The overall goal of this procedure is to quantify adhesion of bacteria onto cultured mammalian cells. This is accomplished by first incubating bacteria with cultured cells for a period of time. Next, the unbound bacteria are washed away.
Then the cells are lysed to recover the adhered bacteria. Finally, the recovered bacteria are plated on Petri dishes. Ultimately, results can be obtained that show the number of bacteria adhered to mammalian cells through counting of the number of colony forming units on the Petri dishes.
This metal can help answer key questions in the bacteria of pathogens this field, such as mechanism of ion and invasion of host cells, giving new insights on the variance of bacteria, pathogens, Generally individuals new tooth dis method with struggle because of lack of consistency in the washing and recovery step of the procedure. To prepare the bacteria used for this study, freshly plate bacteria from glycerol stocks such as esia coli, pathogenic strains as shown here on misogyny, broth, grates and incubate at 37 degrees Celsius. Keep the plates sealed at four degrees Celsius for no longer than two weeks.
The mammalian hep two cells used in the assay are manipulated under a laminar flow hood and maintained in high glucose DMEM with 10%heat inactivated bovine serum, 10 units per milliliter, penicillin, and 10 micrograms per milliliter. Streptomycin at 37 degrees Celsius with 5%carbon dioxide. The cells are ready for an assay typically, once they have almost reached confluence, wash them once with warm dcos phosphate buffered saline to detach the cells.
Incubate them with 0.05%tripsin EDTA for five minutes. Then add fresh, warm, complete medium centrifuge the cell suspension at 2000 RRP M for five minutes. Then resuspend the cell pellets in DPPS and spin them down again.
Resuspend the cells in fresh medium, supplemented with 10%serum without antibiotics at a concentration of two times 10 to the fifth cell per milliliter seed, one milliliter of cell suspension for each cell strain in duplicate wells in the center of a 24 well plate incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide on the same day. Isolate one colony from each bacterial strain and inoculate them into five milliliters of LB broth. Grow overnight at 37 degrees Celsius with vigorous shaking before infecting cells.
Check under an inverted microscope that they're at least 90%confluent and free from contamination. Wash the cells with warm DPBS. Then add one milliliter of fresh medium supplemented with 10%serum.
In addition, add one milliliter of medium to three empty wells. They will be used to determine the total number of bacteria in each inoculum. Next, measure the optical density of diluted bacterial cultures at 600 nanometers.
Then add an aliquot of each strain corresponding to 10 to the sixth colony forming units to one duplicate set of wells containing cells and one well with medium only. Incubate the cells for up to three hours at 37 degrees Celsius with 5%carbon dioxide. Remove the medium from the infected cells using a micro pipette and carefully wash them three times.
With warm DPBS. Check the cells under the microscope for the presence of adherent bacteria to lyce the cells and detach the adhered bacteria. Add 100 microliters of 1%Triton X 100 to each well containing the cells.
Incubate for 10 minutes at room temperature. Then add 900 microliters of LB medium gently homogenize the cell suspensions by pipetting up and down. Prepare tenfold serial dilution of the suspensions of adhered bacteria and inoculum using LB broth and plate 100 microliters from three on LB agar incubate overnight at 37 degrees Celsius the next day.
Count the colonies on the plates and calculate the number of CFUs of adhered bacteria and of the inoculum. By averaging each series of dilution only plates with between 10 to 300 colonies should be counted. This figure represents a game sustaining of bacteria from e coli.
Strain 2 7 87 adhered to hep two cells as viewed under a standard phase contrast microscope. Typical results of CAU counting of adhered bacteria and inoculum from three experiments performed on different days are shown in this table. As can be seen, there are important variations between duplicates and between experiments.
The results can be reported directly as adhered CFU as shown in this graph as can be seen, the pathogenic strain of e coli 2 7 87 does adhere to the hep two cells and deletion of a single gene. Eight A reduces the amount of adhered bacteria more than tenfold. This difference suggests that the level of adhesion of 2 7 87 is significant.
Furthermore, there are tenfold less colony forming units adhered when using a non-pathogenic strain of e Coli C 600 in the same conditions. Thus, we conclude that the pathogenic strain does significantly adhere to epithelial cells and that the adhesion eight A is primarily responsible for this phenotype. Since the inoculum sizes can vary between strains because of differences in growth rates or pipetting errors, it is often advisable to report the results as percentage of adhered bacteria.
The percentage of a adhered bacteria can be calculated by dividing the number of CFUs of adhered bacteria by the number of CFU of the inoculum. As shown here, While attending this procedure, it is important to be consistent in the washing and recovery steps, as well as in the choice of the parameters of infection such as the time or the multiplicity of infection.
