Atomic Force Microscopic Examination of Chromosomes Treated with Trypsin or Ethidium Bromide

Abstract

Trypsin treatment is frequently used during chromosome preparation for removal of cellular contaminants, and ethidium bromide (EB) staining of bands is often used to facilitate high-resolution observations by optical microscopy. However, conventional optical microscopy is unable to visualize potential aberrations of chromosome structures caused by these physicochemical treatments. In this article, we use atomic force microscopy (AFM) in the tapping mode to obtain and analyze high-resolution images of chromosome surface structure damage associated with trypsinization and EB treatment. According to our results, the trypsin-based digestion effects became more severe as incubations increased across a range from 10 to 40 s; a digestion time of 10 to 20 s appeared to be most suitable for observation by AFM. In terms of chromosomal damage induced by EB treatment, addition of EB into the media of cultured human blood cells induced chromosomal breakage in a dose-dependent fashion, and the results indicate centromeric region damnifyed severer than arms. Together, these results indicate that EB staining and the standard chromosomal preparative techniques of trypsinization can induce chromosomal damage that may affect the observed results.