducible.
It also a widely applied both to most of the host cells. This method, there is very straightforward. For the first trial, the conditions like the multiplicity of infection, and also the host bacteria co-incubation time must be optimized.
First harvest the bacterial cultures at an exponential phase by centrifugation at 13, 000 times G for two minutes at room temperature. After washing the cells pallet with one milliliter PBS, resuspend the bacterial palette in one milliliter PBS. Determine the concentrations of bacterial suspension by measuring optical density at 600 nanometers.
Then, for staining the bacterial suspension, add two microliters of the 500 fold concentrated stock green or red staining dye into one milliliter of bacterial suspension to dilute the dye one fold. Incubate the cells at room temperature for 30 minutes with gentle rotation in the dark. After 30 minutes, centrifuge the stained bacteria at 13, 000 times G for two minutes and resuspend the pellet in one milliliter of PBS.
Collect the stained bacterial cells by centrifugation at 13, 000 times G for two minutes. Resuspend the pellet in one milliliter of fresh F12 K medium and measure the optical density of each culture at 600 nanometers. Subsequentially dilute the cultures to the desired concentrations based on the multiplicity of infection and host cell concentration.
For Bacterial Adherence Assay, wash the previously cultured A549 cell mono layers three times by adding 100 microliters of warm PBS to each well and gently pipette up and down. Then discard PBS. Alternatively, after adding PBS, wait for 10 seconds and then remove PBS using vacuum.
To determine the kinetics of bacterial association, overlay the cells with 100 microliters of desired concentrations of bacterial suspension with different multiplicity of infection. Spin down the bacteria and incubate the infected A549 cells at 37 degrees Celsius and 5%carbon dioxide for an additional one hour. Remove the unbound bacteria by washing the mono layers five times with warm PBS as demonstrated.
Next, fix the cells by adding 100 microliters of 4%formaldehyde in all the wells of a 96 well plate on the ice. After 15 minutes, wash the plate three times with PBS to remove the fixation solution. Stain in the nuclei with 50 nanograms per milliliter DAPI stain for 10 minutes at room temperature.
After washing the cells with PBS, cover the infected A549 cells with 100 microliters of PBS to avoid drying and then store the plate at four degrees Celsius for up to two days in the dark or proceed for imaging. To maintain the data integrity, randomly and manually pick five locations of each well to capture the images at 20 times magnification. Capture the fluorescent images of bacteria under GFP channels, A549 cells under DAPI, and for the bacteria stained by the red fluorescent dye use PE Si five channel.
Set the parameters of the rolling ball for smoothing and auto measure the point spread function of image deconvolution based on the objective for achieving better resolution. Measure the fluorescence intensity of the host cells and bacteria. Set the weakest fluorescence intensity of host cells and bacteria as the thresholds for cell count and count all the bacteria proximate to a host cell within 15 micrometers distance as the adherence bacteria.
Once the cells have been counted from all the automated images, analyze critical readouts, such as host cell counts, cell sizes and shapes, total bacterial counts, and the average bacterial account per host cell, which is the most important indicator to determine bacterial adherence. After one hour of co incubation, pseudomonas aeruginosa strain PAO1 adhered to A549 cells in a dose dependent fashion. Similar results were also observed when gram-positive bacteria listeria monocytogenes and its negative control bacillus subtilis.
A representative image of host adherent P aeruginosa segmentation demonstrates the ease of counting bacterial cells using this protocol. The results of the automated analyses include bacterial and host cell counts, the average bacterial counts per host cell and host cell sizes and areas representing the status of host cell health, bacterial adherence level and bacterial cyto toxicity. Apart from A549 cells, HUVECs were also tested for adherence assay.
Serratia rubidaea and streptococcus agalactiae were adherent to HUVEC, but not to A549 cells, and which demonstrates the effective detection and suitability of multiple hosts to study the host bacteria interactions using this method. Host cells should reach around 90 to 100%confluence to minimize the undesirable bacteria adherence to the bottom of the wells. This technique will allowed researchers to explore the new mechanisms, the host-pest interactions.
