In the Schaeffer-Fulton spore stain, how do the spores and vegetative cells appear?

Colourless spores in blue cells

A student stains a smear of Staphylococcus aureus in this order: crystal violet, acetone-alcohol, Gram's iodine, safranin. The cocci will most likely appear:

Pink, like gram-negative bacteria

Purple, as in a correct Gram stain

Bacteriology: staining methods

23 Kerala PSC Lab Technician Gr II practice questions on Bacteriology: staining methods, part of Microbiology. The first 23 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Easy
In the Ziehl-Neelsen method, the slide is heated after it is flooded with:
1. AStrong carbol fuchsin
2. BDilute carbol fuchsin
3. CMethylene blue
4. DCrystal violet
Show answerHide answer
Answer: A · Strong carbol fuchsin
Strong carbol fuchsin is the primary stain, and gentle heating until steam rises helps it penetrate the waxy wall of mycobacteria. The smear is then decolourized with acid and counterstained with methylene blue. Acid-fast bacilli stay red against a blue background. The stain must not boil or dry on the slide.
- B: Dilute carbol fuchsin is a counterstain and is not heated.
- C: Methylene blue is the counterstain and is applied without heat.
- D: Crystal violet is the primary stain of the Gram method.
Question 2 · Easy
A throat swab from a child with a greyish membrane on the tonsils is stained by Albert's method. What is the stain meant to show?
1. AMetachromatic granules
2. BCapsules
3. CFlagella
4. DEndospores
Show answerHide answer
Answer: A · Metachromatic granules
Albert's stain uses toluidine blue and malachite green, followed by Albert's iodine. The volutin (metachromatic) granules of Corynebacterium diphtheriae stain bluish-black and the bacilli stain green, often in Chinese-letter arrangements. Finding such bacilli supports a presumptive diagnosis of diphtheria, but culture and a toxigenicity test are needed to confirm it. Neisser's stain shows the same granules.
- B: Capsules are shown by negative staining or special capsule stains.
- C: C. diphtheriae is non-motile and has no flagella.
- D: C. diphtheriae does not form spores.
Question 3 · Medium
Mycobacteria are not decolourized by acid after carbol fuchsin staining mainly because their cell wall is rich in:
1. ADipicolinic acid
2. BMycolic acids
3. CLipopolysaccharide
4. DTeichoic acids
Show answerHide answer
Answer: B · Mycolic acids
The mycobacterial cell wall contains long-chain mycolic acids and other waxy lipids that keep dyes out. Once heat drives carbol fuchsin in, the dye bound to these lipids resists removal by acid. The bacilli therefore stay red while other bacteria and cells take up the blue counterstain. Nocardia, with shorter mycolic acids, is only weakly acid-fast.
- A: Dipicolinic acid is found in bacterial spores and gives them heat resistance.
- C: Lipopolysaccharide belongs to the outer membrane of gram-negative bacteria.
- D: Teichoic acids are found in gram-positive walls and do not give acid-fastness.
Question 4 · Medium
In the Schaeffer-Fulton spore stain, how do the spores and vegetative cells appear?
1. AColourless spores in blue cells
2. BPurple spores in pink cells
3. CRed spores in green cells
4. DGreen spores in red cells
Show answerHide answer
Answer: D · Green spores in red cells
Malachite green is driven into the tough spore coat by steaming; water then washes it out of the vegetative cells but not the spores. Safranin counterstains the vegetative cells red. The spores therefore look green, whether inside red bacilli or free. In a Gram stain, by contrast, spores appear as unstained gaps in the cell.
- A: Spores look colourless in Gram or simple stains, not in a spore stain.
- B: Purple and pink are the colours of the Gram stain.
- C: This reverses the colours; malachite green stays in the spores.
Question 5 · Hard
A student stains a smear of Staphylococcus aureus in this order: crystal violet, acetone-alcohol, Gram's iodine, safranin. The cocci will most likely appear:
1. ABrown, from the iodine
2. BPink, like gram-negative bacteria
3. CPurple, as in a correct Gram stain
4. DColourless, with no stain
Show answerHide answer
Answer: B · Pink, like gram-negative bacteria
Iodine must be applied before decolourization, because it forms the large crystal violet-iodine complex that the thick gram-positive wall traps. Applied first, acetone-alcohol washes the uncomplexed crystal violet out of the cocci. The iodine then has no dye to fix, and safranin stains the decolourized cells pink. The result is a false gram-negative appearance, which is why the order of reagents matters.
- A: The faint iodine colour is masked by the safranin.
- C: Purple needs the dye-iodine complex to form before decolourization.
- D: Safranin stains the decolourized cells, so they are not colourless.
Question 6 · Easy
In Hiss's capsule stain, the smear is washed after staining with:
1. A20% copper sulphate
2. BLugol's iodine
3. CAcetone-alcohol
4. D3% acid alcohol
Answer and explanation in LearnFree account
Question 7 · Hard
In a Ziehl-Neelsen run, the bacilli on the positive control are red, but on every slide, including the control, the background is pink and the pus cells are red instead of blue. The most likely cause is:
1. ACarbol fuchsin was not heated
2. BDecolourisation was too short
3. CSmears were not heat-fixed
4. DMethylene blue was left on too long
Answer and explanation in LearnFree account
Question 8 · Medium
Gram-positive bacteria keep the crystal violet-iodine complex during decolourisation mainly because:
1. ATheir outer membrane binds the dye firmly
2. BTheir thick peptidoglycan shrinks and traps it
3. CTheir teichoic acid reacts with safranin
4. DTheir mycolic acid holds the dye
Answer and explanation in LearnFree account
Question 9 · Medium
A slit-skin smear for leprosy is stained by the Ziehl-Neelsen method. Why is 5% sulphuric acid used to decolourise it instead of 20%?
1. ASkin smears hold no mycolic acid
2. BM. leprae is gram-negative
3. CStronger acid would damage the skin cells
4. DM. leprae is less acid-fast than M. tuberculosis
Answer and explanation in LearnFree account
Question 10 · Easy
In Neisser's stain, the bodies of Corynebacterium diphtheriae appear:
1. AYellowish-brown
2. BGreen
3. CRed
4. DDeep purple
Answer and explanation in LearnFree account
Question 11 · Easy
Albert's stain A contains toluidine blue and:
1. ABasic fuchsin
2. BMalachite green
3. CCrystal violet
4. DSafranin
Answer and explanation in LearnFree account
Question 12 · Easy
In the Gram stain, the reagent that acts as a mordant is:
1. AAcetone-alcohol
2. BSafranin
3. CGram's iodine
4. DCrystal violet
Answer and explanation in LearnFree account
Question 13 · Easy
In the Ziehl-Neelsen method for sputum, the counterstain used is:
1. AGentian violet
2. BMethylene blue
3. CNeutral red
4. DSafranin
Answer and explanation in LearnFree account
Question 14 · Hard
A Gram-stained smear of an E. coli culture shows purple rods in the thick centre and pink rods at the thin edges. The cause is:
1. AThe iodine was left out
2. BThe safranin was too strong
3. CThe smear is too thick in the centre
4. DThe culture is mixed with gram-positives
Answer and explanation in LearnFree account
Question 15 · Hard
An Albert-stained throat swab shows green bacilli with bluish-black polar granules, in V and L shapes. How should it be reported?
1. AVincent's angina; no further tests
2. BDiagnostic of diphtheria; no culture needed
3. CSuggestive of diphtheria; culture to confirm
4. DNormal throat diphtheroids; reassure
Answer and explanation in LearnFree account
Question 16 · Medium
The Kinyoun method stains acid-fast bacilli without any heating because it:
1. AUses a stronger carbol fuchsin
2. BUses a fluorescent dye instead
3. CUses iodine as a mordant
4. DLeaves out the acid decolouriser
Answer and explanation in LearnFree account
Question 17 · Easy
In a negative stain with nigrosin or India ink, the bacteria appear:
1. AColourless against a dark background
2. BPurple against a pink background
3. CRed against a blue background
4. DDark against a colourless background
Answer and explanation in LearnFree account
Question 18 · Medium
In the Schaeffer-Fulton spore stain, the malachite green is steamed over the smear because:
1. AHeat drives the stain into the tough spore coat
2. BHeat makes the vegetative cells take the stain
3. CHeat removes the stain from vegetative cells
4. DHeat is needed to fix the counterstain
Answer and explanation in LearnFree account
Question 19 · Easy
Heat fixing a dried bacterial smear before staining:
1. ARemoves all fat from the bacterial cell
2. BKills the organisms and sticks them to the slide
3. CStains the capsule and spores
4. DMakes every organism gram positive
Answer and explanation in LearnFree account
Question 20 · Hard
An 18-year-old has fever, neck stiffness and a petechial rash. CSF Gram stain shows many pus cells with intracellular gram-negative kidney-shaped diplococci. The likely organism is:
1. AHaemophilus influenzae
2. BStreptococcus pneumoniae
3. CNeisseria meningitidis
4. DListeria monocytogenes
Answer and explanation in LearnFree account
Question 21 · Medium
In a Gram stain, the Staphylococcus aureus control slide shows pink cocci. The most likely error is:
1. AMissing the safranin step
2. BOver-decolourisation
3. CUsing a very thin smear
4. DToo short a counterstain
Answer and explanation in LearnFree account
Question 22 · Medium
Why are Gram stains done on young (18-24 hour) cultures rather than old ones?
1. AOld cultures are too thick to spread
2. BOld gram-positive cells may stain gram negative
3. CYoung cells take up no counterstain
4. DOld cells lose their capsule
Answer and explanation in LearnFree account
Question 23 · Easy
Under NTEP, how many oil-immersion fields must be examined before a Ziehl-Neelsen sputum smear is reported negative?
1. A20
2. B100
3. C300
4. D50
Answer and explanation in LearnFree account

Bacteriology: staining methods

In the Ziehl-Neelsen method, the slide is heated after it is flooded with:

A throat swab from a child with a greyish membrane on the tonsils is stained by Albert's method. What is the stain meant to show?

Mycobacteria are not decolourized by acid after carbol fuchsin staining mainly because their cell wall is rich in:

In the Schaeffer-Fulton spore stain, how do the spores and vegetative cells appear?

A student stains a smear of Staphylococcus aureus in this order: crystal violet, acetone-alcohol, Gram's iodine, safranin. The cocci will most likely appear:

In Hiss's capsule stain, the smear is washed after staining with:

In a Ziehl-Neelsen run, the bacilli on the positive control are red, but on every slide, including the control, the background is pink and the pus cells are red instead of blue. The most likely cause is:

Gram-positive bacteria keep the crystal violet-iodine complex during decolourisation mainly because:

A slit-skin smear for leprosy is stained by the Ziehl-Neelsen method. Why is 5% sulphuric acid used to decolourise it instead of 20%?

In Neisser's stain, the bodies of Corynebacterium diphtheriae appear:

Albert's stain A contains toluidine blue and:

In the Gram stain, the reagent that acts as a mordant is:

In the Ziehl-Neelsen method for sputum, the counterstain used is:

A Gram-stained smear of an E. coli culture shows purple rods in the thick centre and pink rods at the thin edges. The cause is:

An Albert-stained throat swab shows green bacilli with bluish-black polar granules, in V and L shapes. How should it be reported?

The Kinyoun method stains acid-fast bacilli without any heating because it:

In a negative stain with nigrosin or India ink, the bacteria appear:

In the Schaeffer-Fulton spore stain, the malachite green is steamed over the smear because:

Heat fixing a dried bacterial smear before staining:

An 18-year-old has fever, neck stiffness and a petechial rash. CSF Gram stain shows many pus cells with intracellular gram-negative kidney-shaped diplococci. The likely organism is:

In a Gram stain, the Staphylococcus aureus control slide shows pink cocci. The most likely error is:

Why are Gram stains done on young (18-24 hour) cultures rather than old ones?

Under NTEP, how many oil-immersion fields must be examined before a Ziehl-Neelsen sputum smear is reported negative?