Flotac®
Aims: This commercially available test allows for the quantitative analysis of parasitic eggs, oocysts, and larvae found in animal faeces.
Here we perform the Flotac® basic technique on a sheep faecal sample.
Materials:
- Faecal sample
- Weighing balance/scale
- Flotac®
- Flotation Solution (e.g. Zinc Sulphate, s.g. 1350, that is one of the most used solution for ruminants, but we can use a lot of solutions based on our target)
- 2 500 mL plastic containers
- Graduated cylinder
- Immersion blender
- Fine mesh strainer
- 1 serological pipette
- Centrifuge tubes
- Laboratory centrifuge
- Pasteur/transfer pipettes (volume: 3 mL)
- Reading stand for microscope
- Hand-held tally counter
Procedure: Weigh 10 g of faeces and place it in one of the two plastic containers. Using a graduated cylinder, measure 90 ml of tap water, and add it to the sample (dilution ratio: 1:10). Homogenize the contents with an immersion blender. Filter the solution through a fine mesh strainer (to remove larger debris) and transfer the filtered contents into the second plastic container. While keeping the suspension thoroughly mixed, collect 6 ml of the filtrate with a serological pipette and transfer it into a test tube. Centrifuge the solution for 3 minutes at 1,500 rpm. Discard the supernatant by carefully pouring it off into a sink while keeping the pellet in the bottom of the tube. Refill the tube with floatation solution, restoring the initial volume of 6 mL, and resuspending the pellet into the solution. Assemble the Flotac® device (see figure). Fill the chambers of the device with 5 mL of the resuspended faecal solution. Rotate the Flotac® to the closed position and centrifuge it for 5 minutes at 1,000 rpm. After centrifugation, loosen the locking key and rotate the device to the reading position.
Results:
Place the Flotac® on the microscope, affixing it with the supplied adapter. Examine each reading chamber at 10X magnification objective and count all parasitic form using the hand-held tally clicker to record the number of organisms seen. The total number of eggs counted is then multiplied by a multiplication factor that varies depending on the dilution ratio used. Using the dilution ratio of 1:10, as previously mentioned, would require a multiplication factor of 2. Total # of eggs counted x 2 to obtain the number of eggs per gram of faeces (EPG). This same process may be used for oocysts and larvae to obtain the OPG and LPG, respectively.
N.B.
Each Flotac® device has two separate reading chambers allowing for samples to be processed one of three ways. The basic technique involves processing the sample in duplicate with the same floatation solution. The dual technique uses two different floatation solutions for the sample. Finally, the double technique allows two different samples to be processed at one time with the same floatation solution.
The dilution ratio should be adapted to the expected number of parasitic elements (PE). The count results with a dilution ratio 1:10 are accurate when the number of PE is under 500 per grams of faeces. When the PE per gram are greater than 500 (that frequently occurs in sheep and goats) it is advisable to dilute the sample more. E.g. when the expected PE per gram are 500-1000 the dilution ratio should be 1:20 and the multiplication factor should be changed accordingly (x4)
References:
Cringoli, G., Rinaldi, L., Maurelli, M.P., Utzinger, J., 2010. FLOTAC: new multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat. Protoc. 5, 503-515.