Modified Knotts Test

The Modified Knotts Test is used to detect and identify microfilariae of filarid nematodes in blood. Microfilariae of D. immitis and other nematodes in the blood of hosts can be visualized by chemically lysing the cells in an unclotted blood sample, centrifuging the sample, and collecting and staining the sediment, which will contain the microfilariae.

2% formalin

• Centrifuge and tubes
• Methylene blue
• Microscope + slides

 Add 1ml unclotted EDTA blood to 10 ml 2% formalin and mix.

• NOTE:  Samples for testing are often best taken in the early morning or evening.

2. Centrifuge for 5 min at 1000 to 1500 rpm.
3. Pour off supernatant fluid. Be careful not to disturb the sediment.
4. Mix sediment with equal volume of 1:1000 aqueous Methylene Blue.
5. Examine as a wet mount.

Live microfilariae can also be seen in a direct accumulation of freshly collected, unclotted blood, and in the buffy coat in a microhematocrit tube. The microfilariae, however, cannot be specifically identified using these techniques.

If the dog is infected with only male worms and/or immature or non-reproducing female worms, none of which produce microfilariae, the Modified Knott’s Test will return a negative test result despite a positive infection (occult heartworm infection). Also, since only a very small volume of blood is being tested it is possible to not find any microfilariae in the small sample being tested even though the animal is heartworm positive.

Very rarely in Canada dogs may be infected with the related, non-pathogenic nematode Acanthocheilonema reconditum. The Modified Knott’s test permits differentiation of the two species based on morphology of the microfilariae, but this can be difficult. The heartworm antigen test does not detect A. reconditum. The Modified Knott’s Test is of very limited value for D. immitis in cats because the infection is very rarely patent in these hosts.