Negative Staining- Principle, Procedure and Result Interpretation

Interesting Science Videos

Objectives of Negative Staining

  1. To perform a negative staining procedure.
  2. To understand the benefit obtained from visualizing unstained microorganisms.

Principle of Negative Staining

Negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily discernible against the colored background.

The practical application of negative staining is twofold.

First, since heat fixation is not required and the cells are not subjected to the distorting effects of chemicals and heat, their natural size and shape can be seen.

Second, it is possible to observe bacteria that are difficult to stain, such as some spirilla. Because heat fixation is not done during the staining process, keep in mind that the organisms are not killed and slides should be handled with

Reagent and Equipment’s for Negative Staining

Nigrosin, Microincinerator or Bunsen burner, inoculating loop, staining tray, glass slides, lens paper, and microscope.

Procedure of Negative Staining

  1. Place a small drop of nigrosin close to one end of a clean slide.
  2. Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix.
  3. 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.
  4. Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry.
    Note: Do not heat fix the slide.
  5. Examine the slides under oil immersion.

Result Interpretation of Negative Staining

Negative Staining
Fig: Negative Staining, Bacilli (1000×). © Pearson Education Limited 2018

About Author

Photo of author

Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He is doing his Ph.D. at the Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal. He was awarded the DAAD Research Grant to conduct part of his Ph.D. research work for two years (2019-2021) at Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. Sagar is interested in research on actinobacteria, myxobacteria, and natural products. He is the Research Head of the Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Sagar has more than ten years of experience in blogging, content writing, and SEO. Sagar was awarded the SfAM Communications Award 2015: Professional Communicator Category from the Society for Applied Microbiology (Now: Applied Microbiology International), Cambridge, United Kingdom (UK). Sagar is also the ASM Young Ambassador to Nepal for the American Society for Microbiology since 2023 onwards.

2 thoughts on “Negative Staining- Principle, Procedure and Result Interpretation”

Leave a Comment

This site uses Akismet to reduce spam. Learn how your comment data is processed.