Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture
Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture
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DOI：
10.1016/j.exppara.2008.04.023
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发表时间：
2008-09-01
影响因子：
2.1
通讯作者：
Ryan, Una
中科院分区：
文献类型：
--
作者：
Boxell, Annika;Hijjawi, Nawal;Ryan, Una
隐孢子虫在宿主无细胞培养基中的完整发展于2004年首次被描述，这是隐孢子虫研究的一个重大进展，可以促进隐孢子虫研究的许多方面。目前无宿主细胞培养的一个限制是难以可视化生命周期阶段，因为它们的尺寸非常小，形态上难以识别并且分散在整个培养基中。这与隐孢子虫的传统细胞培养方法相反，隐孢子虫的传统细胞培养方法可以集中在宿主细胞上并观察宿主细胞上的感染灶。在本研究中，我们比较了三种特异性和三种非特异性技术在无细胞培养中观察小隐孢子虫生命周期阶段；使用抗孢子子和抗卵囊壁抗体（Sporo-Glo （TM）和Crypto Cel）进行抗体染色，使用隐孢子虫特异性rRNA寡核苷酸探针和非特异性染料进行荧光原位杂交（FISH）；德克萨斯红，羧基荧光素二乙酸琥珀酰酯（CFSE）和4,6'二氨基-2-苯基吲哚二盐酸盐（DAPI）。结果表明，Sporo-Glo （TM）和Crypto Cel联合染色可轻松可靠地识别所有生命周期阶段。(C) 2008爱思唯尔公司版权所有。
The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptospoiidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo (TM) and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6' diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo (TM) and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages. (C) 2008 Elsevier Inc. All rights reserved.