Knott’s Test
Aims: This test allows for the examination and identification of parasitic species containing microfilarial life stages present in the blood. This is most commonly performed for the identification of microfilariae of Dirofilaria immitis, Dirofilaria repens and Achantocheilonema reconditum in the blood of companion animals.
Materials:
- Whole blood sample (stored in a collection tube with a suitable anticoagulant)
- Distilled water
- 1 15 mL centrifuge tube
- 1 rubber stopper
- Pasteur/transfer pipettes
- Laboratory centrifuge
- Methylene Blue Dye
- Microscope slides and coverslips
Procedure: Add 1 mL of whole blood to 9 mL of distilled water in the centrifuge tube to create a dilution ratio of 1:10. Centrifuge the sample for 5 minutes at 1500 rpm. Using a Pasteur/transfer pipette, remove the supernatant leaving only the pellet in the tube. Add an equal volume of Methylene Blue dye (~1-2 drops) to the sediment. Gently resuspend the pellet into the dye and allow to stand for approximately 15 minutes to allow the dye to penetrate into any microfilariae present.
Results: Collect a small amount of the sediment (~ 50 µL) using a Pasteur pipette and place it on a microscopic slide. Cover the sample with a coverslip and examine under the microscope at 10x magnification. If microfilariae are present, specific identification can be accomplished based on the size (length and width) and morphological features of the anterior and posterior ends of the microfilariae. To see these features and obtain precise measurements, the 20x and 40x objectives are recommended.
References:
Knott Test:
Lindsey LR (1965) The identification of canine microfilariae. Journal of the American Veterinary Medical Association. 146:1106-1114
Mylonakis, M.E., Papadopoulos, E., Koutinas, A.F., Paitaki, C., Leontides, L. (2004) Comparative methodology for the detection and differentiation of circulating microfilariae of Dirofilaria immitis in the dog. Journal of Helminthology. 78(2): 137-140.
Identification of microfilariae:
Magnis, J., Lorentz, S., Guardone, L., Grimm, F., Magi, M., Naucke, T.J., Deplazes, P. (2013) Morphometric analyses of canine blood microfilariae isolated by the Knott’s test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis. Parasites Vectors 6, 48.