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 depressors

• Paper cups

• Cheesecloth

• Wash bottle

• Syringe or other instrument capable or measuring 15 ml

• Test tubes

• Centrifuge

• Sheather’s flotation solution

• Vortex mixer or wooden applicator sticks

• Dropper bottle

• Slide and coverslips

• Compound Microscope

Procedure

1. Use a tongue depressor to weigh 5g of faeces into a paper cup (if there is less than 5g of faeces then weigh out the amount available and record on the data sheet).

2. Add 12ml of tap water to the faeces (if faeces are very dry, allow to soak for 5-10 minutes).

3. Use the tongue depressor to mix the faeces and water until homogeneous.

4. Pull a single layer of cheese cloth over the top of a second paper cup. Squeeze first cup to create a  spout and pour the faecal mixture through the cheese cloth into the second cup.

5. With a wash bottle, rinse the first cup with approximately 3ml of tap water and pour through the cheese cloth filter into the second cup.

6. Pour the filtrate into a 16x125 mm test tube.

7. Centrifuge the test tube for 10 minutes at 1500 rpm.

8. After centrifugation decant the supernatant using a single pouring motion, taking care not to disturb the sediment.

9. Add 4-5 ml Sheather’s solution to the sediment, mix well using a vortex mixer or wooden applicator stick and then fill the test tube to 5 mm from the top and place the tube back in the centrifuge..

10. With a dropper bottle add Sheather’s solution until there is a slightly convex meniscus.

11. Carefully vertically place a 22x22mm coverslip on the meniscus at the top of the tube.

12. Centrifuge the test tube for 10 minutes at 1500 rpm.

13. Lift the coverslip vertically and transfer to a slide. Do not slide the coverslip along the top of the tube.

14. Examine the slide under 100x total magnification. Results are recorded as eggs per 5 grams of faeces.

15.  Calculate eggs per gram of feces by dividing the number of eggs on the slide by 5.  If less than 5 grams of feces was used during the test procedure divide by the actual starting weight.

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.

Flotation 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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