Negative staining of Viruses 50 FAQs And 30 MCQs:
Negative staining of Viruses 50 FAQs:
What is negative staining of viruses?
Negative staining is a microscopy technique where the background is stained, leaving the virus particles unstained. This creates contrast, allowing visualization of viral morphology under an electron microscope.
Why is negative staining used for viruses?
Viruses are too small for light microscopy. Negative staining enhances contrast in electron microscopy, revealing viral shape, surface structures, and internal details.
How does negative staining differ from positive staining?
Negative staining: Background is stained, virus remains unstained.
Positive staining: Virus is directly stained, which may obscure fine details.
What types of microscopes are used for negative staining?
Transmission Electron Microscopes (TEM) are primarily used because they provide high-resolution images of viruses.
What are the advantages of negative staining?
Fast and simple
No heat fixation required (preserves viral structure)
High contrast and resolution
Visualizes surface features (spikes, filaments, etc.)
What stains are used in negative staining?
Common stains:
Uranyl acetate (for enveloped viruses)
Sodium silicotungstate (SST) (for small viral proteins)
Ammonium molybdate (for large viruses like orthomyxoviruses)
Why are heavy metals used in negative staining?
Heavy metals (e.g., uranium, tungsten) scatter electrons, creating contrast in TEM images.
What is the role of uranyl acetate in negative staining?
Binds to lipid envelopes, stabilizing viral membranes.
Provides high contrast but can cause radioactive hazards.
What pH does uranyl acetate have?
Uranyl acetate has a low pH (4.4), making it suitable for enveloped viruses.
What is the disadvantage of sodium silicotungstate (SST)?
It forms large stained molecules, which may reduce image resolution.
Why is ammonium molybdate preferred for large viruses?
It firmly binds to the support film and offers high contrast for large particles like orthomyxoviruses.
Can negative staining be radioactive?
Yes, uranyl acetate contains uranium, which can be radioactive. Proper safety measures (gloves, lab coat, mask) are required.
What support films are used in negative staining?
Carbon films (better results, hydrophilic)
Plastic films (hydrophobic, require frequent cleaning)
How is a carbon film prepared?
Carbon is evaporated onto freshly cleaved mica under high heat and vacuum.
What concentration of virus is needed for staining?
0.05–0.1 mg/ml for most viruses
1 mg/ml for intact viruses (e.g., adenovirus, influenza)
How is the virus sample applied to the grid?
A small drop of virus suspension is placed on a carbon-coated grid.
Excess liquid is wicked away with filter paper.
What happens if too much stain is left on the grid?
The TEM image will show only stain, obscuring the virus.
What happens if too little stain is left?
The virus won’t be properly contrasted, making visualization difficult.
How long does the staining process take?
Typically 30 minutes to 1 hour, including drying time.
Why is air-drying a problem for enveloped viruses?
It can cause flattening and lipid blebbing, distorting the virus structure.
How do viruses appear in negative staining under TEM?
Light (unstained) virus particles
Dark (stained) background
What viral features can be seen with negative staining?
Shape (spherical, helical, complex)
Surface spikes/proteins
Internal structures (if stain penetrates)
How does adenovirus appear in negative staining?
Long fibers (120–350 kDa)
Penton bases (300 kDa)
What does orthomyxovirus (e.g., influenza) look like?
Spiked surface (hemagglutinin & neuraminidase)
What is a moiré pattern artifact?
A false lattice-like pattern caused by superimposed stained layers, leading to misinterpretation.
Why do some viruses appear collapsed in TEM?
Due to one-sided staining, which doesn’t fully support the 3D structure.
What are the applications of negative staining?
Virus identification (morphology, spikes, filaments)
Bacterial flagella studies
Macromolecule analysis (proteins, membranes)
Can negative staining diagnose specific viruses?
It provides morphological clues but may not distinguish closely related viruses without additional tests.
What are the limitations of negative staining?
Flattening of fragile viruses
Possible artifacts (e.g., moiré patterns)
Cannot visualize internal nucleic acids clearly
Why is negative staining disappearing from mainstream use?
Advanced techniques (e.g., cryo-EM, immunolabeling) offer better resolution and specificity.
What safety precautions are needed for uranyl acetate?
Lab coat, gloves, mask, eye protection
Proper waste disposal (radioactive hazard)
How can staining artifacts be minimized?
Proper sample concentration
Optimal stain drying time
Use of carbon films (better adsorption)
What causes poor contrast in negative staining?
Insufficient stain
Incorrect pH of stain
Improper drying
Why do some viruses appear positively stained?
Uranyl acetate can bind to nucleic acids/proteins, causing false positive staining.
How can negative staining be combined with immunolabeling?
Antibody-gold conjugates can be used to tag viral proteins, enhancing diagnostic specificity.
How does negative staining compare to thin-section EM?
Negative staining: Faster, no fixation/sectioning, but 2D only.
Thin-section EM: Shows internal structures but is more complex.
Is negative staining better than freeze-fracture EM?
Negative staining: Simpler, faster, but may distort viruses.
Freeze-fracture: Preserves 3D structure but is more expensive.
Can light microscopy be used for negative staining?
No, viruses are too small. Only electron microscopy provides sufficient resolution.
Why is TEM preferred over SEM for negative staining?
TEM allows higher resolution of viral surface details.
Can negative staining be automated?
Partially, but manual grid preparation is still common for optimal results.
Can negative staining detect virus mutations?
Only if mutations cause morphological changes (e.g., spike protein alterations).
How does negative staining help in vaccine development?
It allows quality control of virus-like particles (VLPs) used in vaccines.
Can negative staining visualize bacteriophages?
Yes, it is commonly used to study phage structure and attachment mechanisms.
What is immuno-negative staining?
Combining antibody labeling with negative staining to identify specific viral proteins.
Can negative staining detect viral entry into cells?
Yes, by capturing virus-cell interaction stages.
Who introduced negative staining?
Brenner and Horne (1959) first described the technique.
Why was negative staining revolutionary?
It allowed rapid, high-contrast imaging of viruses without complex preparation.
What bacteria were studied using negative staining?
Spirilla (hard-to-stain bacteria)
Klebsiella pneumoniae
Staphylococcus aureus
Is negative staining still used in diagnostics?
Yes, but less frequently due to molecular methods (PCR, sequencing).
What is the future of negative staining?
It remains useful for quick preliminary virus identification but may be replaced by cryo-EM for high-resolution studies.
Negative staining of Viruses 30 MCQs :
What is the primary purpose of negative staining in electron microscopy?
A) To stain the virus itself
B) To stain the background while leaving the virus unstained✔
C) To fix the viral nucleic acids
D) To increase virus infectivity
2. Which microscope is most commonly used for negative staining of viruses?
A) Light microscope
B) Scanning Electron Microscope (SEM)
C) Transmission Electron Microscope (TEM)✔
D) Fluorescence microscope
3. Negative staining is particularly useful for studying:
A) Only bacterial cells
B) Only plant tissues
C) Viruses and bacterial surface structures✔
D) Fungal spores
4. What type of stains are used in negative staining?
A) Basic dyes
B) Heavy metal salts (e.g., uranium, tungsten)✔
C) Fluorescent dyes
D) Methylene blue
5. How does negative staining create contrast in TEM images?
A) By absorbing electrons in the stained background✔
B) By staining the virus directly
C) By amplifying viral RNA
D) By using antibodies
6. Which stain is commonly used for enveloped viruses due to its low pH?
A) Sodium silicotungstate (SST)
B) Ammonium molybdate
C) Uranyl acetate✔
D) Phosphotungstic acid
7. What is a disadvantage of uranyl acetate?
A) It produces low contrast
B) It can be radioactive✔
C) It only works for non-enveloped viruses
D) It cannot be filtered
8. Which stain is chemically inert and has a neutral pH?
A) Uranyl acetate
B) Sodium silicotungstate✔
C) Methylene blue
D) Crystal violet
9. Why is ammonium molybdate preferred for large viruses?
A) It binds tightly to the support film✔
B) It is radioactive
C) It stains nucleic acids
D) It works only at high pH
10. What happens if too much stain is left on the grid?
A) The virus appears brighter
B) Only stain is visible, obscuring the virus✔
C) The virus disintegrates
D) The grid becomes hydrophobic
11. What is the ideal concentration range for most viruses in negative staining?
A) 0.05–0.1 mg/ml✔
B) 10–20 mg/ml
C) 1–5 mg/ml
D) 50–100 mg/ml
12. Which support film is preferred for negative staining?
A) Plastic film
B) Carbon film✔
C) Glass slide
D) Gold film
13. What is a major problem during air-drying of enveloped viruses?
A) They multiply rapidly
B) They flatten and lose structure✔
C) They become positively stained
D) They dissolve in the stain
14. How long should uranyl acetate be stirred before use?
A) 1–2 minutes
B) 30 minutes to 1 hour✔
C) 24 hours
D) Not required
15. What is used to remove excess stain from the grid?
A) Heat fixation
B) Filter paper wicking✔
C) Alcohol wash
D) Centrifugation
16. In negative staining, how do viruses appear under TEM?
A) Dark against a light background
B) Light against a dark background✔
C) Fluorescent green
D) Invisible
17. What viral feature is commonly observed in adenovirus after negative staining?
A) Long fibers and penton bases✔
B) Lipid blebs
C) Double-stranded DNA
D) Capsid symmetry
18. What artifact can occur due to superimposed staining layers?
A) Moiré pattern✔
B) Fluorescence quenching
C) Gram-positive reaction
D) Acid-fast staining
19. Why might some viruses appear “collapsed” in TEM?
A) Due to over-staining
B) Due to one-sided staining✔
C) Because of high pH
D) Due to freeze-thawing
20. What does orthomyxovirus (e.g., influenza) show in negative staining?
A) Spiked surface (hemagglutinin)✔
B) Helical nucleic acid
C) Thick cell wall
D) Flagella
21. Which of the following is NOT an application of negative staining?
A) Studying viral morphology
B) Diagnosing bacterial infections
C) Visualizing bacterial flagella
D) Sequencing viral RNA✔
22. What is a limitation of negative staining?
A) It requires live viruses
B) It can distort fragile viruses✔
C) It cannot be used with TEM
D) It only works for fungi
23. Why is negative staining disappearing from mainstream use?
A) It is too expensive
B) Advanced techniques (e.g., cryo-EM) offer better resolution✔
C) It cannot stain bacteria
D) It requires radioactive stains
24. Which safety precaution is necessary when handling uranyl acetate?
A) Using a fume hood
B) Wearing gloves and a lab coat✔
C) Avoiding water contact
D) Keeping it frozen
25. What is “immuno-negative staining”?
A) Combining antibodies with negative staining✔
B) A type of Gram staining
C) A fluorescence technique
D) A DNA sequencing method
26. Who first introduced negative staining?
A) Robert Koch
B) Louis Pasteur
C) Brenner and Horne (1959)✔
D) Antonie van Leeuwenhoek
27. Which virus is best studied with ammonium molybdate staining?
A) HIV
B) Influenza (Orthomyxovirus)✔
C) Rabies
D) Hepatitis B
28. What is the pH of uranyl acetate?
A) 4.4 (acidic)✔
B) 7.0 (neutral)
C) 9.2 (basic)
D) 2.0 (highly acidic)
29. Why is carbon film preferred over plastic film?
A) It is hydrophobic
B) It provides better adsorption and contrast✔
C) It is cheaper
D) It fluoresces under UV
30. Negative staining is most useful for:
A) Quantifying viral load
B) Rapid preliminary virus identification✔
C) DNA extraction
D) Culturing viruses
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