Negative Staining: Exercise Notes

Negative Staining

Background and Principles

• Mechanism: The negative stain technique operates by staining the background surrounding the bacteria instead of staining the bacteria themselves. Consequently, the bacteria appear clear or colorless against a stained background.

• Types of Stains Used:

  • Colloidal Stains: Examples include India ink and nigrosin.

    • These stains contain particles, such as those in India ink, which are approximately $1.0 ext{-}\\mu ext{m}$ in size. These particles are too large to enter the bacterial cells, thus staining only the surrounding background.

  • Negatively Charged Chromophores: Examples include eosin.

    • The chromophore (the color-bearing part of the stain) is negatively charged.

    • Bacterial cells typically have a net negative charge on their surface, especially at neutral pH.

    • Due to electrostatic repulsion, the negatively charged chromophore is repelled by the negatively charged cell, preventing the stain from entering or adhering to the cell.

    • The chemical reaction for sodium eosinate releasing its chromophore is: $\text{Sodium eosinate} \rightleftharpoons \text{Na}^+ + \text{Eosin}^-$ (Chromophore) with particle sizes ranging from $0.2 ext{ – }1 ext{ }\mu ext{m}$.

• Nigrosin as the Chosen Stain: For this exercise, a colloidal suspension of nigrosin is utilized.

  • Nigrosin particles do not enter the bacterial cells.

  • At a pH of $7$ (neutral pH), nigrosin particles are negatively charged.

  • This negative charge causes the nigrosin particles to be repelled by the negatively charged bacterial cell surface, maintaining a small distance from the cells and thus creating a clear halo around them.

• Advantages of Negative Staining:

  • Minimal Distortion: No heat-fixing or strong chemicals are used in this procedure. This minimizes the distortion of bacterial cell morphology and size, providing a more accurate representation of the natural state of the bacteria compared to other staining methods.

• Applications and Usefulness:

  • Particularly useful when other staining techniques do not clearly reveal cell morphology or size.

  • Coccobacilli: Negative staining is especially valuable for observing coccobacilli, which are short, oval-shaped bacilli that are notoriously difficult to stain effectively with other routine methods.

Materials Required

• Nigrosin (colloidal suspension)

• Clean slides ($6$)

• Distilled water

• Sterile toothpicks

• Cultures:

  • Bacillus subtilis

  • Staphylococcus epidermidis

  • An Unknown Culture

Techniques Required

• Compound light microscopy (as covered in Exercise 1)

• Smear preparation (as covered in Exercise 5)

Investigative Exercise Procedure

1. Slide Preparation: Ensure all slides are thoroughly clean and grease-free before starting.

2. Preparing the Negative Stain Smear (Refer to Figure 6.1 for visual guidance):

   • Initial Drop Placement: Place a small drop or loopful of nigrosin near one end of a clean slide.

   • Culture Emulsification:

     • For cultures from solid media: Add a loopful of distilled water to the nigrosin drop first. Then, emulsify a small amount of the bacterial culture from the solid medium into this nigrosin-water drop.

     • For broth cultures: Mix a loopful of the broth culture directly into the drop of nigrosin.

   • Critical step: At this point, do not spread the drop and do not let it air-dry naturally.

   • Spreading the Smear: Using the end edge of a second clean slide, gently draw it across the surface of the first slide until it contacts the mixed drop. The drop will spread along the edge of the top slide. Then, push the top slide horizontally to the left along the entire surface of the bottom slide, effectively spreading the drop into a thin film.

     • The resulting smear should vary in thickness, ranging from opaque black (heavy stain) to gray (thinner stain).

     • The angle at which the spreading slide is held will determine the final thickness of the smear.

   • Air-Drying: Allow the smear to air-dry completely. Do not heat-fix the slide at any point, as this would distort the bacterial cells.

   • Repeat for Other Culture: Prepare a negative stain of a second culture (e.g., Staphylococcus epidermidis) by repeating the previous steps.

3. Microscopic Examination:

   • Examine the stained slides microscopically using the low-power, high-dry, and oil immersion objectives (refer to Figure 6.2 and Figure 6.3).

   • Morphology Interpretation: If the observed field shows a few short rods alongside small cocci, the overall morphology should be interpreted as rod-shaped. The apparent cocci are likely short rods viewed from their ends or very small bacilli that are products of cell division.

   • Optimal Smear Observation:

     • Too heavy a smear results in cracks in the stain (Figure 6.3a).

     • The desired view should show colorless cells clearly visible against the stained background (Figure 6.3b).

     • Areas with too little stain will not provide good contrast (Figure 6.3c).

4. Unknown Culture: Repeat step 2 to prepare a negative stain of the unknown culture provided.

5. BSL-2 (Biosafety Level 2): For specific BSL-2 protocols, such as preparing a negative stain for a potentially hazardous organism, the initial step might involve placing a small drop of nigrosin and a loopful of water at the end of a slide as a general preparatory measure. Be aware of safety guidelines for handling materials at this level.