The overall goal of the following experiment is to develop a cheap time-lapse micros cinematography assay with sufficient rigor to generate reproducible and quantifiable results. This is achieved by employing the use of inexpensive reusable silicon gaskets to construct agros media plates or microscope slides. Results are obtained that show these plate complexes remain hydrated and sufficiently oxygenated for more than a week over a variety of media types and agar concentrations.
In addition, because time-lapse micro cinematography is a slow process, the use of eight inexpensive microscopes helps to accelerate data acquisition. Hello, I'm Ryan Taylor from the laboratory of Dr.Roy Welch from the Department of Biology at Syracuse University. Today we'll show you a procedure for making time-lapse movies of bacterial biofilms in our lab, we'll use this procedure to study multicellular behavior of MTIs biofilms.
So let's get started. To begin this procedure, measure the cell density of an overnight mix Occus sanus culture with a clut meter transfer one milliliter of the culture into a 2.5 milliliter centrifuge tube pellet wash and resuspend the bacteria in TPM media as described in the accompanying written protocol, pipet and vortex to completely resuspend the pellet, which may take a while. Next, add 0.5 grams of aros to 50 milliliters of TPM, which is the assay media.
Also add 0.5 grams of aros to 50 milliliters of C-T-T-Y-E, which is the nutritive disc media autoclave to sterilize. Then maintain the media at 55 degrees Celsius in an incubator or water bath. While constructing the tracking assay.
Plate complexes proceed to construct the nutritive disc first flame sterilizer glass microscope. Slide and place a sterile 0.5 millimeter thick silicone rubber gasket on top of the slide. Pipettes around 300 microliters of C TT YE media aros into the well created by the gasket on the slide.
The C-T-T-Y-E media agro should mound up flames sterilize another slide and set it down at an angle on top of the C-T-T-Y-E media aros. Setting the slide down at an angle prevents bubble formation. Once the slide is in place, plump the complex together with mini binder clips one on each side.
Place the clipped complex at four degrees Celsius and allow the C-T-T-Y-E media agro to set. This usually takes about five minutes. Once the agro is set, one can prepare the plate complexes used in the tracking assay.
Start by preparing the slides for constructing the assay complexes. For each complex flame sterilized two glass microscope slides. Place an autoclave gasket on top of one of the slides to form the bottom of the assay.
The other slide is used for flattening the agros and as a support slide. The top of the assay is a cover slip. First wrap a microscope slide with labeling tape, then flame sterilizer glass cover slip and place it on top of the wrapped slide sterilized.
Slide up the wrapped slide supports the cover slip, keeping it from cracking. Place an autoclave gasket on top of the flame sterilized cover slip with forceps. Press down on the gaskets on the bottom slide and the cover slip to make sure they form a seal with the glass.
Otherwise, the media a arrays may dry out. Set the slide gasket and the cover slip gasket assemblies aside with the sterilized sides up from here on work only on one complex at a time to prevent the media agros from solidifying, which would result in poor movie quality. Begin by placing the nutritive disc.
Remove the nutritive disc complex from four degrees Celsius. Remove the bind eclipse and prior part the slides exposing the now solidified C-T-T-Y-E aros using a micro sampling pipette with a 100 microliter glass disposable tip. Remove a one millimeter diameter nutritive disc from the C-T-T-Y-E media agro as well and place it in the well created by the gasket on the cover slip.
Then retrieve the TPM media agros from the incubator and pipette 300 microliters into the well created by the gasket on the cover slip and containing the nutritive disc. The TPM media Agros should mound up to flatten the T-P-M-S-A medium aros Place one of the flame sterilized slides without a gasket at an angle on top of the TPM media aros. Placing the slide at an angle helps prevent bubble formation.
Clamp the complex together with mini binder clips one on each side. Place the clipped complex at four degrees Celsius and allow the media arose to set. This usually takes about five minutes.
Once the media arose has set, remove the complex from four degrees Celsius. Remove the binder clips and squeeze the end of the complex. To loosen the tape wrapped slide, remove the tape wrapped slide and place it on the bench for further use.
Then using a pair of forceps as a wedge. Separate the cover slip gasket media agros complex from the support slide and discard the support slide. Do not use a prying motion to separate the cover slip gasket complex since this could result in the cover slip braking and or the media aray sticking to the support slide.
After removing the support slide, place the cover slip gasket media aray complex onto the tape wrapped slide with the gasket side up. Place this complex next to a burner to allow all visible moisture to evaporate from the newly exposed media agros surface. Do not let the media agros dry for longer than one minute.
As this could impact the swarm behavior of M Zant, proceed to plate the cells on the newly prepared assay complex. Once the excess moisture has evaporated from the cover slip gasket media agros complex aspirate 0.5 microliters of the concentrated cells for depositing on the media. Aeros depress the pipette tip before approaching the media agros.
A drop of cells appears at the bottom of the pipette tip, making it easier to deposit the cells without the tip touching the media agros. A depression on the agros surface can change the behavior of the mz the swarm. Move the pipette straight down to dispense the cells approximately one millimeter away from the embedded nutritive disc.
Then pull the pipette straight back up. In this way, one ensures that the swarm is circular. Once the cells are deposited, place the complex next to the burner to allow the cells spot to dry for no more than 20 seconds.
After drying, align the slide gasket assembly forming the bottom of the complex with the gasket on the cover. Slip gasket media acro cell complex forming the top of the complex. Gently press together to form a seal.
Clean the surfaces of the slide in the cover slip with a kim wipe to remove any residue in order to prevent condensation. Place the complex on the heated microscope stage immediately after cleaning the slides with the slide down and the cover slip up. If condensation has formed, then let the slide complex sit on the heated stage for several seconds before starting the image acquisition software.
In order to prepare the movie, choose the appropriate objective, two x four x or 10 x on the microscope. Turn on the camera and the microscope and check the light levels to make sure the light is not too intense. Start up the computer and open the image acquisition program.
Click the live image window and focus the microscope. The image appears similar to the one shown here. Start acquiring images.
Make sure to check the focus regularly during the first hour as the media agar tends to settle causing the focus to drift. Once image acquisition is complete, transfer acquired images for storage. Break down the slide complex by soaking in 90%ethanol overnight or TOLA the gaskets for reuse.
This time lapse movie shows em subjected to the tracking assay described here. Capture rate is one image per minute at a magnification of 20 x playback rate is 60 images per second. This time lapse movie shows a population of eant the cells where 1%of the cells are expressing GFP.
The movie was compiled by merging alternating phase contrast and fluorescent images. The assay was performed on C-T-T-Y-E in 1%Aros capture rate is one image per minute as a magnification of 100 x. This time-lapse movie shows the gliding motility of M Anthes.
This assay was performed on C-T-T-Y-E in 1%Aros capture rate is one image per minute at a magnification of 100 x. This TimeLapse movie shows P OSA twitching motility. This assay was performed on LB in 1%Aros capture rate is one image per minute.
At a magnification of 20 x playback rate is 60 images per second. This time lapse movie shows Smar senses swarming motility. This assay was performed on LB and 1%Aris capture rate is one image per minute.
At a magnification of 20 x playback rate is 60 images per second. This time lapse movie shows EGT sliding motility. This assay was performed on LB in 0.5%Tag rose capture rate is one image per minute as a magnification of 20 x playback rate is 120 images per second because of the fragility of 0.5%arose in lv.
This assay employs the use of the larger gaskets seen in figure one B.We've just shown you how to generate time-lapse movies of bacterial biofilms. When doing this procedure. It's important to remember to take your time and be as precise as possible to achieve consistent results.
So that's it. Thanks for watching and good luck with your experiments.
