Negative Stain, Atlas of Negative Stain, Bacterial Cellular Morphology and Simple Stains, A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
Purpose
The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. A primary example is the spirochete Treponema, which is distorted by the heat-fixing of other staining techniques. Also, where determining them accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage.
Principle
The negative staining technique uses a dye solution in which the chromogen is acidic and carries a negative charge. (An acidic chromogen gives up a hydrogen ion, which leaves it with a negative charge.) The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background (Figure 5-4). Examples of acidic staining solutions used in negative stains are Nigrosin and Congo red (Figures 5-5 and 5-6).
5-4 CHEMISTRY OF ACIDIC STAINS Acidic stains have a negatively
charged chromogen (●–) that is repelled by negatively charged cells.
Thus, the background is colored and the cell remains transparent.
5-5 A NIGROSIN NEGATIVE STAIN Notice that the Bacillus megaterium
cells are unstained against a dark background. Cell dimensions are
1.2–1.5 µm wide by 2.0–5.0 µm long. The small, irregularly-shaped white
spots are bubbles or other artifacts.
5-6 A NEGATIVE STAIN WITH CONGO RED Compare this negative
stain of Bacillus megaterium to Figure 5-5. B. megaterium is a soil
organism. Cell dimensions are 1.2–1.5 µm wide by 2.0–5.0 µm long.
Suggested Reading
- Michael J. Leboffe & Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
COMMENTS