Question
Asked 25th Oct, 2016
Can anyone fix my sucrose flotation protocol for Cryptosporidium flotation?
Hi,
I did sucrose flotation for recovery of Cryptosporidium oocyst from water sample. However, there was no oocyst detected after staining. Staining is not the problem I suppose because previously the staining worked well.
I prepared the Sucrose solution by adding 128 g Sucrose into 100 mL distilled water. I heated and stirred the solution to make sure they dissolve. The specific gravity is 1.266 as shown in a paper (See attachment).
I added water sample into a 15-mL Falcon tube and added sugar solution until 12-mL volume. I vortexed the sample and took some portion from point A (See photo) and proceed with staining.
Also I tried from point B and C. But no oocyst detected. Can anyone help me to fix my step? Does anyone have better protocol? Please assist me. I really appreciate.
All Answers (1)
Centrifuge is the preferred method for detecting parasite eggs in fecal samples in veterinary medicine.
If you have a swinging head centrifuge you can create a convex meniscus with surface tension and spin with the cover slip already on. there should be no air bubbles between the solution and the glass slide cover slip. If you have a stationary head, you can fill your tube about 80% of the way, spin then add enough solution to create meniscus. We were taught in school to always spin samples at 1,200 rpm (280 g) for five minutes, then let stand for 10min, with the cover slip over the tube. Gently life the cover slip off, and place on slide for examination. I'm assuming that you want to collect the oocysts, so perhaps scrape with small spatula into collection container?
Sheather’s Sucrose (sp gravity 1.275) is used preferentially to identify potential infections with Toxocara sp, Ancylostoma sp., Trichuris sp., Capillaria sp. and Isospora sp. in fecal samples from dogs and cats.
Sheather’s Sucrose 454 grams sugar, dissolved in 355 ml water. 6 ml formalin to prevent mold growth.
Centrifugation is especially necessary when the flotation procedure is performed using a viscous solution like Sheather’s Sucrose. The downward force created by the centrifugal spinning enhances the buoyancy of the eggs in the viscous solution and drives them to the surface meniscus where they are concentrated and result in greater parasite recovery.
recipe taken from http://www.vet.utk.edu/.
Minor note, yes the centrifuges will get contaminated and proper cleaning is required after every use.
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