Laboratory Analysis of Protozoan Cysts, Helminth Ova, and Helminth Larvae and Parasite Burdens

    The zinc sulfate flotation technique and the Stoll dilution egg count method are clinical methods to determine the presence or absence of parasitic organisms and also the approximate parasite burden.  The zinc sulfate flotation technique breaks up feces and using differential specific gravities, isolates the protozoan cysts, and helminth ova and larvae.  The Stoll dilution method approximates the number of worms present by determining the daily egg capsule output.

ZINC SULFATE FLOTATION

1.  Mix one to a few grams of feces with 10 times its volume of distilled water.
2.  Strain 10 ml through gauze into a centrifuge tube.
3.   Centrifuge at 2500 RPM for one minute.
4.  Discard the supernatant and add 2-3 ml of water to the sediment, breaking up the sediment.
5.  Repeat this procedure until the supernatent is nearly clear. (usually 2-4 washings).
6.  Pour off the supernatant and mix the sediment with 2 ml of zinc sulfate (38.6 g/100 ml of distilled water).  Fill the centrifuge tube to within 5 mm of the top.
7.  Centrifuge again for 5 minutes.
8.  Let the sample stand for 5 minutes.
9.  Using a bacteriological loop, remove several loopfuls and examine microscopically.
10.  Add a drop of Lugol's iodine to stain.
11.  Examine for protozoan cysts and helminth egg capsules and larvae.

STOLL DILUTION EGG COUNT

1.  Calibrate a 75 ml or 100 ml Erlenmeyer flask to a 56 ml mark and a 60 ml mark or use a Stoll Flask if one is available.
2.  Fill the flask to the 56 ml mark with 0.1 N NaOH and add feces until the volume reaches the 60 ml mark.
3.  Add about 10 glass beads, stopper and shake until mixed thoroughly.  You may have to let the mixture stand overnight to soften the feces if it is dry or hard.
4.  Shake the flask and immediately before settling, pipette 0.15 ml onto a wide slide and cover with a 22x40 mm cover slip. 
5.  Count all the ova (egg capsules) of a single type of parasite.  If more than one organism is represented, count all the ova of each parasite.
6.  Multiply that number by 100 to estimate the number of ova (egg capsules)/ml or nearly equivalent to ova/gram.
7.  Determine the weight of feces/day the host deposits and from that a number can be determined to approximate the daily output of ova (egg capsules) by that host.
8.  The daily output divided by the number of ova (egg capsules) a single worm produces daily provides an approximation of the number of ova-producing worms (females) present.