Chen Fu, Huang Ke-he
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Jun;24(3):219-22.
To explore an applicable method for isolation and purification of Cryptosporidium parvum oocysts with high purity, recovery and vigor from mouse feces.
Four techniques were used for isolating and purifying C. parvum oocysts from mouse feces: modified saturated saline flotation, percoll gradient centrifugation, CsCl gradient centrifugation and the classical discontinuous sucrose gradient centrifugation. Oocysts received from the methods were used respectively to infect in vitro bovine fallopian tube epithelial cells (BFTE) and the development of the oocysts was examined under microscope after 48 h and 72 h cultivation.
The number of oocysts received by the classical discontinuous sucrose gradient centrifugation [(2.86 +/- 0.08) x 10(7)] was significantly higher than that of percoll gradient centrifugation [(1.52 +/- 0.08) x 10(7)] (P<0.01) and CsCl gradient centrifugation [(2.46 +/- 0.13) x 10(7)] (P<0.05), but similar to that of the modified saturated saline flotation [(2.88 +/- 0.15) x 10(7)]. No significant difference was found on the number of oocysts by BFTE cultivation at 48 and 72 hours post-inoculation(P>0.05). Oocysts received from CsCl gradient centrifugation showed higher purity than those by discontinuous sucrose gradient centrifugation.
In comparison to the classical discontinuous sucrose gradient centrifugation, operation of the modified saturated saline flotation is easier and faster, and the purity of oocysts isolated by CsCl gradient centrifugation is higher.
探索一种从鼠粪中分离纯化高纯度、高回收率及高活力微小隐孢子虫卵囊的适用方法。
采用四种技术从鼠粪中分离纯化微小隐孢子虫卵囊：改良饱和盐水漂浮法、聚蔗糖密度梯度离心法、氯化铯密度梯度离心法和经典的不连续蔗糖密度梯度离心法。分别用这些方法获得的卵囊感染体外培养的牛输卵管上皮细胞（BFTE），接种48小时和72小时后在显微镜下观察卵囊的发育情况。
经典的不连续蔗糖密度梯度离心法获得的卵囊数量[（2.86±0.08）×10⁷]显著高于聚蔗糖密度梯度离心法[（1.52±0.08）×10⁷]（P<0.01）和氯化铯密度梯度离心法[（2.46±0.13）×10⁷]（P<0.05），但与改良饱和盐水漂浮法[（2.88±0.15）×10⁷]相近。接种后48小时和72小时BFTE培养获得的卵囊数量差异无统计学意义（P>0.05）。氯化铯密度梯度离心法获得的卵囊纯度高于不连续蔗糖密度梯度离心法。
与经典的不连续蔗糖密度梯度离心法相比，改良饱和盐水漂浮法操作更简便、快速，氯化铯密度梯度离心法分离的卵囊纯度更高。
