The overall goal of this procedure is to use a fluorescence microscope to follow sulfate in e coli in real time after infection by phage lambda. Start by creating a crude lysate of phage lambda. Then purify the phage and infect e coli by phage lambda in a tube.
Continue to follow bacterial cell fate under the microscope. Ultimately, results can show in real time different cell fats in e coli through the expression of different fluorescent proteins associated with cell growth or death. Generally, one check to this method is in the creation of a purified front and labeled FI stock.
To avoid fige sharing during preparation and purification, also optimize the imaging parameters under the microscope to allow a healthy cell growth Inoculate. An overnight culture of E 3 92 Lambda LZ one PPL star D into LBM. Media at cell density of OD 600 around 0.6 induce the lygen in a 42 degree Celsius water bath shaker.
Continue to incubate at 37 degrees Celsius until lysis is evident. The culture becomes clear. Now add 2%chloroform, and then incubate for 15 minutes at room temperature.
Next, transfer the phage lysate to 250 milliliter bottles and centrifuge the culture. Then recover the supernatant containing the phage particles and perform a second centrifugation to remove visible debris with standard phage titration protocols. Measure the phage concentration, then purify the phage through a cesium chloride gradient and assay the activity with DPI for the preparation of aro slabs.
Clean six microscope slides with 70%ethanol. Arrange five slides as shown and secure with tape. Now pour a solution of 1.5%aros in medium onto the secured slides.
Position the last slide on top, carefully avoiding air bubbles. Place a weight on top and allow AROS to cool for about 30 minutes. Remove the four slides on the side and package the slab of slides with cling wrap.
Using an overnight culture of transformed bacteria as inoculum, grow a culture in LBMM pellet one milliliter of bacteria and resus. Suspend the cells into 20 microliters ice cold. LBMM with a wide pipette tip.
Add 20 microliters of purified phage. Gently mix and incubate first on ice for 30 minutes to allow phage absorption. Then in a 35 degree Celsius water bath for five minutes.
To trigger phage DNA injection, separate any cell aggregates by mixing. Dilute the mixture one to 10 into LBMM. Now place an LBM agro slab.
On a cover slip, add one microliter of the phage cell mixture. Once absorbed, gently position another cover slip on top. Start by acquiring an image set through the YFP channel for the initial timeframe.
Take a series of 15 images at 200 nanometer Z axis intervals. In addition, take a single in-focus image through the phase contrast and m cherry channels. Then acquire a time-lapse movie of the post infection cell fate.
To minimize bleaching and phototoxicity during the time lapse movie, use a single Z position image per channel per time point image, the sample in phase contrast YFP and M cherry channels at time, intervals of 10 minutes for around four hours. Begin analysis of the images with a manual count of phages, and also record phage location and the cell length. In the initial timeframe, record the cell fates of lytic, lysogenic, or uninfected.
Also document the lysis time and any other desired information by playing the time-lapse movie, extract more quantitative information like fluorescence level over time in individual cells by using automated cell recognition and lineage tracing algorithms. Typically, the plaques of the fluorescently labeled phages are significantly smaller than those of wild type and the YFP and DAPI signals of a successfully purified phage co localize. These data combine image sets of phase contrast YFP and M cherry channels, and the corresponding overlaid images from a time-lapse movie.
The individual phages are clearly visible at the initial timeframe. Here two cells are seen each infected by a single phage, and one cell is infected by three phages. Typically, a number of phages are seen on the cell surface while other phages are unabsorbed.
Over time, the infection outcome becomes distinguishable. 80 minutes later, the two cells infected by single phages have each entered the lytic pathway. With intracellular production of new phages, the cell infected by three phages has gone into the lysogenic pathway marked by production of m Cherry from the pre-motor.
After watching this video, you should have a good understanding of how to create a purified phage stock to infect e co bacteria by phage lambda, and perform lifestyle imaging under the microscope in real time.
