Negative Staining- Principle, Procedure and Result Interpretation

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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
care.

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

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Sagar Aryal, PhD

Dr. Sagar Aryal is a microbiologist, researcher, and science communicator from Nepal. He is also co-founder and Research Head of the Department of Natural Products at the Kathmandu Research Institute for Biological Sciences (KRIBS), where he leads research on natural product discovery and microbial biodiversity. Dr. Aryal earned his Ph.D. in Microbiology from Tribhuvan University, Kathmandu, Nepal, in 2025, supported by a DAAD Research Grant and a University Grants Commission (UGC) Nepal Ph.D. Fellowship. His research interests include Actinomycetes, Myxobacteria, microbial taxonomy, and natural products, particularly the discovery of bioactive compounds with potential pharmaceutical applications. He isolated and identified Nocardia nepalensis, a novel bacterial species from Nepal, contributing to understanding microbial diversity in the region. Dr. Aryal has published more than 16 research articles in international journals and contributed three book chapters to established academic publications. He has presented scientific research findings and developed international research collaborations through visits to more than 18 countries across Asia, Europe, North America, and Oceania. From 2023 to 2025, he served as the American Society for Microbiology (ASM) Young Ambassador to Nepal. Since December 2025, he has served as an Editorial Board Member of Scientific Reports, a Nature Portfolio journal. In recognition of his scientific contributions, Dr. Aryal received the NAST Science and Technology Youth Award 2025 from the Nepal Academy of Science and Technology. He previously received the Frs. Locke and Stiller Research Award (LSRA) for the 2017-2018 cycle from St. Xavier's College, Kathmandu, and a Nepal Academy of Science and Technology (NAST) Research Grant for 2017-2018. Alongside his scientific career, Dr. Aryal has more than 15 years of experience in blogging, content writing, and search engine optimization (SEO).