细胞计数板的使用.docVIP

For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.　　 A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts. 　　 To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.　 It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed　　 Suspensions should be dilute enough so that the cells or other p