The overall goal of this procedure is to micro inject and localize borrelia berg de ferry cells in the tick gut. This is accomplished by first preparing the glass capillary needles and filling them with beberg de ferry cells. Next, the beberg defer cells are transferred into the tick gut via microinjection.
The tick is then dissected and the gut tissues are removed. Finally, immunofluorescence labeling of beberg defer in the tick gut is performed. Ultimately, results can be obtained that demonstrate localization of beberg to ferry in the tick gut using confocal microscopy.
Hi, I'm Dr.Ruth Paul Paul, assistant professor at the University of Maryland in the Department of Veterinary Medicine. Today two of my lab member Toro Cairo, a postdoctoral fellow and Adam Coleman, a graduate student, will show you the procedure for artificial infection of ticks via microinjection that we routinely do in the laboratory. After microinjection, we'll dissect the tick and image the bacteria in the tick gut using confocal CE microscope.
The main advantage of tick microinjection of a generation of natural infected ticks is a duration of the procedure. Microinjection shorten the whole procedure from weeks hours as approximately five weeks are required to generate infected ticks by natural feeding process. This technique can also be adapted to inject other useful reagents such as double stranded RNA for RNAi or antisera for specific antibody interference inside of the tick.
To begin fabricate several microinjection needles by heating and pulling one millimeter glass capillary tubes in a glass micropipet pull device. Carefully remove the fragile capillary tubes. Store the pooled needles with tip facing upward on adhesive tape in a Petri dish.
Next, grow b berg de ferry in BSK culture. Medium to a concentration of about 10 to the seven cells per milliliter pellet b berg de ferry by centrifugation at 3000 times G for 10 minutes. At room temperature, remove the snat and completely resuspend the pellet in BSK medium by gently passing through a 200 microliter sterile microtip to a final concentration of 10 to the nine cells per milliliter.
Since the cells are resuspended at high cell density and could clump together, the cell suspension should be used immediately for microinjection to prepare ticks, place clear double-sided adhesive tape on a glass slide working on a sticky mat, carefully remove ticks from the container Using a small brush place the required number of ticks to be injected on the adhesive tape ventral side up facing the same direction. Load five microliters of the beberg de ferry culture into the pooled capillary needle. Using a 20 microliter micro load of pipet tip, inspect the needle for trapped air bubbles and reload the needle with bacterial culture.
If necessary, view the tick under the dissecting binocular microscope and focus on the tick's anal aperture area. Gently touch the tip of the capillary needle in order to break the tube where the diameter is slightly smaller than that of the tick's anal aperture forming the microinjection needle. This step is critical as any attempt to inject ticks with non appropriate needle tips causes injury and possible death.
To the injected tick. Place the immobilized ticks under the dissecting binocular microscope and focus on the anal aperture, which is covered by two moveable anal plates. Using fine forceps, gently touch and apply very mild pressure to any area near the anal aperture.
This will allow separation of the anal plates and opening of the anal pore that connects to the gut. Carefully insert the tip of the needle slightly into the anal aperture through the forced opening of the anal plates. Needle insertion should be kept to a minimum as the glass tip could damage the semi-transparent hind gut that connects to the rectum.
Using a micro injector equipped with an automated foot control inject beberg de ferry solution using the following parameters. 1000 Hector Pascals injection pressure 0.2 seconds injection time, and eight Hector Pascal's compensation pressure. Each tick receives a single injection following microinjection.
The tick should show signs of movement or crawl in response to stimuli similar to before injection. To dissect ticks for immunofluorescence microscopy, first place a droplet of phosphate buffered saline on a clean glass slide for dissection and another on a poly L lysine coated slide For microscopy, prepare another glass slide with double-sided adhesive tape. Next, remove the tick from the container and place it on double-sided adhesive tape.
Using a small brush under the dissection microscope view and focus in on the tick, place a sharp razor blade on the tick between the first and second pairs of legs. Press down firmly cutting the tick in two pieces, exposing the abdomen immediately submerged the abdomen in a droplet of PBS using fine forceps. Grab the dorsal and ventral exoskeleton around the site of the cut.
Carefully pull the dorsal shield up and away from the tick exposing the brownish colored gut diverticular. Be careful to keep the dissecting ticks under the PBS at all times. The semi translucent sali gland bundles are located on either side of the anterior region of the gut and can be removed at this point if desired.
Holding the remaining exoskeleton in place with forceps, carefully pull the gut out from the abdomen. Carefully clean the gut by removing any trapped tissues such as the trachia. Using the tip of fine forceps, quickly transfer the tick gut into a droplet of PBS on a poly L lysine glass slide.
Separate the gut into smaller pieces using fine blades or by gently pressing with the tip of fine forceps. Carefully aspirate excess PBS around the gut tissues. Allow the gut tissues to air dry ed room temperature.
Fix the tick gut by submerging it in acetone for 10 minutes. Allow it to air dry it room temperature slides can be stored at this step for months at minus 20 degrees Celsius in an airtight container. For immunofluorescent labeling of the gut, draw a circle around the fixed tick gut with a pap pen or other hydrophobic barrier lining device.
This will help to retain the staining solutions during subsequent incubation steps. Cover the tick gut with one or a few drops of blocking buffer for 30 minutes. At room temperature.
The serum used for blocking depends on the source of the host animal for the antibody. Do not allow the slide to dry from this point forward. Slowly aspirate the blocking buffer.
Incubate the gut with appropriate primary and or secondary antibody solutions. We use fluorescein, isothiocyanate, or FXI labeled anti B berg. Defer antibody at a one to 100 dilution in blocking buffer for one hour.
At room temperature, cover the container with aluminum foil to limit exposure to light. Remove the antibody solution by gentle aspiration. Incubate the gut with a fluorescent dye to label tick tissues such as DPI or propidium iodide.
We normally use 20 micrograms per milliliter peridium iodide in PBS for five minutes at room temperature, wash three times with 0.05%tween 20 in PBS for five minutes. Finally, mount the slide in buffered glycerol containing an Antifa reagent and carefully cover with a glass cover. Slip slides can be stored at this step for a few months at four degrees Celsius in an airtight container image and localized b berg de ferry under a confocal microscope.
A tick microinjection is shown here with a needle filled with kumasi brilliant blue for visibility. Fine forceps are used to apply gentle pressure to the body, which allows separation of the anal plates and opening of the anal pore for microinjection. Here, the anterior region of a gut diverticulum is shown with beberg ferry within the gut.
The gut nuclei and spirochetes are labeled with propidium iodide or fitzy conjugated anti beberg ferry respectively. Once mastered, we have found that 10 ticks can be micro injected power with naturally a hundred thousand surviving and infected with fer. While ticks can be used immediately for other experiments, we generally wait one or two days to make sure the ticks survive the procedure before proceeding.
After watching this video, you should have a good understanding of how to generate artificial infected ticks by micro and localized barrier bug do within the tick gut using confocal immuno flows. Micro Working with ticks and fine glass needles filled with a human pathogen such as brelia brior fry can be hazardous while you're working. Be conscious of the locations of ticks and needles and practice biosafety level two precautions at all times.
Thank you for watching and good luck with your experiments.
