Experiment 2: Negative Staining

Negative Staining provides a more detailed assessment of a microbe's morphology, and helps prevent any distortion of the microbe's ultrastructure by the staining procedure. It also makes the outline of the cells highly visible.

Nigrosin and bacterial cells are negatively charged. Therefore, negative staining works because like charges repel each other, preventing the cells from taking up the stain.

Procedure

1. Over a sink or disposable plastic container, place 1 - 2 drops of nigrosin near the frosted end of the dried bacterial slide.
2. Place the clean slide at about a 45° angle next to the nigrosin and gently spread the dye over the bacterial specimen.
3. Air dry the dye-stained sample for approximately 10 minutes.
4. If you have a microscope available, observe the stained slide under increasing the magnification and record what you see at each magnification in Table 2. If there is no microscope is available, refer to Figures 11 and 12.
5. Place your slide in a disposable plastic container, and pour bleach over the surface until the sample is completely covered/saturated. Allow the bleach to soak for approximately 20 minutes and then rinse the bleach down the sink with running water.
6. Wrap the bacterial slide in Parafilm™ and dispose of it in the trash.

Figure 11: Negative stain image of Ebola virus. This image reveals some of the ultrastructural morphology. Image courtesy of the CDC: Public Health Image Library.	Figure 12: Negative stain image of Marburg virions (not bacteria). Viewed at 100,000X. Image courtesy of the CDC: Public Health Image Library.