Qualitative Faecal Centrifugation-Flotation

This technique is usually applied to faeces, or sometimes intestinal contents, to recover eggs of helminth parasites and cysts and oocysts of intestinal protozoa.

If faeces are thoroughly mixed with a flotation solution of relatively high specific gravity (e.g. sugar at SG 1.27), filtered and left to stand the lighter weight eggs, cysts and oocysts will float to the surface of the fecal suspension. They can then be recovered and examined microscopically.  Centrifugation of the fecal suspension significantly improves the efficiency of the flotation procedure by bringing more eggs to the surface of the suspension.

• High Density Flotation Solution (Sheathers sugar flotation solution used at WCVM)

• Centrifuge

• Centrifuge tubes

• Paper cups

• Tongue depressors

• Cheesecloth or tea strainer

• Microscope slides and coverslips

• Compound Microscope

1. Weigh out (estimate) 1-2 grams of faeces and mix with 10ml of flotation solution (Sheather’s flotation solution) in a paper cup, stir until faeces is uniformly suspended in liquid.

2. Filter the fecal suspension through a single layer of cheesecloth into a second cup, squeeze cheesecloth plus contents into the paper cup (using tongue depressor) and then discard the cheesecloth and faeces.

3. Pour filtrate containing eggs, cysts and oocysts from second cup into centrifuge tube to about 1 cm from the top and place tube into centrifuge.

4. Using a dropper bottle or eye dropper fill tube with sugar solution to form a slight convex meniscus.

5. Very carefully VERTICALLY place a coverslip on top of the meniscus ensuring that there is minimal dripping of solution down the outside of the tube.

6. Centrifuge at 1500 rpm for 10 minutes (make sure the centrifuge is balanced); eggs, cysts and oocysts rise and debris sinks.

7. Very carefully remove the coverslip by lifting vertically and place on a microscope slide (Note: if >50% of the space beneath the coverslip in the tube is occupied by
air bubbles, start again).

8. Examine the preparation at 100x total magnification for the presence of eggs, cysts and oocysts.

1.Weigh out (estimate) 1-2 grams of faeces and mix with 10 ml of sugar solution (Sheather’s flotation solution) in a paper cup, stir until faeces is uniformly suspended
in liquid.

2. Filter the faecal suspension through a single layer of cheesecloth into a second cup, squeeze cheesecloth and contents into the paper cup (using tongue depressor)
and then discard the cheesecloth and faeces.

3. Pour filtrate containing eggs, cysts and oocysts from second cup into centrifuge tube to about 2 cm from the top and place tube into centrifuge. (DO NOT place a
coverslip on the tube).

4. Centrifuge at 1500 rpm for 5 minutes (make sure the centrifuge is balanced) – eggs, cysts and oocysts rise and debris sinks.

5. Remove the test tube from the centrifuge and using a dropper bottle or an eye dropper fill the tube to the top with sugar solution so that a slight convex meniscus is
formed.

6. Very carefully VERTICALLY place a coverslip on top of the meniscus ensuring that there is minimal dripping of solution down the outside of the tube.

7. Let stand for 10 minutes.

8. Very carefully remove the coverslip by lifting vertically and place on a microscope slide (Note: if >50% of the space beneath the coverslip in the tube is occupied by
air bubbles, start again).

9. Examine the preparation at 100x total magnification for the presence of eggs, cysts and oocysts.

Details of the methods used may vary with laboratory, and different flotation solutions are used for different parasites: Sheather’s sugar solution works very well for the detection of all important helminth eggs (nematodes, cestodes and trematodes) and lungworm larvae; zinc sulphate (1.18 SG) allows Giardia cysts to be detected.

This technique minus centrifugation is still sometimes used, and there are commercial kits available. Several studies have shown, however, that the lack of centrifugation significantly reduces test sensitivity.

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
• Check the specific gravity with a hydrometer and adjust accordingly
• Also available commercially through WDDC

Zinc Sulfate solution: 1.18 SG (good for Giardia)

• 386 grams of ZnSO₄ crystals (ZnSO₄ Heptahydrate 99-103%)
• 1 liter of water
• Mix thoroughly
• Check the specific gravity with a hydrometer and adjust accordingly

Saturated Salt Solution: 1.20 S.G.

• 400g Sodium Chloride
• 1 liter of water
• Dissolve salt and water in the top of a double boiler or with gentle heat
• Check the specific gravity with a hydrometer and adjust accordingly

Sodium Nitrate solution: 1.20 SG (commercial solution used with the Fecalyzer and Ovassay kits)

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.