2. PURPOSE OF NEGATIVE STAINING?
■ The main purpose of Negative staining is to study the morphological
shape, size and arrangement of the bacteria cells that is difficult to stain.
eg: Spirilla. It can also be used to stain cells that are too delicate to be
heat-fixed.
■ Why is it called a negative stain?
■ Because it does not stain the bacterial cells directly, instead, it stains
the background; it stains the actual glass slide. Because it is also uses
a negatively charged dye.
■ Why bacterial cell unstained in negative staining?
■ Due to repulsion between the negative charges of the stains and the
negatively charged bacterial wall, the dye stains the background.
3. What is an advantage of negative staining?
■ The advantages of the negative stain include the use of only one stain
and the absence of heat fixation of the sample. Negative staining
employs the use of an acidic stain and, due to repulsion between the
negative charges of the stain and the bacterial surface, the dye
will not penetrate the cell.
■ The negative stain is particularly useful for determining cell size and
arrangement. It can also be used to stain cells that are too delicate to
be heat-fixed.
4. Principle of Negative Staining
■ Negative staining requires an acidic dye such as India Ink or Nigrosin.
■ India Ink or Nigrosin is an acidic stain. This means that the stain readily
gives up a hydrogen ion (proton) and the chromophore of the dye
becomes negatively charged. Since the surface of most bacterial
cells is negatively charged, the cell surface repels the stain. The glass
of the slide will stain, but the bacterial cells will not. The bacteria will show
up as clear spots against a dark background.
■ Background:dark
■ Cell:bright
5. Requirement of Negative Staining
Nigrosin, Microincinerator or Bunsen burner, inoculating loop, staining
tray, glass slides, lens paper, and microscope.
Procedure of Negative Staining
Place a small drop of nigrosin close to one end of a clean slide.
Place a slide against the drop of suspended organisms at a 45° angle and
allow the drop to spread along the edge of the applied slide.
Using aseptic technique, place a loopful of inoculum from the bacterial
culture in the drop of nigrosin and mix.
Push the slide away from the drop of suspended organisms to form a thin
smear. Air-dry.
Note: Do not heat fix the slide.
Examine the slides under oil immersion.
