Sporulation Assay
Because of the morphologic similarity between freshly passed, unsporulated Cyclospora oocysts and blue-green algae (cyanobacterium-like bodies), it has been advocated that to confirm the diagnosis of cyclosporiasis, unfixed oocysts should be examined over a 2- to 3-week period for evidence of sporulation.  This is accomplished by placing an aliquot of fresh stool in 2.5% potassium dichromate (which reduces bacterial overgrowth), and keeping it under observation for sporulation of the oocysts.*

*As the ability of laboratorians to accurately diagnose cyclosporiasis has improved, the need to do sporulation assays has decreased.

Procedure:
Mix stool with 2-3 volumes of potassium dichromate (depending on the stool consistency) and agitate specimen occasionally during the incubation period.  Leave an air space over the specimen, for example by turning the specimen tube on its side to increase surface area, to promote aeration.  At room temperature, oocysts generally sporulate in 5-14 days, although the percentage of oocysts that sporulate can vary.  If the number of oocysts is small, the material held in potassium dichromate can be centrifuged and the pellet examined.  Prior to examination, the potassium dichromate solution should be diluted (it quenches fluorescence): add to the specimen an equal volume of distilled/deionized water; or remove the dichromate by centrifugation and washing (use distilled/deionized water or various solutions such as PBS, culture medium).  Optimal examination of the specimen, using a wet mount, would include epifluorescence (to identify the oocysts) followed by DIC, phase contrast microscopy, or conventional bright-field microscopy (to identify the sporocysts/sporozoites).  Sporulated mature oocysts contain two sporocysts, each of which contains two sporozoites. (Because the sporozoites are tightly packed inside the sporocysts, they are difficult to visualize.)

A

A: Sporulation of Cyclospora oocysts.  The sequence shows, as observed by DIC microscopy of wet mounts: an oocyst passed in fresh stool (Day 0); sporulated oocysts at days 5 (Day 5) and 10 (Day 10), which both contain 2 sporocysts; and a ruptured oocyst (Rupture), with a sporocyst still inside the oocyst and the other sporocyst just outside—the coiled sporozoites are barely visible inside the sporocysts.