sem样品处理方法.doc

SEM样品处理方法
Electron microscopy.
Exponential-phase cultures of different M. smegmatis strains were harvested, washed three times with M cacodylate buffer, treated with 1% osmium tetroxide for 1 h at room temperature, centrifuged, and the collected cells were fixed with M cacodylate buffer containing 2% glutaraldehyde for 2 h at room temperature. Cells were again collected after centrifugation and fixed overnight with M cacodylate buffer containing 1% osmium tetroxide at 4 ℃. The cells were dehydrated using graded ethanol (10 %, 30 %, 50 %, 70% and 100 %), mounted on microscope stubs and sputter coated on the sputter coater. The samples were then examined under a Leica S440 scanning electron microscope.
. Scanning electron microscope(SEM)
For SEM analysis, cells were fixed %(v/v) glutaraldehyde for 2–3 h. The fixed cells were washed with - buffered saline(PBS)() for three times(10mineach). Subsequently, ethanol concentration gradient(v/v)of 50%,70%, 80%, 90%and100%was used to dehydrate the fixed cells in a sequential way. Finally, the resulted cell samples were further dehydrated by two more100% ethanol treatments. After that, tertiary butyl alcohol mixed with ethanol in a ratio of 1:1and pure tertiary butyl alcohol was applied to achieve metathesis of ethanol in the cells. At last, cells were mixed
in tertiary butyl alcohol and