Purpose

This technique is usually applied to faeces, or sometimes intestinal contents, to recover and count eggs of helminth parasites and cysts and oocysts of intestinal protozoa. The resulting data, usually recorded as eggs per gram of feces, provides an estimate of parasite infection intensity within a host.

Principle

This flotation technique is conducted on a known weight of faeces and uses known volumes of flotation and other solutions. Parasite stages are separated from faecal debris and counted . Using appropriate calculations the egg counts can be converted to a number of eggs per gram of feces. This number can be used to estimate relative parasite infection burden.

Equipment

• Scale
• Tongue depressor
• Paper cups
• Cheese cloth or Tea Strainer
• Test tubes
• Sheather’s solution
• Funnel
• Centrifuge
• Dropper bottle
• Slide and coverslips
• Microscope

Procedure

1.Fill a 15 ml test tube with 10 ml of Sheathers solution.
2.Weigh 3 grams of faeces and place into a paper cup.
3.Pour the Sheathers solution from the test tube into the cup and mix well using a tongue depressor.
4.Place a funnel into the test tube, place a strainer or cheesecloth into the funnel and pour the fecal-sugar solution mixture through the strainer into the test tube. Using a tongue depressor, squeeze the liquid out of the faeces that is left in the strainer.
5.Centrifuge the tube in a fixed-head centrifuge for 2 to 4 minutes at approximately 1500 rpm.
6.Fill the tube to just over the top with Sheathers solution to form a slight convex meniscus and place a cover slip on to the meniscus.
7.Let sit for 10 minutes then remove the cover slip vertically and place on a slide.
8.Examine the entire cover slip and count the number of eggs..
9.Calculate Eggs Per Gram (E.P.G.) by dividing the total count by 3 (the starting weight of sample in grams)

•    This procedure is for use with a fixed head centrifuge, but can be modified for use with a swinging head centrifuge. After step 4 the test tubes can be filled to just over the top with Sheathers solution to form a slight convex meniscus and a cover slip can be placed on the the meniscus.  The tube can be centrifuged for 10 minutes and then the coverslip can be removed, placed on a slide and examined for eggs.
•    Advantages: this test is quite sensitive since it counts all the eggs in 3 grams of faeces and minimal specialized equipment is necessary

Variations

Variations of the Wisconsin egg count are possible.  Reducing the starting sample weight from animals with high fecal egg counts is common.  Varying liquid volumes to accommodate different centrifuge capacities is also possible.

Flotatiion solutions

The usual flotation solution used for this technique is Sheathers flotation solution. 

Sheather’s sugar solution: 1.27 SG

• 454 g granulated sugar
• 355 ml tap water
• 6 ml formaldehyde
• Dissolve sugar and water in the top of a double boiler or with gentle heat

It is also available commercially through WDDC.

Problems

The most common problem encountered with using the Wisconsin egg count is when there are heavy infections.  In those cases there may be too many eggs to count accurately using the standard technique.

If the faeces are not properly stored between collection and examination, nematode eggs may hatch, releasing larvae, and any of the parasite stages in the faeces may degenerate, making detection and identification difficult or impossible. Problems may also arise from inadequate mixing of faeces and sugar solution or from careless transfer of the coverslip to and/or from the centrifuge tube, causing the eggs to temporarily sink or fluid to run down the outside of the tube.

For some nematodes, cestodes and protozoa, identification to genus or species is not possible on the basis of egg, cyst or oocyst structure. Also, eggs, cysts or oocysts present in small numbers in the faeces, or present in the faeces only sporadically, may not be detected during the flotation procedure or, very rarely, may be missed during microscopic examination.

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