Gram Staining 50 FAQs and 25 MCQs
Gram Staining 50 FAQs
What is Gram staining?
Gram staining is a differential bacterial staining technique used to classify bacteria into Gram-positive and Gram-negative based on their cell wall composition.
Who developed Gram staining?
Danish bacteriologist Hans Christian Gram in 1882.
Why is Gram staining important?
It helps quickly identify bacterial infections, guides antibiotic treatment, and is the first step in bacterial identification.
What are the two main types of bacteria identified by Gram staining?
Gram-positive (purple) and Gram-negative (pink/red).
What is the principle behind Gram staining?
Differences in bacterial cell wall structure (peptidoglycan thickness and lipid content) determine stain retention.
Can Gram staining diagnose all bacterial infections?
No, it doesn’t work for bacteria without cell walls (e.g., Mycoplasma) or very small bacteria (e.g., Chlamydia).
What are the limitations of Gram staining?
False results due to over-/under-decolorization, old cultures, or thick smears; cannot identify specific species.
What reagents are used in Gram staining?
Crystal violet (primary stain), Gram’s iodine (mordant), decolorizer (ethanol/acetone), and safranin/carbol fuchsin (counterstain).
Why is iodine used in Gram staining?
It forms a complex with crystal violet, trapping it in Gram-positive cell walls.
What is the role of the decolorizer?
It dissolves lipids in Gram-negative cell walls, washing out the primary stain.
How long should each staining step last?
Typically 30–60 seconds per step, but decolorization requires careful timing (5–10 seconds).
Why is safranin used as a counterstain?
To stain decolorized Gram-negative bacteria pink/red for visibility.
Can carbol fuchsin replace safranin?
Yes, it’s an alternative counterstain, especially for faintly staining Gram-negative bacteria.
How do you prepare a bacterial smear for Gram staining?
Spread a thin bacterial sample on a slide, air-dry, and heat-fix to adhere cells.
What happens if the smear is too thick?
It may retain excess stain, leading to false Gram-positive results.
Why is heat fixation necessary?
It kills bacteria and adheres them to the slide to prevent washing off during staining.
What microscope magnification is used to view Gram stains?
100× oil immersion lens after initial 10× and 40× checks.
How do you differentiate Gram-positive and Gram-negative bacteria under a microscope?
Gram-positive: Purple/blue; Gram-negative: Pink/red.
What does a “Gram-variable” result mean?
Bacteria that stain irregularly (mix of pink/purple) due to cell wall variability.
What are common Gram-positive cocci?
Staphylococcus, Streptococcus, and Enterococcus.
What are common Gram-negative bacilli?
E. coli, Pseudomonas, Klebsiella, and Proteus.
What does “gram-negative diplococci” suggest?
Possibly Neisseria gonorrhoeae or N. meningitidis.
How do Staphylococcus and Streptococcus appear differently on Gram stain?
Staphylococcus: Clusters; Streptococcus: Chains.
What bacteria are acid-fast and won’t Gram stain well?
Mycobacterium spp. (require Ziehl-Neelsen stain).
Why might Gram-positive bacteria appear Gram-negative?
Over-decolorization, old cultures, or antibiotic treatment.
What does the presence of white blood cells (WBCs) in a Gram stain indicate?
Likely an active bacterial infection.
When is Gram staining ordered?
For suspected bacterial infections (e.g., UTIs, pneumonia, meningitis).
What infections are diagnosed using Gram staining?
Pneumonia (S. pneumoniae), UTIs (E. coli), gonorrhea (Neisseria), etc.
Can Gram staining detect fungi?
Yes, yeasts (e.g., Candida) may appear Gram-positive.
How does Gram staining guide antibiotic therapy?
Gram-positive vs. Gram-negative results help narrow antibiotic choices (e.g., vancomycin for Gram-positive).
Why is Gram staining faster than a culture?
It provides results in minutes/hours, while cultures take days.
What samples can be Gram-stained?
Sputum, blood, urine, CSF, wound swabs, and bodily fluids.
How is a urine sample prepared for Gram staining?
Midstream urine is centrifuged, and the sediment is smeared.
What does a Gram stain of CSF indicate?
Bacterial meningitis (e.g., S. pneumoniae or N. meningitidis).
What causes false Gram-negative results?
Over-decolorization or old Gram-positive cultures.
What causes false Gram-positive results?
Under-decolorization or thick smears.
Why might no bacteria be seen on a Gram stain?
Low bacterial load, prior antibiotics, or improper sample collection.
How does antibiotic use affect Gram staining?
It may alter bacterial cell walls, leading to atypical staining.
Why are some Gram-negative bacteria faintly stained?
Campylobacter or Brucella may require carbol fuchsin for better visibility.
What if the Gram stain shows contamination?
Squamous epithelial cells suggest poor sample collection (e.g., saliva in sputum).
How is crystal violet prepared for Gram staining?
Mixed with ammonium oxalate and ethanol
Can expired reagents affect results?
Yes, crystal violet may form precipitates; filtering is recommended.
Why is immersion oil used?
To improve resolution under the 100× objective lens.
What are alternative decolorizers?
Acetone alone or ethanol (95%), but 50:50 acetone-ethanol is common.
How should slides be stored after staining?
Air-dried and kept away from light to prevent fading.
What is the role of peptidoglycan in Gram staining?
Thick peptidoglycan in Gram-positive bacteria traps the CV-I complex.
Why do Gram-negative bacteria lose the primary stain?
Their thin peptidoglycan and outer membrane are disrupted by decolorizer.
What modifications exist for Gram staining?
Burke’s (aniline-oil decolorizer) and Atkin’s methods.
How does Mycobacterium differ in staining?
Its waxy cell wall requires acid-fast staining (e.g., Ziehl-Neelsen).
Can Gram staining identify spores?
Yes, Bacillus and Clostridium spores may appear as clear ovals within stained cells.
Gram Staining 25 MCQs
- Who developed the Gram staining technique?
a) Robert Koch
b) Louis Pasteur
c) Hans Christian Gram✔
d) Alexander Fleming - Gram staining is a type of:
a) Simple staining
b) Differential staining✔
c) Negative staining
d) Acid-fast staining - Gram staining helps differentiate bacteria based on:
a) Shape
b) Cell wall composition✔
c) Motility
d) Spore formation - Which of the following is NOT a step in Gram staining?
a) Decolorization
b) Counterstaining
c) Heat fixation at 100°C✔
d) Mordant application - Gram staining is most useful for identifying:
a) Viruses
b) Bacteria✔
c) Fungi
d) Parasites
- The primary stain in Gram staining is:
a) Safranin
b) Crystal violet✔
c) Methylene blue
d) Carbol fuchsin - Gram’s iodine acts as a:
a) Decolorizer
b) Mordant✔
c) Counterstain
d) Fixative - The decolorizer used in Gram staining is:
a) Acid-alcohol
b) Acetone/ethanol✔
c) Hydrogen peroxide
d) Formalin - The counterstain in Gram staining is:
a) Crystal violet
b) Safranin✔
c) Methylene blue
d) Malachite green - Over-decolorization can lead to:
a) False Gram-negative results✔
b) False Gram-positive results
c) No effect
d) Enhanced staining
- Gram-positive bacteria appear:
a) Pink
b) Purple✔
c) Red
d) Colorless - Gram-negative bacteria appear:
a) Pink/red✔
b) Purple
c) Blue
d) Green - Gram-positive bacteria have:
a) Thick peptidoglycan layer✔
b) Thin peptidoglycan layer
c) No peptidoglycan
d) Outer membrane - Gram-negative bacteria have:
a) Thick peptidoglycan
b) Outer membrane with LPS✔
c) No cell wall
d) Teichoic acids - Which structure is absent in Gram-negative bacteria?
a) Teichoic acids✔
b) Lipopolysaccharides (LPS)
c) Porins
d) Periplasmic space
- A Gram stain of sputum shows purple cocci in clusters. The likely organism is:
a) Streptococcus pneumoniae
b) Staphylococcus aureus✔
c) Escherichia coli
d) Pseudomonas aeruginosa - Gram-negative diplococci in CSF suggest:
a) Neisseria meningitidis✔
b) Streptococcus pyogenes
c) Haemophilus influenzae
d) Mycobacterium tuberculosis - A UTI sample shows pink rods. The likely pathogen is:
a) Staphylococcus saprophyticus
b) Escherichia coli✔
c) Enterococcus faecalis
d) Streptococcus agalactiae - Which bacteria are NOT reliably stained by Gram staining?
a) Mycoplasma pneumoniae✔
b) Staphylococcus aureus
c) Escherichia coli
d) Pseudomonas aeruginosa - Gram staining is LEAST useful for diagnosing:
a) Bacterial pneumonia
b) Viral influenza✔
c) Urinary tract infection
d) Meningitis
- A smear that is too thick may cause:
a) False Gram-positive results✔
b) False Gram-negative results
c) No effect
d) Over-decolorization - Old cultures may appear Gram-negative due to:
a) Loss of peptidoglycan integrity✔
b) Increased lipid content
c) Over-staining
d) Contamination - Under-decolorization leads to:
a) False Gram-positive results✔
b) False Gram-negative results
c) No effect
d) Smear washing off - Carbol fuchsin is preferred over safranin for:
a) Gram-positive bacteria
b) Faintly staining Gram-negative bacteria✔
c) Acid-fast bacteria
d) Capsule staining - Gram staining cannot identify:
a) Viruses ✔
b) Staphylococcus
c) Escherichia coli
d) Candida albicans
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