Negative Staining – Principle, Procedure, Result

What is Negative Staining?

• Negative staining is a well-established technique in microscopy, particularly useful in diagnostic applications. Unlike positive staining methods that directly colorize the specimen, negative staining involves staining the background surrounding the specimen, leaving the specimen itself unstained and visible. This approach is beneficial for observing delicate structures like bacterial cells without altering their natural morphology through harsh staining or heat fixing processes.
• The technique works by utilizing optically opaque, negatively charged dyes such as Nigrosin, India ink, Eosin, or Congo red. These dyes are repelled by the negatively charged bacterial cell walls, preventing them from penetrating the cells. As a result, the bacterial cells appear as clear zones against a darkly stained background. This contrast enhances the visibility of the cells and allows for detailed observation of their size, shape, and arrangement.
• Negative staining is particularly advantageous in cases where traditional staining methods may not adequately highlight certain bacteria, such as spirochetes, or when preserving the natural structure of the cells is crucial for accurate analysis. Unlike heat fixation, which can alter bacterial morphology by affecting cell structures like capsules and slime layers, negative staining preserves these features intact.
• Overall, negative staining is a valuable tool in microscopy for its ability to provide clear, high-contrast images of bacteria and other microscopic specimens, making it a preferred method in various diagnostic and research settings where detailed observation of cell morphology is essential.

Objective of Negative Staining

• The technique is used to observe microorganisms or other specimens under a microscope without altering their natural morphology. It is particularly useful for delicate structures or organisms that are difficult to stain with traditional methods.

Principle of Negative Staining

Negative staining is based on the principle of using acidic stains like India ink or Nigrosin. These stains have a negatively charged chromogen that does not penetrate the bacterial cells due to the cells’ own negative surface charge. As a result, the cells remain unstained and appear as clear spots against a darkly stained background.

To perform negative staining, a thin smear of the specimen is prepared on a microscope slide. Unlike traditional staining methods that require heat fixation, which can distort the cells, negative staining avoids this step. Instead, the acidic dye is applied directly to the specimen. The negatively charged dye molecules are repelled by the similarly charged bacterial cell surfaces. Consequently, the dye stains the background of the slide but does not enter the bacterial cells themselves. This creates a high contrast image where the unstained bacterial cells stand out clearly against the stained background.

Negative staining is particularly advantageous in microscopy for two main reasons:

1. Preservation of Natural Cell Shape: By avoiding heat fixation and intrusive chemical treatments, negative staining allows for the observation of bacteria in their natural size and shape. This preservation is critical for accurately studying the morphology and structure of microorganisms.
2. Visualization of Hard-to-Stain Bacteria: Some bacteria, such as certain spirilla, are notoriously difficult to stain using conventional methods. Negative staining provides a viable alternative by highlighting these organisms without the need for intensive staining protocols that might compromise their visibility.

Requirement for Negative Staining

Reagents

1. Nigrosin: Nigrosin is a common acidic dye used in negative staining. It possesses a negatively charged chromogen, which is repelled by the similarly charged bacterial cell walls, resulting in a stained background while leaving the cells clear.

Equipment

1. Microincinerator or Bunsen Burner: While negative staining typically does not require heat fixation, a microincinerator or Bunsen burner may be used for sterilizing the inoculating loop before and after use to prevent contamination.
2. Inoculating Loop: This tool is essential for transferring the bacterial sample onto the slide. The loop is sterilized using the microincinerator or Bunsen burner to ensure a clean transfer.
3. Staining Tray: A staining tray is used to contain any spills and to facilitate easy handling of slides during the staining process. It helps maintain a clean and organized workspace.
4. Glass Slides: Glass slides serve as the surface on which the specimen is prepared. They must be clean and free from any contaminants to ensure accurate staining results.
5. Lens Paper: Lens paper is used for cleaning the microscope lenses. It is non-abrasive and ensures that the lenses remain clear and free from scratches.
6. Microscope: A microscope is essential for observing the stained specimen. High-quality optics are necessary to achieve the best possible resolution and contrast in the images.

Step-by-Step Procedure of Negative Staining

1. Prepare the Slide: Begin with a clean, flame-sterilized microscope slide. Place a small drop of Nigrosin, an acidic dye, near one end of the slide. The drop should be small—more than a loopful but less than a drop from a dropper.
2. Inoculate the Stain: Using aseptic technique, sterilize an inoculating loop by passing it through a microincinerator or Bunsen burner flame. Once cooled, pick a small amount of bacterial culture from a slant or broth and mix it into the drop of Nigrosin on the slide. Ensure the culture is well-dispersed within the stain without spreading the drop.
3. Spread the Stain: Take another clean slide and rest one end of it on the center of the slide with the Nigrosin-bacterial mixture. Tilt the clean slide to form an acute angle (about 45 degrees) and draw it back until it touches the drop. This will cause the drop to spread along the edge of the second slide.
4. Form the Smear: Maintaining the acute angle, push the clean slide away from the drop, dragging the Nigrosin mixture along the surface of the primary slide. This action should produce a thin, even smear of the stain containing the bacteria.
5. Air Dry: Allow the smear to air dry naturally. Do not heat fix the slide, as this could distort the morphology of the bacterial cells.
6. Examine Under Microscope: Once the smear is dry, place the slide on the microscope stage. Use oil immersion to focus on a thin area of the smear. Under the microscope, the unstained bacterial cells will appear as clear shapes against a dark gray or black background created by the Nigrosin stain.

Key Considerations

• Aseptic Technique: It is crucial to maintain aseptic conditions throughout the procedure to avoid contamination.
• No Heat Fixation: Heat fixation is deliberately avoided in negative staining to preserve the natural size, shape, and arrangement of the microorganisms.
• Oil Immersion: Using oil immersion improves the resolution and clarity of the observed microorganisms, making it easier to study their morphology.

Step-by-Step Procedure to view in Transmission Electron Microscope (TEM)

1. Prepare the Grid: Start by holding a coated grid film-side up using a pair of self-clamping forceps. The grid serves as the support for the specimen and needs to be handled with care to avoid contamination.
2. Prepare the Stain and Sample Mixture: Prepare a 1:1 mixture of your sample and a negative stain. Commonly used stains include 2% uranyl acetate or 2% sodium or potassium phosphotungstate, with a pH of 7.4. These stains help to provide contrast by surrounding the specimen with an electron-dense medium.
3. Apply the Sample to the Grid: Add approximately 5µl of the stain-sample mixture to the grid. It is important to note that smaller particles tend to adsorb to the grid surface more rapidly than larger particles, so the mixture must be adequately homogeneous.
4. Alternative Fixation Method: Alternatively, if the sample requires fixation, mix it with a fixative before applying it to the grid. This step can enhance the preservation of delicate structures.
5. Incubation: Allow the sample to incubate on the grid for 30 to 90 seconds. This incubation period ensures that the particles have enough time to adhere to the grid and interact with the stain.
6. Remove Excess Liquid: After incubation, remove the excess liquid from the grid. This can be done by gently touching the torn edge of a piece of filter paper to the grid, which will wick away the excess stain without disturbing the sample.
7. Air Dry: Allow the grid to air dry completely. Proper drying is crucial to prevent artifacts that can arise from residual moisture during TEM observation.
8. Examine in the TEM: Once the grid is dry, it can be inserted into the TEM for examination. Carefully place the grid in the microscope and adjust the settings to obtain the best possible resolution and contrast.

Key Considerations

• Cleanliness: Ensure all tools and surfaces are clean to prevent contamination.
• Timing: The incubation time can vary depending on the sample and stain used; monitoring this step is critical for optimal results.
• Handling: Handle the grid with care at all times to avoid damage to the sample or grid.

Observation 

Under microscope, the bacterial cells appear as a clear transparent body of variable size and shape (If you are using a mixture of bacterial culture) against a dark background.

Result Interpretation of Negative Staining 

Importance of Negative Staining

1. Negative staining method is used to stain cells that are too delicate to be heat-fixed. 
2. Used to prepare biological samples for electron microscopy.
3. Used to observe viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power.
4. Used for the examination and identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H) phases by Negative staining transmission electron microscopy.
5. Can be used to observe the bacterial capsule.
6. There are several bacterial cells (Spirillum, Spirochetes etc.), that are difficult to stain by other staining techniques, but this negative staining method can be used to stained them easily.

References

• ScienceDirect. (n.d.). Negative staining. Retrieved from https://www.sciencedirect.com/topics/nursing-and-health-professions/negative-staining#:~:text=Negative%20staining%20is%20performed%20for,the%20glass%20background%20containing%20cells.
• ScienceDirect. (n.d.). Negative staining. Retrieved from https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/negative-staining
• Kuo, W. T., & Huang, K. Y. (2013). Negative staining of proteins. Biomolecules, 3(3), 665-681. DOI: 10.3390/biom3030665
• Microbiology Info. (n.d.). Negative Staining: Principle, Reagents, Procedure and Result. Retrieved from https://microbiologyinfo.com/negative-staining-principle-reagents-procedure-and-result/
• University of Massachusetts Medical School. (n.d.). Negative Staining. Retrieved from https://www.umassmed.edu/cemf/Negative-Staining/
• University of Rochester Medical Center. (n.d.). Negative Staining Electron Microscopy. Retrieved from https://www.urmc.rochester.edu/research/electron-microscope/services/protocols-techniques/negative-staining-electron-microscopy.aspx
• Liu, J., et al. (2009). Negative staining of cells. Journal of Microscopy, 236(2), 142-148. DOI: 10.1111/j.1365-2818.2009.03245.x
• Microbiology Research. (n.d.). Negative Staining. Retrieved from https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-29-3-503?crawler=true
• IUBEM Center. (n.d.). Negative Staining Procedures. Retrieved from https://iubemcenter.indiana.edu/equipment/tips-and-help/negative-staining-procedures.html
• AAT Bio. (n.d.). What is the difference between negative and positive staining? Retrieved from https://www.aatbio.com/resources/faq-frequently-asked-questions/what-is-the-difference-between-negative-and-positive-staining
• Current Protocols. (n.d.). Negative Staining. Retrieved from https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mca03fs15
• IntechOpen. (n.d.). Negative Staining. Retrieved from https://www.intechopen.com/chapters/48639