The overall goal of this procedure is to image live cells through growth and division using fluorescence microscopy so that the effect of cell history and ancestry on cellular development of bacteria can be studied. This is accomplished by first transferring the cells from liquid medium with relatively high nutrient concentrations to liquid starvation medium. Next, a microscopic slide on which the bacterial cells will grow into a micro colony.
Monolayer is prepared. Then a time lapse fluorescence microscopy movie is recorded. Finally, the movie is processed and the data is analyzed.
Ultimately, results can be obtained that show development of a single bacterial cell through time-lapse fluorescence microscopy. The main advantage of this technique over existing techniques such as flow cytometry and standard microscopy, is that it allows the monitoring of protein dynamics, cellular behavior, and development of single cells in time, demonstrating the procedure will be in cone, a PhD student from my lab To prepare b tuus inoculate cells from minus 80 degrees Celsius, stocks in 10 milliliters of time-lapse microscopy or TLM medium in sterile shake flasks supplemented with antibiotics if necessary, grow the cells overnight at 30 degrees Celsius and 225 RPM in a sterile shake flask the following morning to loot the cells one to 10 in Prewarm, chemically defined medium or CDM without antibiotics, grow the bee subtlest cells to mid exponential phase, which takes approximately four hours. Measure the absorbance of the culture at 600 nanometers and dilute the cells to an approximate a 600 of 0.035.
Using CDM. This OD ensures that single cells with appropriate spacing between cells are spotted on the microscope Slide for time-lapse microscopy. One hour before cells reach mid exponential growth.
Prepare the microscope slide by cleaning two glass slides with 70%ethanol and water. Remove one of the plastic covers from a gene frame, being careful not to cause disassembly of the plastic cover on the other side of the frame. Attach the gene frame in the middle of one of the glass slides by first sticking it to one side, then guiding the rest with a fingernail.
Use a microwave to dissolve 150 milligrams of high resolution low melting aros. In 10 milliliters of CDM, ensure the aros is fully dissolved to prevent background fluorescence. Pipette 500 microliters of the warm agro CDM into the middle of the gene frame, making sure that the whole area including the borders is fully covered.
Working quickly to prevent excessive drying of the aros. Place the second glass slide on the Aros CDM filled gene frame trying to avoid air bubbles. Place the sandwich in a horizontal position at four degrees Celsius for 45 minutes to allow the agros to solidify sufficiently.
Once the aros has solidified carefully, slide off the upper glass. Use a razor blade to cut out agro strips of about five millimeters in width on which the bacteria will grow. A maximum of three strips can be used per slide, separated by about four millimeter spaces on either side.
These spaces will provide air which is essential for Blu growth. Carefully remove the second and final plastic cover from the gene frame to expose the sticky side. Load single cells in about 2.5 microliters of liquid on the solid medium, beginning at the top of the agros pad without touching it.
With the pipette tip, allow the medium to spread evenly by tilting the slide up and down. Place a clean microscope cover on the gene frame to completely attach the cover slip. Use a fingernail to apply pressure So after the cells have been loaded.
It is crucial to wait for the liquid to dry long enough so that you don't get swimming cells and growth in multiple layers. However, if the cells are dried for too long, then they will have problems growing into a micro cholon monolayer. So the time required to dry your slides depends on the experiment and cannot be measured.
And consequently, one of course needs to get a feeling for it To prevent auto-focus problems during the first hours of the time-lapse experiment prewarm the slide for one hour at 30 degrees Celsius to prepare for time-lapse microscopy. Prewarm the environmental chamber at least two hours. Before the start of the experiment, select the appropriate objective filters and dichroic mirror according to your experimental setup.
For long experiments, make sure that a UV filter is placed between the light source and the sample. In addition, if possible, block some of the excitation light using neutral density filters to minimize exposure. Program the experiment according to the specific experimental setup.
It is wise to determine the amount of light required for specific constructs as well as the auto-focus settings for other time-lapse microscopes or bacteria prior to the actual experiment. Shorter exposure times and lower levels of excitation light will minimize bleaching and phototoxicity Use scopic light for the auto-focus function. Finally, place the prepared slide in the prewarm environmental chamber of the microscope and monitor the outgrowth of single cells into a micro colony monolayer at 30 degrees Celsius.
A successful time-lapse fluorescence experiment will produce a micro colony monolayer completely located within the field of view. At the end of the experiment in this movie, bright field is on the left and fluorescence can be seen. On the right.
Here is an example of a B Subtles micro colony in which some cells grew on top of one another, making it difficult to trace back their history accurately as well as measure their fluorescence levels correctly. After watching this video, you should have a good understanding of how to successfully prepare a microscopic sample for time-lapse microscopy and how to perform a time-lapse fluorescence microscopy. Experiment with BACE cells.
