The overall goal of this procedure is to collect, tick, hemo, lymph salivary glands and saliva from actively feeding ex ODI scapularis ticks. This is accomplished by first amputating the tick's leg at the distal joint to expel hemo limp. Next salivary glands are collected by dissecting the tick in phosphate buffered saline, locating the salivary glands and transferring them into a clean pool of PBS.
Saliva is collected by applying pilocarpine onto the ticks do dorsum of the micro pipette, inserting the tick's ome into a pooled capillary tube and allowing the tick to salivate into the tube. Ultimately, results can be obtained that show microorganisms present in the hemo lymph or salivary glands via immunofluorescence or protein. Protein interactions with salivary proteins among other results.
Generally, individuals new to hemo lymph collection will struggle because it is difficult to immobilize the tick without rupturing the midgut, thereby contaminating the hemo lymph. Generally, individuals may struggle with saliva collection because not all ticks will salivate. Therefore, it is important to attempt saliva collection from ticks that are at or near repletion.
Generally, individuals new to salivary gland collection will struggle because the salivary glands are difficult to locate amongst the tick debris. And once located, these salivary glands are easily ruptured and lost during the wash steps. After gently removing actively feeding ticks from an animal host, place them in 3%topical hydrogen peroxide for five minutes.
Transfer the ticks to 70%ethanol for 10 minutes to surface, sterilize them. Remove the ticks from 70%ethanol by pouring them onto biles paper with a pap pen. Draw a circle onto a sane coated microscope.
Slide and place a tick within the pat pen circle under a dissecting microscope. Use forceps to gently push down on the tick's dorsum. Display the tick's legs and immobilize it.
Then with a fine point disposable scalpel amputate the tick's leg or legs at the distal joint. After the leg or legs are cut, continue to apply pressure to the tick's dorsum for the hema lymph to secrete outta the legs. Onto the slide.
Gently move the tick around on the slide to spread the hema lymph to remove the salivary glands from a tick spot. Several 25 microliter pulls of phosphate buffered saline onto a microscope. Slide and place a tick into one of the PBS pools under the dissecting microscope.
Use fine tipped forceps to stabilize the tick by holding the mouth parts or the rear of the tick. Insert the fine tipped forceps into the rear of the tick and slice up the tick's dorsum to expose the organs. Find the pair of salivary glands located bilaterally alongside the legs of the tick.
If the glands are not visible amongst the tick debris, move the main tick portion still containing the glands to a fresh pool of PBS to reduce the disruption and loss of the salivary glands with fine tipped forceps. Remove the salivary glands from the tick and place them in a fresh pool of PBS. Transfer the glands to another clean 25 microliter PBS pool and gently repeat this wash.
Step three to four more times to remove any external microorganisms and tick debris. Place the glands into a clean pool of PBS on a sane coated slide. After removing a nearly engorged tick from an animal host, tape it to one end of a glass microscope slide.
The tape should be placed approximately three quarters of the way up. The tick's dorsum toward the head, leaving the tick's mouth parts exposed where the tape meets the anterior edge of the tick's dorsal surface pipette five microliters of pilocarpine solution. Allow the tape to wick the pilocarpine over the tick's dorsum without allowing the pilocarpine to come in contact with the tick's mouth parts.
Place a piece of non-toxic modeling clay onto the microscope. Slide approximately one inch from the tick's mouth parts using fine tip forceps. Break off the tip of a pulled capillary tube to the desired diameter.
Then while viewing the tick under a dissecting microscope, gently fit the tick's hyper stone into the capillary tube, allowing the maxillary pulps to reside alongside the outside of the capillary tube. Press the opposite end of the capillary tube into the modeling clay to hold the capillary tube in place. Next place the mounted salivating tick inside a dark chamber with high humidity.
Tilt the slide so that the hyper stone points to the bottom of the container, allowing gravity to aid in saliva collection. Place the container at room temperature and closely monitor the salivating ticks for the first hour. Remove the capillary tube from the ticks hyper stone and collect saliva as it is generated.
Using a pasta pipette bulb by expelling it outta the capillary tubes. Replace the capillary tube onto the hyper stone and check accumulation every hour for at least four hours and continue to collect saliva as it is generated. If the ticks are not salivating or if more saliva is required.
Induce salivation by using the capillary tube to massage the hyper stone. Finally, add 0.1 volumes of protease inhibitor cocktail to the saliva and store at minus 80 degrees Celsius until needed. This figure shows an example of uncontaminated hemo lymph.
Once the legs are amputated, a clear fluid is secreted. If the midgut is ruptured, the hemo lymph appears cloudy and is considered contaminated as shown here. Shown here is an immunofluorescence confocal image of salivary glands stained for Borrelia burgdorferi with 10 micrograms per milliliter.
Fluorescent isonate conjugated rabbit antib burgdorferi arrows denote beberg Dre in salivary glands. After watching this video, you will have a good understanding how to dissect an engorged tick for salivary glands and collect hema lymph. You'll also have an understanding how to collect saliva from an exo scapularis adult female.
