Our research aims to understand the tripartite interactions between bacteriophages, bacteria, and the mammalian immune system within the context of the gut, gnotobiotic mouse gut. We hope to gain insight into these interactions under health and disease conditions to better understand the importance of the gut microbiome. Recent developments have found that bacteriophages can be detected by the mammalian immune system.
It has been suggested that the human gut phageome may be dysregulated in disease conditions such as inflammatory bowel disorder and immune stimulation by phages could exacerbate disease phenotypes. Our goal is to provide others with a standardized protocol for producing a T4 phage lysate for inoculation and enumeration in gnotobiotic mouse models. These protocols can be built upon and modified for use with other bacteriophage bacteria pairs To begin, culture Escherichia coli in 5 milliliters of Luria-Bertani or LB medium in a sterile polystyrene or glass culture tube.
Incubate the tube overnight at 37 degrees Celsius with shaking at 200 RPM. Subculture the overnight grown culture 1-50 in 100 milliliters LB in a conical flask. Incubate the culture at 37 degrees Celsius and 200 RPM for approximately 1.5 hours.
Next, add 100 microliters of the high titer T4 phage stock into the E.coli culture and incubate at 37 degrees Celsius with shaking for three hours. Once the T4 phage lysate is clear, collect them in a 50-milliliter conical tube. Centrifuge the lysate set 4, 000 G for 20 minutes to pellet any remaining bacteria and cellular debris.
Filter sterilize the resulting supernatant using a 0.22-micron nylon filter and collect the filtrate to a new 50 milliliter tube. Add 0.1 volume of chloroform to the filtered T4 phage lysate to kill the remaining bacteria and prevent bacterial growth. Vortex briefly and incubate the lysate at room temperature for 10 minutes.
Centrifuge the lysate at 4, 000 G for 5 minutes to separate the chloroform from the lysate. Using a serological pipette, carefully transfer the top lysate layer into a new 50-milliliter tube. Add 13 milliliters of phage lysate to the upper reservoir of 100 kilodalton centrifugal filter device.
Concentrate the lysate at 4, 000 G for 5 minutes or until most of the lysate has passed through the filter. Using a P200 or P1000 pipette, gently pipette the remaining 2 milliliters of lysate up and down within the upper reservoir to unclog the filter membrane. Discard the filtrate from the lower reservoir into a waste container.
Repeat concentration and retain approximately 2 milliliters of concentrated phage in the upper reservoir. After the last spin, gently pipette the remaining lysate up and down in the upper reservoir. To wash the phage lysate, add 12 milliliters of saline magnesium or SM buffer to the upper reservoir and centrifuge at 4, 000 G for 10 minutes.
After the second wash, resuspend the remaining 2 milliliters of lysate in the SM buffer to a final volume of 10 milliliters. Transfer the lysate to a 50-milliliter tube and store it at 4 degrees Celsius. To remove contaminating endotoxins, add 0.4 volumes of 1-octanol to the total volume of lysate.
Seal the lid of the tube with sealing film and shake at 120 RPM for 1 hour, followed by incubation at 4 degrees Celsius for 1.5 hours without shaking. Centrifuge to separate the endotoxin-cleared lysate from the 1-octanol. Carefully remove as much 1-octanol as possible using a P1000 pipette and discard it into the appropriate waste container.
Using an 18-gauge needle and 10-milliliter syringe, collect the phage lysate beneath the remaining 1-octanol layer. Transfer 1 milliliter aliquots of T4 phage lysate into sterile 1.5 milliliter microcentrifuge tubes. Speed vacuum the tubes with lids open at 4, 000 G to evaporate residual 1-octanol from the lysate.
Prepare a serial dilution of phage lysate in factors of 10. Then spot 5 microliters of each dilution onto the LB agar plates and incubate the plates overnight at 37 degrees Celsius. The next day, count the plaques to calculate plaque forming units or PFU.
Dilute the resulting lysate in SM buffer to the desired concentration and re-titer to confirm. Store the concentrated lysate at 4 degrees Celsius until use. To begin, collect fecal pellets from T4 phage-inoculated Escherichia coli monocolonized mice into a sterile pre-weighed microcentrifuge tube.
Record the final weight of the tube and calculate the sample weight by subtracting the initial tube weight. Add 1 milliliter of sterile SM buffer to the tube and vortex at maximum speed to homogenize the fecal pellets. Record the volume of SM buffer added to each sample for colony forming units or CFU or PFU per gram calculations.
Prepare a series of eight serial dilutions of 20 microliters of each sample in 180 microliters SM buffer. Spot 5 microliters of each dilution onto LB agar plate containing E.coli or only LB agar plates to determine T4 phage and E.coli concentration. Allow each spot to dry before inverting the plates and incubating overnight at 37 degrees Celsius.
The next day, select the dilution with 3-30 countable plaques per spot. Count and record the number of plaques and dilutions used. Divide the number of plaques by volume plated in each spot to give PFU per microliter.
Multiply this by the dilution factor and the volume of SM buffer added to each sample for PFU per sample. Finally, divide by the sample weight to obtain PFU per gram of sample. The levels of T4 phage and E.coli detected in mice injected with 2 by 10 to the 6th PFU of T4 phage or a vehicle lysate demonstrated the coexistence of T4 phage and E.Coli without depletion of either population over four weeks.
Lower doses of T4 phage did not affect colonization compared to 2 by 10 to the 6th PFU, and no dose dependent effect on E.coli levels was observed.
