Negative staining




Principle

Negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain is a negatively charged chromogen that cannot penetrate into the bacterial cell due to the negative charge on the surface of bacterial cells. Therefore, unstained cells are easily visible against the colored background. A negative stain has two advantages. First, as heat fixation is not done and the cells are not subjected to any chemical or heat distortion so we can observe the natural shape and size of bacterial cells. Secondly, we can easily observe the bacterial cells that are difficult to visualize.

Materials 

24h agar culture of Micrococcus luteus, Bacillus cereus, Nigrosin, slides, inoculation loop, staining rack, Bunsen burner, microscope.

Procedure 

  1. Place a small drop of nigrosin stain close to one end of the clean glass slide.
  2. Place a loopful culture of bacterial cells in the nigrosin drop and mix with inoculation loop.
  3. With the help of second slide edge placed at a 45° angle push the mixture to the other end of the slide to form a uniform smear.
  4.  Air-dry the smear.
  5. Examine under the oil immersion lens of Microscope

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