The overall goal of this procedure is to examine the ability of a probiotic streptococcus cell levarius to inhibit pneumococcal adherence to an epithelial cell line. This is accomplished by first preparing CCL 23 epithelial cell monolayers. In a 24 well plate next slovar and pneumococci are added to the monolayer in sequence and the cells are incubated.
In the final step, the wells are washed to remove the non-adherent bacteria. The CCL 23 epithelial cells are lysed and the well contents are harvested. Ultimately, the impact of salivarius on pneumococcal adherence can be evaluated by quantifying the number of cell associated pneumococci.
This method helps answer key questions in the infectious diseases field, such as whether probiotics can inhibit the adherence of bacterial pathogens such as the pneumococcus. We first had the idea for this method when we began investigating the potential use of lactobacillus hypnosis, GG, and other probiotics in the respiratory tract. Demonstrating this procedure will be Jane Manning and Zen to PhD students from our laboratory On the day before the experiment split to culture of confluence, CCL 23 epithelial cells, and then dilute them in Prewarm media to a 150, 000 cells per milliliter concentration in a sterile 50 milliliter tube.
Next seed, one milliliter of the cells per well in a treated 24 well tissue culture plate and incubate the cells overnight. Then using a 10 microliter loop inoculate, 10 to 12 well separated colonies of each bacterial culture into a 10 milliliter aliquot of Todd Hewitt broth plus 0.5%yeast extract. Briefly vortex and incubate the cultures for 14 to 16 hours on the day of the experiment.
Vortex each of the bacterial cultures for three to five seconds, then add 100 microliters of pneumococci and 100 microliters of salivarius into labeled prewarm tubes, containing 10 milliliters of broth. Vortex the inoculated tubes for three to five seconds and incubate at 37 degrees Celsius to grow the cultures to mid log phase. After confirming at least 80%co fluency of the CCL 23 cell culture, use the transfer pipette to remove the media from each well of the 24 well plate tilting the plate slightly to avoid disturbing the cell monolayer then wash wells twice by adding approximately 500 microliters of pre prewarm PBS and gently removing the liquid with a transfer pipette.
Tilt the plate during the washes to help preserve the monolayer after the second wash. Add 500 microliters of prewarm media to each well and return the plate to the incubator while preparing the bacterial cultures. First, measure the optical density of one milliliter of the levarius culture at absorbent 600.
Then transfer five milliliters of the Sal Levarius culture into a 10 milliliter tube and centrifuge to tube for four minutes at 1, 870 Gs at room temperature. After discarding the snat Resus, suspend the pellet in 200 to 1000 microliters of point 85%sodium chloride depending on the OD reading to a final concentration of approximately 1.5 times 10 to the ninth colony forming units per milliliter. Adding the correct number of bacteria is the trickiest part of the experiment as the actual number of bacteria added is not determined until colony counting is performed the following day.
Now transfer the resuspended salivary inoculum to a micro fuge tube labeled high. Dilute the high concentration at one to 10 and one to 100 in more point 85%sodium chloride to make medium and low concentrations of alvar for the assay vortex tubes in between dilutions to create the heparin control wells. Remove 40 microliters of media from each control well and add 50 microliters of heparin to a final concentration of 100 units per milliliter per well.
Add 10 microliters of point, 85%sodium chloride to the CCL 23 cells only, and the CCL 23 cells plus pneumococci only wells completely vortex all SIV areas concentrations. Then add 10 microliters of undiluted one to 10 and one to 100 siv various concentrations to the appropriate wells to promote bacterial contact with the cell. Monolayer centrifuge the plate for three minutes at 114 Gs at room temperature and then incubate the plate for an hour while the cells are incubating serially dilute the salivary stock in point 85%sodium chloride and use a sterile spreader to disperse 100 microliters of the 10 to the minus five, 10 to the minus six and 10 to the minus seven dilutions in duplicate on horse blood agar plates.
Culture the plates in an inverted position overnight towards the end of the monolayer as salvers incubation. Measure the optical density of the pneumococcal culture and then spin down one milliliter of the cells. Resus suspend the pellet in 300 to 2000 microliters of point 85%sodium chloride depending on the OD reading to a final concentration of approximately 150 million colony forming units per milliliter.
Then add 10 microliters of undiluted pneumococci to all the wells except the negative control wells centrifuge the 24 well plate again and then incubate the co cultures for three hours during the incubation step. Serially dilute and plate the pneumococcal stock in a similar manner to salivarius. After three hours of incubation, check the cells under the microscope for any cytopathic effect.
Now remove the media from each well and gently wash the co cultures three times with one XPBS to remove the non-adherent bacteria, taking care not to disturb the monolayers. Then lys the cells with 200 microliters of 0.1%Dig toin for seven minutes at 37 degrees Celsius and 5%carbon dioxide. Next, add 800 microliters of THB to each.
Well pipette up and down a few times and harvest the cell suspensions from each well into labeled micro centrifuge tubes. Finally, serially dilute the adherent pneumococci and then count the colonies the following day on the appropriate dilution plates. To quantify the pneumococci and alvar in this table, the results from a representative experiment in which the pneumococci were added to CCL 23 cells one hour after the addition of slovar are shown.
Pneumococcal adherence was quantified by both viable count and lit. A-Q-P-C-R results were consistent between the two methods for both the absolute number of bacteria and the percent adherence. Normalized to the number of adherent pneumococci in the wells containing pneumococci alone.
Heparin is used as a control as it is known to inhibit pneumococcal adherence. Therefore, the reduction of the adherence by approximately 40%or more in the pneumococci plus heparin samples compared to pneumococcal alone is indicative of a successful assay. As salivary inhibits pneumococcal adherence in a dose dependent manner where the high concentration of salivarius of approximately 10 salivarius to one pneumococcus resulted in the lowest pneumococcal adherence.
These graft demonstrate the adherence of pneumococci to the CCL 23 cells when salivarius is added, one hour before the pneumococci concurrently, or one hour after the pneumococci. Alvar is more effective at inhibiting pneumococcal adherence when added prior to the pneumococci, as both the high and medium doses of Alvar significantly reduced pneumococcal adherence in the pre edition assay. Whereas the Coit edition and post edition assays only the high doses of Slovar inhibited pneumococcal adherence.
So after watching this video, you should have a good understanding of how to perform an in vitro assay to measure the inhibition of pneumococcal adherence by the probiotic streptococcus cell areas.
