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Fecal float question (sorry, gotta ask)

Fecal float question (sorry, gotta ask) - Molecular Biology Techniques

Fecal float question (sorry, gotta ask) - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 08-28-2013, 02:25 PM
Pipette Filler
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Default Fecal float question (sorry, gotta ask)



Hi all,

I'm looking for advice on fecal float techniques. I have my own little flock of pet chickens, and I'd like to do my own fecal analyses instead of paying for it every time someone has a runny butt. My questions are:

1. When looking for coccidiosis oocytes, should I use a fecal float method?
2. To use a fecal float method, should I use a sodium nitrate solution?
3. Is a microscope of 40x enough to detect oocytes?

Thank you - I took some microbiology courses in university for electives, believe it or not, but that was 600 years ago so please forgive my memory lapse.

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Old 09-01-2013, 02:49 PM
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Default Re: Fecal float question (sorry, gotta ask)

Introduction
A large number of different procedures are available for demonstrating coccidia oocysts in poultry faeces. The most widely used principle for concentration of parasite oocysts is flotation. As most nematode eggs, cestode eggs and coccidia oocysts have a specific gravity which is lower than that of plant residues in the faeces, the oocysts may be separated from other faecal particles by mixing the faeces with a fluid at s.g. 1.28 (saturated NaCl + glucose) in which the oocysts float. Sugar solutions should be prepared with a preservative (e.g. formalin) to retard bacterial or yeast growth, since digestion of the sugar molecules will lower the specific gravity. Sugar solutions are less expensive to make, do not distort eggs or oocysts to the same degree as salt solutions, and will not crystallize rapidly. The latter advantages mean that prepared slides may be refrigerated for days prior to examination, without loss of parasite structural integrity. Flotation solutions should be compared through side-by-side preparations using known positive samples.
Test tube flotation procedures
Suspend 0.9 - 1.1 g (measure with pre-calibrated teaspoon) faeces "hot and steamy" (old feces dry out and some eggs and cysts will be damaged) in 10 ml of flotation fluid saturated NaCl+glucose with specific gravity of 1.28 (dissolve 350 g NaCl in 300 ml of tap water; dissolve 500 g of glucose in 600 ml tap water, mix two solutions and add 6 ml of formaldehyde at 37%, check gravity with hydrometer and results were adjusted for temperature by adding or subtracting 0.001 specific-gravity units for each 3C above or below 20C, respectively, and finally fill to 1 liter with tap water) and mixed thoroughly with a stirring device immediately after homogenizing with wood skewer. The faecal suspension poured through a tea strainer or a single layer of cotton gauze into plastic container 2, the retained faecal debris discarded, and poured the strained faecal suspension from plastic container 2 into a test tube immediately. The faecal suspension poured through a tea strainer or a single layer of cotton gauze, the retained faecal debris that are discarded, into plastic cup and poured immediately the strained faecal suspension into 12 ml a test tube. Test tube was placed in a vertical position in a test tube rack, the test tube was topped up with the faecal suspension, so that it has a convex meniscus at the top, leave to stand for 5 minutes, so that the faeces settles, then place the slide on top, being careful not to get too many bubbles. (Air bubbles come up like round black balls). For this reason I leave the faeces to stand for 5 minutes before placing the slide on top is that it gives time for any blood or large matter in the faeces to settle, which will make the slide easier to read. This is particularly helpful when testing dark samples. The test tube was leaved for about 20 minutes. The coccidia oocysts floated and thus accumulated just beneath the cover slip, the cover slip was lifted off vertically from the tube together with the adhering flotation fluid. Some of the accumulated coccidian oocysts are within the adhering fluid, and the cover slip is transferred very carefully in order to retain as many oocysts as possible. The cover slip is placed on a microscope slide, and the sample was examined microscopically at 40x magnification using a light microscope. Remember, coccidia must have a definite centre if not sporalating, and more if sporalating. Unsporalating oocysts have a round centre that looks like crystal glass. Unsporalated oocysts are not infective. They have to go back into the ground and be ingested before sporalating. The sporalated oocysts are those that have come out of the ground and are ready to infect an animal. The pet is considered uninfected when have been examined at least 3 faeces sample in different days. Watch out for pollens and plant material because coccidian they are to them similar. To differentiate both mix 2 drops of dye pollens solution (5 ml glycerol, 10 ml 95% ethanol, 15 ml dH2O and 2 drops of aqueous saturated basic fuchsin) with 2 drops of feces and therefore to observe at microscope with 40x objective and to notice possible pollens components that becomes colored in clear purple and not dark red.
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Old 09-04-2013, 09:42 PM
Pipette Filler
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Default Re: Fecal float question (sorry, gotta ask)

Thank you Zagami - that's awesome information. Really appreciate it.
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