[Isolation of Cryptosporidium parvum oocysts from fecal samples--the combination of ether extraction and discontinuous sucrose gradients]

Abstract

A calf and 50 mice were infected with Cryptosporidium parvum, and their fecal materials were collected and treated with ether extraction (EE), followed by discontinuous sucrose gradients (DSG) method. EE method was to remove some of fat or lipid from feces. Sediments were washed by centrifugation (1,500 x g for 10 min., 3 times) in phosphate-buffered saline and then these washed sediments were sieved sequentially through stainless steel screens with a final mesh of 250 (61 microns porosity) to remove other debris. After sieving, the materials were suspended in 2.5% potassium dichromate solution. Oocysts were counted by using a hemocytometer and the recovery rate of pure oocysts was calculated on the basis of the count. Following centrifugation (1,500 x g for 30 min.) by DSG method, most oocysts were recovered at the interface between a gravity of 1.103 and 1.064. The recovery rates of pure oocysts from the fecal suspension of the calf (3.8 x 10(7)/ml) and the mouse (3.2 x 10(6)/ml) treated with EE method were 81.6% and 51.6%, respectively. It is suggested that the recovery rate was dependent on the number of oocysts in each suspension treated with EE method. To get the 50% recovery rate, there must be more than 2 x 10(6) oocysts per ml of the fecal suspension treated with EE method. By the combination of the two methods it was possible to isolate C. parvum oocysts from normal feces of the calf and mouse as well as from diarrheic feces.