INSITU DETECTION OF MYCOPLASMA CONTAMINATION IN CELL-CULTURES BY FLUORESCENT HOECHST-33258 STAIN
INSITU DETECTION OF MYCOPLASMA CONTAMINATION IN CELL-CULTURES BY FLUORESCENT HOECHST-33258 STAIN
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DOI：
10.1016/0014-4827(77)90089-1
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发表时间：
1977-01-01
影响因子：
3.7
通讯作者：
CHEN, TR
中科院分区：
文献类型：
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作者：
CHEN, TR
报道了一种简单、快速且易于重复的常规实验室技术，用于检测细胞培养物中的支原体污染。生长在盖玻片上的细胞直接用 Carnoy 固定、风干、用 DNA 特异性荧光 Hoechst 33258 染色并在显微镜下检查。所有被支原体感染的培养物在核外和细胞间隙中都具有易于辨别的、小的、形态均匀的、明亮的荧光体，而未污染的对照培养物中核外背景呈现均匀的黑色。为了探究检测支原体的灵敏度，用连续稀释的细胞或从猪鼻支原体感染的培养物中收集的培养基的等分试样感染对照培养物。通过增加样本量可以轻松降低最低感染率（感染后 4-24 小时，采样约 1000 个细胞/培养物，为 0.40%），因为即使感染 1 个支原体的细胞也能被识别出来。这些支原体能抵抗 2500 rpm 离心 30 分钟，并且很容易通过 0.22 μm 孔径的滤膜过滤。令人惊讶的是，感染后 24 小时的感染率为 0.63%，在 120 小时内每个宿主细胞被数百个支原体污染达到 100%。
A simple, fast and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy''s, air-dried, stained with DNA-specific fluorescent Hoechst 33258 and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling .apprx. 1000 cells/culture, 4-24 h after infection) scored can easily be lowered by increasing sample size since a cell infected with even 1 mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2500 rpm for 30 min and easily filtered through 0.22 .mu.m pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundred mycoplasmas per host cell within 120 h.