HOW TO DO AMPHIBIAN FECAL EXAMS
© Paul W. Rust – NW AMPHIBIAN RESCUE
PERMISSION FOR USE ON FROG FORUM GRANTED
THE FLOAT METHOD Making the flotation solution.
The first thing we need to understand is the relationship between solution density and whatever we are trying to float in it. This relationship has to do with specific gravity. Specific gravity is the density of a liquid compared to water and is sometimes referred to as relative density. Pure water has a specific gravity of 1. The density of all other solutions are compared to water. If an item has a density of less than 1 it will float in water, if it has a density of more than 1 it will sink in water.
Parasite eggs have a density of 1.1 to 1.2 and will sink in water, therefore we need to create a solution that has a density of 1.2 or more. Debris in the fecal matter has a density of 1.3 and will float along with the eggs obscuring and deforming them if the solution density is too high.
What we need to do is dissolve a sugar or a salt in water to raise the specific gravity to just over 1.2. Sugar can be used but it is sticky and it is difficult to get the specific gravity above 1.2 which means that larger eggs will not float. However, I use a sugar solution when I use centrifugation. Table salt can also be used but it is very corrosive to lab equipment and forms crystals that can distort the eggs. Another problem with table salt is that you can never get the specific gravity above 1.2 so again you have the problem of larger eggs not floating.
The best thing to use is Magnesium Sulfate (Epsom Salt). It is not expensive and works very well for a flotation solution because it has a specific gravity of 1.29. This makes it dense enough to float all the eggs and not so dense that it will float debris.
Start by pouring whatever amount of distilled water that you are going to store as flotation into a clean sauce pan and heat to a slow boil. Now pour a little Epsom Salt in and stir it around until it dissolves. Keep doing this until you can no longer get the salt to dissolve. You should see some salt crystals in the bottom of the pan. Let the solution cool down some and then carefully pour it into a glass jar that can be closed air-tight. Let it sit overnight and in the morning the crystals will have dissolved and it will be super-saturated. Keep the bottle sealed and it will last for months.
Preparing the sample.
Our samples are going to be very small so we need scaled down equipment to make this work. I do enclosure testing because I treat for parasites on a regular schedule, If I detect a problem i will then target individual animals. Collect as many fresh droppings as you can find and deposit them in a small glass beaker. Using an eyedropper add enough float solution to half fill a small test tube and dissolve the feces. Now use a popsicle stick or glass stir rod to smash and stir the whole mess to mix it all up. Now strain the mixture through cheesecloth or let it sit for a few minutes and collect the fluid from the top. Now fill a small test tube almost to the top with the strained fluid. Set the test tube in a holder and using the eyedropper carefully fill the test tube with float solution until you have created a convex meniscus, fancy talk for a slight bulge of fluid above the test tube. Follow the pictures below to see how it all looks.
Gently place a microscope cover slip straight down on top of the test tube. Some liquid will run down the test tube. Let it sit undisturbed for about 15-20 minutes to give the microscopic parasite eggs time to float to the surface. The eggs will stick to the cover slip when they reach the top. Don't wait too long or the float solution will begin to dry and distort the eggs.
After the correct amount of time has passed, lift the cover slip straight up and place on a glass microscope slide. Push down gently with your figernail on the cover slip to remove excess liquid. Now the sample is ready for inspection under a microscope.
Searching for eggs under a microscope.
Place the slide on the microscope stage and bring the sample into focus using the 10X objective. I use an inverted microscope with no mechanical stage so I can quickly scan the sample and my slide is upside down. Your microscope likely has a mechanical stage so get used to how it works and your slide will be right side up.
Remember that it will be like looking into a mirror, left is right and up is down. Scan the slide using a pattern similar to this; starting from the top left corner move the slide over then down, over then up, over then down, over then up... continue until you reach the end of the sample. As you go along you are looking for eggs as you will not see the actual parasites except in a few cases you might see a worm. Each time you see an egg make a tick mark on a piece of paper to keep track of how many there are. Now, what do parasite eggs look like? Good question.
Identifying parasite eggs.
In this section when I say "identifying parasite eggs" I mean recognizing the eggs from the other stuff. We don't care one bit about species identification. If we can see them under 10X we are dealing with worm eggs. When I go looking for protozoa I use the 40X objective lense and higher and a centrifuge. I am not going to get into that because most people do not have a centrifuge or the necessary equipment that goes with that method. This is only a quick check for parasites to see if further investigation is needed. As you scan the slide you are looking for oval shaped eggs with opaque centers. You are going to see a lot of junk and air bubbles that look like eggs. Air bubbles are perfectly round with a very dark edge and bright center. After a little practice you will be able to see the eggs very easy. Buy a book like "Understanding Reptile Parasites" and study the pictures to help you find eggs. How many eggs is too many? Another good question.
Because we are dealing with such small samples I consider anything over 10 eggs to be too many. Remember that you want some parasites in the intestines and you also want the animal to retain it's natural defenses against parasites so don't treat for parasites if you only have a few. For less than 10 eggs just call it a day and test again next month. You need to test again even though the sample was acceptable because it might not have been shedding when you tested but could still have a heavy parasite load, this is why I randomly test also. If you get 20 or more, do another fecal exam in a few days with fresh samples and see what happens. If you get 3 exams over 20 then you need to quarantine one animal at a time and do individual fecal exams to isolate the infected animal, they are all likely infected. Once you identify the infected animals you need to treat those animals if you are prepared to do so, or get them to a vet for diagnosis and treatment.
Notes.
I do a three day run of Panacur at the beginning of every month as a parasite maintenance measure. I always have some parasite eggs in my exams but that is what I want. For the average frog keeper I would say that after you decide that you have an infected animal let a vet do the rest.
Equipment.
As far as supplies go, you can get everything you need from a student science supply store online pretty cheap. You don't need an expensive microscope, you only need 10X and maybe 40X objectives. All basic microscopes have these. You can get one for around $100.00. I would recommend that you try and get a stereo microscope so you aren't squinting into one little objective and getting a headache. One of the best mid level microscopes is the American Optical AO-150. It has a wide and clear field of view and stereo objectives. These are older model laboratory microscopes and very well built. They are about $300.00. I still use mine now and then to verify something I thought I saw with my main microscope.
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