To infect salmonella cultures with bacteria phages dilute an overnight culture of salmonella enterica in a final volume of five milliliters of LB and add 0.1 milliliters of previously prepared bacteria phage lysate to it. Then incubate at 37 degrees Celsius and 200 RPM for 24 hours. In a 50-milliliter conical centrifuge tube, dilute the bacterial culture containing phages 100 times in five milliliters of 0.22 micron-filtered LB.Centrifuge the suspension for 10 minutes at 2, 377g.
Carefully decant the supernatant, leaving some volume at the bottom to ensure the presence of cells. Wash the bacterial cells with 10 milliliters of 0.22 micron-filtered LB medium. After repeating the centrifugation and washing three times, resuspend the pellet in five milliliters of the filtered LB.Employ a vacuum filtration system to filter 50 microliters of the obtained bacterial suspension through a sterile 0.45 micron filter.
Rinse the filter with 100 milliliters of the filtered LB.To facilitate phage release from bacterial cells, incubate the filter containing the cells without disassembling the system. Rinse the filter again, as demonstrated previously, using the vacuum filtration system. To recover the bacterial cells from the filter using sterile forceps, transfer the filter to a flask.
Then add two milliliters of 2x LB over the filter and pipette several times to release the cells from the filter. Centrifuge one milliliter of this suspension for two minutes at 10, 354g. After discarding the supernatant, wash the cells three times with one milliliter of filtered LB medium.
Then resuspend the cells in one milliliter of 2x LB.Next, mix commercial salmonella enterica lipopolysaccharide, or LPS, with 20 microliters of the bacterial suspension in one milliliter of filtered LB.Incubate the mix for two hours at 37 degrees Celsius with shaking at 200 RPM to prevent bacteria phage reinfection. After that, transfer one milliliter of culture to a microcentrifuge tube and centrifuge the suspension for two minutes at 10, 354g. Wash the pellet three times with one milliliter of filtered LB medium.
After washing, resuspend the pellet in one milliliter of 2x LB.Add 20 microliters of this mix to one milliliter of filtered LB, containing 0.8 milligrams per milliliter of commercial LPS. And incubate the mixture at 37 degrees Celsius and 200 RPM for two hours. Next, pellet one milliliter of culture in a microcentrifuge tube by centrifugation for two minutes at 10, 354g.
After washing the cells three times with filtered LB, resuspend them in one milliliter of filtered LB.Using a vacuum filtration system, pass one milliliter of bacterial suspension through a sterile 0.45 micron filter and rinse the filter using 100 milliliters of filtered LB.Transfer the filter to a flask using sterile forceps. Add two milliliters of 2x LB over the filter and pipette several times to release cells from the filter. Centrifuge one milliliter of cells for two minutes at 10, 354g before washing three times with filtered LB.Resuspend the collected cells in one milliliter of 2x LB.To prepare the phage-free inoculum, add 100 microliters of cells from the bacterial suspension to 900 microliters of LB containing 0.45 milligrams per milliliter LPS.
Incubate at 37 degrees Celsius for 24 hours with shaking at 200 RPM. Use this as the aliquot to confirm the absence of phage reinfection. By performing titrations at different stages of the protocol, it was observed that repeated washing and filtering were not sufficient to eliminate bacteria phages from cultures.
However, the number of phages decreased as soon as a step of incubation with commercial LPS was employed. The crucial step for the complete removal of bacteria phages in the bacterial culture was the second incubation with commercial LPS.
