I want to mount the body on slides for morphological comparison as voucher specimen.
You can follow the protocol we developed for the detection of vector-borne pathogens (Maggi et al Ticks and Tick-borne Diseases 1 (2010) 168–171)
We use ammonium hydroxide at 20%, 500 ul to adults and 100ul to nymphs and larvae's. Crush them in that solution with a micropipette tip of 1000ul, bent the tip so the liquid does not rise. boil to 100ºC during 20 min, and then with the cap open until the volume evaporated to half! Good luck!
My only suggestion is to first dissect the tick under a drop or two of PBS (can be done only for nymphs and adults ) by snipping along the edge of the ticks body with its dorsal side facing outward (from the bottom towards the head). Then to open the cuticular flap that is created (just like opening the back of a car) removing the organs, placing the open flap back and then in most instances, you would still have the whole tick for mounting on slides. The organs can then be used for DNA isolation using any of the commercial kits for isolation.
You should contact Lorenza Beati at Georgia Southern. She has developed a protocol for extracting DNA from ticks without destroying the specimen. You may try Qiagen extraction kit, I have heard of many researchers making a small incision in the cuticle before soaking the specimen overnight in an incubator. For the rest of the extraction protocol you remove the animal and use just the slurry solution for your extraction.
I have had great results using Zygem extraction kits from Prepgem to extract DNA from whole fleas. This procedure is specifically designed for insects, reduces plastics and therefore contamination. Total cost is about less than $2.00 USD per sample including disposables.
We use to crush ticks gently with a tip of a micropipette or cut with a scalpel blade and use commercial isolation kits (e.g. Qiagen, Macherey Nagel).
Jainder, your place Gohana is so near to our university, I can dissect out internal organs of the tick with minimum damage to the tick cuticle. You can have the DNA as well as preserve the specimen.
which espécimen you want to keep, larvae nymph or adult, if it is the adult you can use the fluid obtained from punctioning the back of the tick with a micro syringe. there is no way to do it with larvae. However you can use a non phenol technique to extract dan from larvae by smashing the larvae within an eppendorf tuba, just by adding 10 - 20 ul of water or TE, and boíl for 5 minutes. we use this technique to amplify targets from single larvae.
The best protocol is crushing ticks(larvae, Nymphs and adults) using liquid nitrogen (ticks suspended in liquid nitrogen in ependorf tube and put the bottom of tube on a suitable container filled with liquid nitrogen ). Rapid crushing of tick in liquid nitrogen is critical to get a very good powder of tick, otherwise, it will be time wasting. The powder will be ready to add any lysis buffer made manually or from any available DNA extraction kit .
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