Wee S H, Lee C G, Kim B S, Joo H D, Kang S W
Veterinary Research Institute, Chonnam National University, Kwangju, Korea.
Korean J Parasitol. 1994 Mar;32(1):7-12. doi: 10.3347/kjp.1994.32.1.7.
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.
将一头小牛和50只小鼠感染微小隐孢子虫，收集它们的粪便材料，先用乙醚萃取法（EE）处理，然后采用不连续蔗糖梯度（DSG）法。EE法是为了去除粪便中的一些脂肪或脂质。沉淀物用磷酸盐缓冲盐水离心洗涤（1500×g，10分钟，共3次），然后将这些洗涤后的沉淀物依次通过最终孔径为250（孔隙率61微米）的不锈钢筛网以去除其他杂质。筛分后，将材料悬浮于2.5%重铬酸钾溶液中。使用血细胞计数器对卵囊进行计数，并根据计数结果计算纯卵囊的回收率。采用DSG法离心（1500×g，30分钟）后，大多数卵囊在比重为1.103和1.064的界面处回收。用EE法处理的小牛粪便悬液（3.8×10⁷/ml）和小鼠粪便悬液（3.2×10⁶/ml）中纯卵囊的回收率分别为81.6%和51.6%。结果表明，回收率取决于用EE法处理的每个悬液中的卵囊数量。要达到50%的回收率，用EE法处理的粪便悬液每毫升中必须有超过2×10⁶个卵囊。通过这两种方法的结合，有可能从小牛和小鼠的正常粪便以及腹泻粪便中分离出微小隐孢子虫卵囊。
