negative staining

Buccal Epithelial Cells with Adherent Bacterial Cells Revealed by Negative Staining

Instructions

This Photo Courtesy of Cliff Drury, Biology Dept. Altoona Area High School

Using a tongue depressor, scrape the inside of the cheek so as to remove buccal epithelial cells.
Smear these on one end of a clean microscope slide into a drop of nigrosine dye.
Using a spreading technique similar to that for preparing blood smears, spread the cell/nigrosine mixture the length of the slide so as to produce a feathering effect where the smear film runs out.
Allow the negatively stained slide to air dry and then examine the buccal cells under the oil immersion lens.

Observations and Analysis

Students may see large angular cheek epithelial cells with many adherent bacterial cells, as single cells or micro colonies.
This is also an ideal preparation to illustrate the difference in size between typical procaryotic and eucaryotic cells.

Extensions

In an interesting modification of this technique, the viability of the buccal epithelial cells can be determined by the ability of viable cells to exclude the due trypan blue. The buccal cells are placed in a small culture tube or well slide and mixed with an equal volume of trypan blue dye (0.01%). The mixture is incubated for 10 min at room temperature and then placed in a counting chamber and examined under brightfield microscopy. Cells which exclude the trypan blue can be considered viable (Denyer et al. 1993). An additional portion of the trypan blue stained suspension can now be negative stained as above for the visualization of bacterial cells.

Reference:

Denyer, S.P., G.W. Hanlon, M.C. Davies and S.P. Gorman, 1993. IN Denyer, S.P., S.P. Gorman and M. Sussman, Microbial Biofilms: Formation and Control, Blackwell Scientific Publications, Oxford.