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 glass beaker 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 beaker. Let the solution cool to room temperature. Notice that the volume of the solution has doubled after it has been super-saturated with the Epsom salt. There will be a lot of salt crystals on the bottom of the beaker and a film covering the surface, pour the solution through cheesecloth into a glass jar that can be closed air-tight. 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 float solution to 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. For clarity I used clear liquid for the photos.
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 fingernail 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 until you reach the edge of the cover slip, then down, move back the other direction until you reach the edge of the cover slip again, then down, continue until you reach the end of the sample. As you go along you are looking for eggs, oocysts, and worm larvae, as well as non-parasitic objects that will be present in the sample. If you see something interesting, switch to the 40X objective to identify it. Each time you see an egg, oocyst, or worm larvae, make a notation and a tick mark on a piece of paper to keep track of what you find and how many there are. Remember to return to the 10X objective and continue your search. I like to begin by finding air bubbles, they are perfectly round and have very dark, thick outlines and clear centers. The organisms we are trying to locate are floating at the top of the sample, so are the air bubbles. If you focus sharply on an air bubble before you begin you will likely be at the correct level in the solution to find parasites.
IDENTIFYING PARASITES IN THE SAMPLE
Beginning the search
The effects of salt solutions on parasite shape should be understood. Hypertonic solutions draw water from the bodies of larval parasites and parasitic protozoa killing them and distorting their outlines to such an extent that they may no longer look like typical parasitic species. This can make these organisms difficult to identify. The fecal exam must be done as soon as possible after it is prepared to minimize the time that the organisms are exposed to the distorting solution.
When you begin scanning the slide, don't get too excited about finding any particular parasite and stop searching. You need to scan the entire slide because the parasite that you have found may be present but not the parasite responsible for any clinical symptoms, therefore you could miss the parasite actually causing the problem. That being said, never assume that a parasite found during the exam is the sole parasite responsible for the animals symptoms. If you treat for a parasite, monitor your animal for some time to see that it has made a full recovery, if not, you may have another infection or multiple infections.
Because we are dealing with such small samples I consider any population over 10 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 so don't treat for parasites if you only have a few. For less than 10 of any given type of parasite 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 10 or more, do another fecal exam in a few days with fresh samples and see what happens. If you get 3 exams over 10 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.
Getting down to business
It is generally believed that all amphibians living in the wild have parasites present. However, the relationship between parasite and host in the wild is completely different than in captive animals. This is because in the wild the animal is not confined to a small space and the environmental concentration of parasites is low. In captivity, especially in unsanitary conditions, the concentration of parasites can be overwhelming. If you then add the stress of poor husbandry like incorrect temperature, lighting, humidity, nutritional supplementation, and overcrowding, the parasite load in the animal can become too great and it will succumb to the infestation and die.
In this section I am going to discuss the more common parasites that you are likely to find during your exam and a final section where I will show you examples of non-parasitic objects that can be mistaken for parasites. Keep in mind that this is for reference only and a diagnosis for treatment should be made by a veterinarian familiar with amphibians. It is also a good idea to buy some books and get familiar with what these parasites look like, the more exposure you have with them, the easier they will be to find.
Enteric Parasites (Intestinal)
Protozoa
The concentration and type of protozoa is influenced by many factors, these include the health of the individual animal, conditions in the intestinal tract, pH, hydration and many others. It can also depend on the predation of one type of protozoa on another. You can make identification easier by studying the type of protozoa that you are likely to encounter in the species of animal that you are testing.
Flagellates:
The most likely organism to cause problems in captive populations is Chilomastix quadrii , frequently found in frogs. Tritrichomonas and Trichomonas are common in the intestine of salamanders, frogs and toads. Giardia cysts are frequently seen in amphibian fecal exams. Retortamonad and diplomonad flagellates have been found in poison dart frogs.
Ciliates:
Balantidium species are common in herbivorous turtles and lizards. Balantidium is the only ciliated protozoan known to infect humans and includes B.coli among it's members. Contaminated water is the most common mechanism of transmission. They are usually not considered pathogenic unless present in very large numbers. However, the conditions in captivity can swing the balance in favor of the parasite and I would recommend treating for them. I only mention this parasite because Nyctotherus, another very large ciliate, has been found in anurans and their cysts are easily mistaken for trematode eggs.
Opalinids:
These organisms resemble flagellates and ciliates except they do not have a cell mouth and have only one type of nuclei. Zelleriella have been found in dendrobatid frogs.
Amoeba:
This is a highly pathogenic parasite in amphibians. Symptoms include anorexia, weight loss, blood or mucus in the feces, vomiting, green discoloration to the urine, or swelling of the body. Several species of pathogenic amoeba have been responsible for fatal infections.
Coccidia:
Isospora and Eimeria are the coccidia most recognized in the fecal specimens of infected amphibians. Symptoms include listlessness, anorexia, regurgitation, and intestinal hemorrhage.
Helminths
More commonly referred to as parasitic worms. These are worm-like organisms that live and feed off of living hosts, receiving nourishment and protection while damaging the hosts' nutrient absorption, causing weakness and disease.
Nematodes: (roundworms) Cylindrical
Rhabdias (amphibian lungworm) is found in the hosts lungs and may cause respiratory distress. Strongyloides can produce diarrhea and respiratory distress as infective larvae migrate through the lungs. Symptoms of both parasites include anorexia, weight loss and debilitation. The life cycle is direct and the parasite can be transmitted by ingestion of eggs, larva, or even direct skin penetration. These parasites have a free-living form, making proper and complete enclosure sanitation a necessity.
Cestodes: (tapeworms) Segmented plane.
These tapeworms are hermaphroditic and non-host specific. Transmission is by ingestion of an intermediate host. Plerocercoids (infective larva) of Diphyllobothrium are frequently found in tadpoles feeding on crustaceans.
Trematodes: (flukes) Unsegmented plane.
Eggs in the fecal sample or oral mucus are indicators that these flukes are present. Some Monogenea are external or urinary bladder parasites of frogs, tadpoles and newts. Digenean trematodes (flatworms) often form metacercariae (encysted larva) in the skin, eye or various organs.
Bacteria
You will always find millions of bacteria on a fecal float exam.They are natural residents of the colon and feces and are of little or no diagnostic significance in a fecal float examination.
Below is a series of photos I took from some of my animal fecal float exams. I will continue to add to this list as I find more.
Coccidia Oocyst
Nematode Egg
Tapeworm Egg
Distorted Nematode Against An Air Bubble
Tapeworm Egg
Non parasitic objects and artifacts that may be seen during a fecal float examination.
Fecal matter
Pollen
Plant matter
Bubbles
Bacteria
Flotation medium crystals
Hair
Plant Matter
Plant Matter
Air Bubbles
Air Bubble
Nematode Egg In Bacterial "Soup"
Float Medium "Salt" Crystal
Float Medium "Salt" Crystal
Ingested Hair And Air Bubble
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 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 eyepiece 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 microscopes.
Article and photos are ©2010 Paul W. Rust. All rights reserved. This article may not be reproduced or published in part or in whole without written permission from Paul W. Rust.
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