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I would like to analysis tortoise poo, in order to see whether there are parasites or not. Can someone recommend me a document or explain the technique to me?

I don't mind if the document is in spanish or english.

thanks!

I wrote a long reply to this last night, but it must have gone astray for some odd reason...

I'll try to answer it again later tonight when I get some time...

Chris.

thx anyway. I'll be waiting

What you need to do is called a fecal flotation. This works by using a test tube of fluid with a very high specific gravity to float worm eggs and oocysts to the surface where they stick to a coverslip. It's a relatively simple procedure and needs very little equipment, however a lab will have things like a centrifuge which can make the results more accurate.

If you google "fecal flotation" you'll get lots of information but here is the basics of the process:

Make a super-saturated solution of magnesium sulphate (epsom salts, from the supermarket).

Collect some fresh poo. It's normal to use a 2 or 3g sample for each test however for tortoises you might have to settle for less. Weigh the poo if possible and keep a record in this case. The number of eggs or oocysts found in a given weight of sample gives you the parasite load for that animal.

Place the poo in a small beaker and add some floation solution just to moisten. Mix the poo up until it's not lumpy then add a bit more flotation solution, but keep the volume slightly less than that required to fill a test tube. Leave it two minutes then mix it again and pour it through a fine mesh filter into another beaker.

Pour the strained solution into a test tube, then top the test tube up with more flotation solution. You need to fill the test tube right up to the rim until you get a meniscus (dome) of liquid at the top. Do NOT overflow the test tube otherwise you could spoil the test.

Now place a clean cover slip over the test tube so that the solution contacts the coverslip. Things like worm eggs will float to the surface and stick to the coverslip. Leave the coverslip in place for 30 minutes. Some sources you read will say 15 or 20 minutes however this is NOT enough.

At the end of thirty minutes lift the coverslip straight off the test tube so that it retains the drop of solution that was at the top of the tube. If you do this wrong (lifting at an angle for eg.) you can leave the worm eggs etc in the test tube.

Place the coverslip drop side down on a clean slide, making sure there are no air bubbles (yeah right!)


Believe it or not, that was the easy bit. The really hard bit is finding, counting, and identifying worm eggs and encysted protists such as coccidia. You'll need to find some photographs of the types of parasites you are likely to find to help with identification. These are likely to be four major types of parasites: Cestodes, Nematodes, Trematodes, and protists such as coccidia.

Coccidia is smaller while the worm eggs are generally much larger. There are many Trematodes that infect mulluscs and some of them are thought to also infect vertabrates such as turtles and tortoises, presumably through the food chain. There's also a Tapeworm (cestode) that can use reptiles for part of it's life cycle and can be passed on to cats, dogs, and humans. It's called spirometra erinacei, or the zipper tapeworm.

It's also possible you'll be seeing the infective larval stage of some of these worms which use reptiles as part of their life cycle, so you need to exercise some caution.

The technique to count parasites is to scan the slide methodically and keep notes of numbers and types of parasites. You'll need to use 100X (total) magnification for the scanning, with perhaps 400X to help with identification. I have a 60X objective (600X total mag) that I find very useful for this purpose however 400X is fine.

A mechanical stage is useful for scanning the slide because it's easy to stay on one track. Scan the slide back and forth in strips until you've scanned the entire slide.

You'll have to ask questions if I haven't made something clear, I'm not sure I've done a good job of explaining the process tonight. A list of minimum lab equipment would probably be useful:

2 beakers
stirring rod or stick
flotation solution
fine mesh filter
test tube (16 or 18mm are good)
Test tube rack
coverslips, slides etc.

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Chris.

As Dr Chris said, finding the parasite may be the hard part, but finding the parasites with the microscope is a matter of technique. I don't know what type of microscope you have or how much experience you have. If you have a microscope with an adjustable condenser and an iris diaphragm that will work the best. By lowering the condenser you will increase the contrast of the specimen. It also works if you reduce the size of the aperature. This is not the best way but it does help in finding the specimen. You can always adjust the illumination after you have found the specimen. If you have a phase contrast microscope then the problem of finding an unstained specimen goes
away and viewing becomes much easier. g2b2

Not "Dr", just plain old 'Chris' will do. I hadn't made any assumptions about sssss's experience with the microscope, or even what type of 'scope he has. No doubt it will come clear as he asks more questions, or tells us about his experiences. No matter what that may be I'm happy to help as much as I can.

I must admit I'm curious as to whether they are pet tortoises or animals in an open environment. Pet tortroises are likely to have less parasites than those living in the wild simply because it's a closed system with a clean food supply, however a small system (ie tank) with a heavy parasite infestation would be a serious problem.

I'm currently working on developing a staining technique to help with counting worm eggs, specifically strongyloids such as those that affect livestock. I'd expect this to be useful to farmers and hobbyists who do their own testing and use a student grade light microscope; progress is slow due to time constraints however.

Chris.

I have to confess: this will be my first experiment with a microscope, so thank you Chris for being so descriptive. I am a frustrated vet (I chose computer engineering, instead) but I have at home my grandfather microscope. He was a chemist. He did blood analysis with that microscope, so I hope it will be enough for my 'experiment'

I want to see my pet tortoises poo. My first analysis of feces only included:
1) Is it runny?
2) Does it have stones?

Now I want to improve, and as I have the microscope... the logical step to take is learning how to analyze feces like a prof.

I'll tell you my results and put photographs as I take them.

I am sure I have parasites, as the Iguana (yes, i have an iguana too) threw out a tapeworm. For the record, i am already going to the vet and taking medication. Well, not me, just my fauna.

Well it looks like you're in for an adventure if this is a new hobby then. A tank with tortoises is a whole environment to explore, not just poo.

If the scope was used for blood analysis it's probably quite enough to analyse poo. More modern scopes tend to make things easier in a number of ways, such as being more parfocal and parcentral, and things like mechanical stages were more rare. An older scope may benefit from some modern wide field eyepieces, which makes scanning slides a bit easier. No need to worry about that just yet though.

If you haven't been directed there already, you might want to have a look around the microscopy-UK site which has lots of information for microscopy hobbiests:

Microscopy-UK full menu of microscopy and microscopes on the web

Chris

It's a great site, but a bit overwhelming... so much information!!! I'll peruse through it later, it's not a matter of 5 minutes, so I just can't in my working hours

Well there is a lot of information to assimilate, it's going to take a little time to get over the steep learning curve.

I'm just about to start experimenting with a salt/sugar floatation solution. It has a higher specific gravity and may work better for different kinds of eggs. Here's a message I just sent to a friend who's also been working on floatations:

============================================
I've just made up a new batch of floatation solution, but used an idea from the Food & Agriculture Organisation web site. They describe salt/sugar solutions using sodium chloride and ordinary sugar, presumably because they're (relatively) easily available in third world countries. SG is 1.28

I decided to see if I could improve it by using epsom salts and fructose.

300ml water
120gm MgSO4
100gm Fructose

Dissolve the MgSO4 in water SG=1.2 (super-saturated solution). Add the Fructose and mix. SG = 1.28

That's the same as the sodium chloride/sugar solution however I don't think the sugar is in saturated solution. Viscosity is very low though, so I don't want to add any more fructose at this stage. The reason we decided not to use just sugar originally was because the higher viscosity requires the use of a centrifuge, however this solution looks workable for ordinary floatations. The stickiness/mess was also a concern however having been working with fructose as a mountant I don't think it's going to be a real issue. From my work with fructose mountant I've also found that the crystalisation/evaporation was very low, so much so that it's impossible to get the coverslip to stay in place (on it's own) even if you leave it for days. I'm hoping there'll be some advantages to this solution such as better drop (and egg) retention on the coverslip due to the slightly higher viscosity, and reduced evaporation and crystalisation which will allow more observation time, and the possibility of making a semi-permanent slide for study/photography.

The higher SG may make it easier to float Toxascaris and flukes, and possibly even tape worm if they are present. The solution we were using is fine for strongyloids and coccidia as you know, but borderline or insufficient for others.

Just a last note on sugar-only solutions: Their use is mainly in the lab with the use of a centrifuge, because eggs collected this way are still viable and can be cultured. Salt solutions tend to affect the viability of the eggs, and presumably coccidia.

Chris.
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I haven't had a chance to try the new solution yet, maybe this week...

Chris.