Negative staining is primarily used to study the morphological shape, size, and arrangement of bacterial cells that are difficult to stain, such as Spirilla.
It can stain cells that are too delicate to undergo heat-fixing.
The technique is used to prepare biological samples for electron microscopy.
Negative staining allows visualization of viruses, bacteria, bacterial flagella, biological membrane structures, and proteins or protein aggregates, which all have low electron-scattering power.
It is employed for the study and identification of aqueous lipid aggregates, including lamellar liposomes (le), inverted spherical micelles (M), and inverted hexagonal HII cylindrical (H) phases, through negative staining transmission electron microscopy.
Principle of Negative Staining
The principle of negative staining is based on the use of acidic dyes, such as India Ink or Nigrosin.
These dyes are acidic, meaning they can readily donate a hydrogen ion (proton), which causes the chromophore of the dye to carry a negative charge.
Most bacterial cell surfaces are also negatively charged, so they repel the negatively charged dye.
As a result, the background (glass slide or surrounding medium) absorbs the stain and appears dark, while the bacterial cells remain unstained and appear as clear, bright spots against this dark background.
This technique allows precise observation of the true size, shape, and arrangement of bacterial cells without distortion that can occur from heat-fixing or harsh staining procedures.
Negative staining is particularly useful for visualizing delicate structures like bacterial capsules, flagella, and other fine cellular appendages that are difficult to see with standard staining methods.
It is also commonly applied in electron microscopy to enhance contrast for studying viruses, protein aggregates, and lipid structures, as it highlights specimens with low electron-scattering power.
Reagents of Negative Staining
India Ink – commonly used as an acidic stain for negative staining.
Nigrosin – another acidic dye frequently employed for negative staining.
Nigrosin solution – prepared as 100 g/L Nigrosin with 5 mL/L Formalin in water, used as a staining reagent.
Procedure of Negative Staining
Place a very small drop of Nigrosin on one end of a well-cleaned and flamed slide. The drop should be larger than a loopful but smaller than a free-falling drop from the dropper.
Using an inoculating loop, collect a small amount of culture from the slant and mix it gently into the drop of stain without spreading the drop.
Take another clean slide and use it to spread the drop of stain containing the organism.
Rest one end of the clean slide on the center of the slide with the stain. Tilt it at an acute angle toward the drop, touching the drop so it spreads along the edge of the spreader slide.
Maintain a small acute angle between the slides and push the spreader slide toward the opposite end of the slide, dragging the drop behind it to produce a broad, even, thin smear.
Allow the smear to air dry without applying heat.
Focus on a thin area under an oil immersion microscope and observe the unstained cells appearing as clear shapes surrounded by the gray-stained background.
Procedure to view in Transmission Electron Microscope (TEM)
Hold a TEM-coated grid with the film side facing up using self-clamping forceps to ensure stability and prevent contamination.
Prepare a 1:1 mixture of the biological sample and a negative stain, such as 2% uranyl acetate or 2% sodium or potassium phosphotungstate at pH 7.4.
Apply approximately 5 µL of the mixture onto the grid. Note that smaller particles tend to adsorb to the grid surface more quickly than larger particles, enhancing contrast.
Alternatively, the sample can first be mixed with a fixative before being applied to the grid, followed by negative staining, to preserve delicate structures.
Allow the mixture to incubate on the grid for 30–90 seconds so the stain surrounds the sample and enhances contrast.
Remove excess liquid carefully using the torn edge of a piece of filter paper, taking care not to disturb the sample.
Let the grid air dry completely to prevent artifacts or uneven staining.
Examine the prepared grid under a transmission electron microscope (TEM) to visualize viruses, proteins, bacterial structures, or other fine biological specimens as bright images against a dark, electron-dense background.
Negative staining in TEM provides high-contrast images while preserving the structural integrity of delicate and low-electron-scattering biological specimens, making it ideal for structural studies at the nanoscale.
Results of Negative Staining
In negative staining, bacterial cells or other specimens appear as unstained, bright, or transparent structures against a darkly stained background, because the acidic dye is repelled by the negatively charged cell surface.
The true size, shape, and arrangement of the cells are clearly visible since the cells are not distorted by heat-fixing or harsh chemical stains.
Delicate structures such as bacterial capsules, flagella, and fine appendages can be observed in detail.
In electron microscopy, viruses, protein aggregates, and lipid structures appear as well-defined bright particles contrasted against a dense, dark background.
The technique allows identification of structural features like morphology, aggregation patterns, and surface characteristics, aiding in microbial characterization and research.
Negative staining provides a rapid and simple method for assessing sample quality, particle size, and structural integrity before further microscopic or analytical studies.
References
YashRoy, R.C. (1990). Lamellar dispersion and phase separation of chloroplast membrane lipids by negative staining electron microscopy. Journal of Biosciences, 15(2), 93–98.
ScienceDirect Topics. (n.d.). Negative Staining. ScienceDirect. Retrieved from https://www.sciencedirect.com/topics/medicine-and-dentistry/negative-stain
Harvard Medical School. (n.d.). Negative Staining - Electron Microscopy. Harvard Medical School. Retrieved from https://electron-microscopy.hms.harvard.edu/negative-staining
University of Wisconsin-Madison. (2021). Negative Stain Grid Preparation. Cryo-EM Facility. Retrieved from https://cryoem.wisc.edu/wp-content/uploads/sites/341/2021/01/Negative_Stain_Grid_Preparation_21Jan2021.pdf
McGill University. (n.d.). Negative Staining. Facility for Electron Microscopy Research. Retrieved from https://www.mcgill.ca/femr/resources/protocols/negative-staining-0
University of Rochester Medical Center. (n.d.). Negative Staining Electron Microscopy - Protocols/Techniques. University of Rochester. Retrieved from https://www.urmc.rochester.edu/research/electron-microscope/services/protocols-techniques/negative-staining-electron-microscopy.aspx
Cambridge Advanced Imaging Centre. (n.d.). Transmission Electron Microscopy (TEM) Applications. Cambridge University. Retrieved from https://caic.bio.cam.ac.uk/electron-microscopy/tem/tem-applications/transmission-electron-microscopy-tem-applications
Creative Biostructure. (n.d.). Negative Staining Transmission Electron Microscopy Service. Creative Biostructure. Retrieved from https://www.creative-biostructure.com/negative-staining-transmission-electron-microscopy-service.htm
Wiley Analytical Science. (n.d.). Negative Staining Electron Microscopy. Wiley Analytical Science. Retrieved from https://analyticalscience.wiley.com/content/article-do/negative-staining-electron-microscopy
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