JoVE 실험 백과사전
미생물학
0 조회수 • 2:57 분 • July 1st, 2026
Take a tube containing fecal pellets from a mouse with its intestine infected with E. coli and T4 bacteriophages.
The phages interact with E. coli, deliver their genetic material, and replicate within the bacteria.
The infected bacteria lyse, releasing new phages that infect nearby E. coli.
Some E. coli escape phage infection, allowing both phages and bacteria to coexist and appear in fecal pellets.
To assess their coexistence, add a buffer to the fecal pellets and vortex the mixture to release phages and bacteria into suspension.
Prepare serial dilutions of the fecal suspension.
Spot each dilution onto a nutrient agar plate to quantify E. coli, and onto an agar plate with cultured E. coli to quantify phages.
Incubate the plates. The bacteria form colonies, while the phages lyse bacteria to form plaques.
The appearance of both bacterial colonies and phage plaques confirms their coexistence within the host.
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.