Author Fred Lewis

Introduction

Numerous procedures in a schistosomiasis laboratory necessitate counting cercariae. One such procedure is the exposure of small mammals such as mice to cercariae for infection, where it is important to obtain an accurate estimate of the number of cercariae to which each mouse is exposed. The following procedure is written for S. mansoni cercariae, but it works well for counting S. haematobium cercariae as well.

Equipment

Dissecting microscope

Materials and reagents

Suspension of S. mansoni cercariae

Iodine solution (4 g potassium iodide and 2 g iodine dissolved in 100 ml distilled water)

Adjustable Eppendorf pipette (200 µl +)

Aged tap water

60 mm diameter, scored plastic petri dishes

Procedure

·         Withdraw 200 µl of a cercarial suspension. It is important to mix the suspension gently to evenly distribute cercariae before withdrawing aliquots.

·         Place the aliquot in a scored, 60 mm diameter petri dish.

·         Add a sufficient amount of aged tap water to cover the bottom of the petri dish.

·         Add 1-2 drops of the iodine solution, mix and let stand for about 1 minute. This allows the stained cercariae to settle to the bottom of the dish.

·         Under a dissecting microscope count the heavily stained cercariae (photo – stained cercaria). Note: The cercariae will be killed by the iodine solution, and are, therefore, more easily counted.

Follow-up comments/recommendations

Although variations of the above procedure may be used, we emphasize the importance of counting several aliquots of the cercarial suspension and taking an average of those counts. Because consistently obtaining an even suspension of cercariae is difficult, an accurate estimate of the number of cercariae per ml can be made only by taking multiple counts. Note: If the original cercarial suspension is approximately 2000/ml and above, the cercariae tend to clump together, making an accurate count even more difficult to achieve. The suspension should then be further diluted with aged tap water to prevent clumping and improve counting accuracy.

 

References

Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).  Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.