VLD 411: Veterinary Clinical Biochemistry and Laboratory Diagnosis - I (0+1)

KNOTT'S CONCENTRATION METHOD

• This is the standard test used to screen for microfilariae in blood. It includes lysis of erythrocytes, fixation and staining of larvae.

Procedure

• Mix 1 ml heparinized or EDTA treated blood with 9 ml of 2% formalin in a 15 ml tube; mix by inverting the tube several times after closing the tube with your thumb.
• Centrifuge the tube for 5 min at 1500 rpm, then discard the supernatant.
• Add a few drops of 0.1% methylene blue or 0.2% malalachite green solution to the sediment and mix well.
• Remove a drop of stained sediment with a pipette, spread it on a slide and add a coverslip.
• Examine the complete sediment drop by drop, under the microscope.

Result

• Microfilariae appear as elongate and cylindrical with purple shaded internal structures in a blood smear stained with Leishman's or Giemsa's technique. While using Knott's technique, they appear either blue or green following the stain used.   
• Note that heparinized (EDTA-treated) and formalinized blood samples are suitable for mailing to a laboratory for identification of microfilariae.