A newly trained technologist performs Ziehl-Neelsen stains on three sputum smears and the daily control slide, which carries an acid-fast Mycobacterium species and non-acid-fast Escherichia coli. The log shows: smears heat-fixed on a 70 °C slide warmer for 2 hours; carbol fuchsin flooded on for 5 minutes at room temperature, because the burner for the staining rack had been removed; 3% acid-alcohol applied until no more color ran off; methylene blue for 1 minute. On the control slide, both the mycobacteria and the E. coli are blue. No acid-fast bacilli are seen on the patient smears. The most likely cause of the control failure is:

Carbol fuchsin was applied without heat

The smears were heat-fixed for too long

The acid-alcohol decolorizer was too weak

Methylene blue was left on too long

In the Gram stain, Gram's iodine is applied after crystal violet in order to:

Bind the dye into a complex inside the cell

Fix the bacteria firmly to the glass slide

Counterstain cells that have lost the dye

Remove the dye from gram-negative cells

A Gram stain shows irregular purple granules of uneven size scattered over the background and on top of cells. The most likely cause is:

Crystal violet precipitate in old or unfiltered stain

Over-heating of the smear during fixation

Gram-positive cocci contaminating the slide

Over-decolorization of a thick smear

On an auramine-rhodamine smear read by fluorescence microscopy, acid-fast bacilli appear as:

Yellow-orange rods on a dark background

Bright orange rods on a green background

Purple rods on a pink background

Why may gram-positive bacteria from old cultures, or from a patient on a cell wall-active antibiotic, stain pink?

Their damaged walls cannot hold the dye complex

They form spores that take up only safranin

Their capsules block entry of crystal violet

They gain a lipid-rich outer membrane as they age

Stains: Gram, acid-fast, modified acid-fast

15 ASCP MLS practice questions on Stains: Gram, acid-fast, modified acid-fast, part of Microbiology. The first 15 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 · Hard
A newly trained technologist performs Ziehl-Neelsen stains on three sputum smears and the daily control slide, which carries an acid-fast Mycobacterium species and non-acid-fast Escherichia coli. The log shows: smears heat-fixed on a 70 °C slide warmer for 2 hours; carbol fuchsin flooded on for 5 minutes at room temperature, because the burner for the staining rack had been removed; 3% acid-alcohol applied until no more color ran off; methylene blue for 1 minute. On the control slide, both the mycobacteria and the E. coli are blue. No acid-fast bacilli are seen on the patient smears. The most likely cause of the control failure is:
1. AThe smears were heat-fixed for too long
2. BThe acid-alcohol decolorizer was too weak
3. CCarbol fuchsin was applied without heat
4. DMethylene blue was left on too long
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Answer: C · Carbol fuchsin was applied without heat
Ziehl-Neelsen is the hot acid-fast method: the slide is heated until it steams so that carbol fuchsin can penetrate the waxy mycolic acid layer of the cell wall. Applied cold for 5 minutes, the primary stain never entered the mycobacteria, so the decolorizer removed what little they held and they took up the blue counterstain like E. coli. A weak decolorizer causes the opposite error, leaving non-acid-fast cells red. Heat fixing at 65 to 75 °C for 2 hours is standard for AFB smears. The patient smears must be restained correctly, or by the Kinyoun cold method with its stronger reagent, and reported only after the control passes.
- A: Heat fixing at 65-75 °C for 2 hours is the standard for AFB smears.
- B: A weak decolorizer leaves non-acid-fast cells red; it does not turn acid-fast bacilli blue.
- D: A long counterstain does not remove carbol fuchsin from properly stained acid-fast bacilli.
Question 2 · Easy
In the Gram stain, Gram's iodine is applied after crystal violet in order to:
1. AFix the bacteria firmly to the glass slide
2. BCounterstain cells that have lost the dye
3. CBind the dye into a complex inside the cell
4. DRemove the dye from gram-negative cells
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Answer: C · Bind the dye into a complex inside the cell
Iodine acts as a mordant: it combines with crystal violet to form a large crystal violet-iodine complex inside the cell. In gram-positive cells this complex is trapped in the thick peptidoglycan when the decolorizer dehydrates the wall. Gram-negative cells lose the complex and take up safranin. If the iodine is weak or left out, gram-positive cells can lose the dye and look pink.
- A: Smears are fixed by heat or methanol before staining begins.
- B: Safranin is the counterstain that colors decolorized cells.
- D: Removing the dye is the job of the alcohol or acetone decolorizer.
Question 3 · Medium
A Gram stain shows irregular purple granules of uneven size scattered over the background and on top of cells. The most likely cause is:
1. AOver-heating of the smear during fixation
2. BGram-positive cocci contaminating the slide
3. COver-decolorization of a thick smear
4. DCrystal violet precipitate in old or unfiltered stain
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Answer: D · Crystal violet precipitate in old or unfiltered stain
Crystal violet can form crystals as it ages or evaporates, and these settle on the slide as purple granules. They can be mistaken for gram-positive cocci, but they vary in size, have no regular shape and lie on top of everything. Filtering or replacing the stain and rinsing well prevent the artifact. True cocci are round and uniform, often in pairs, chains or clusters.
- A: Overheating distorts cells but does not deposit purple granules.
- B: Contaminating cocci would be uniform round cells, not irregular granules.
- C: Over-decolorization removes purple dye rather than adding granules.
Question 4 · Easy
On an auramine-rhodamine smear read by fluorescence microscopy, acid-fast bacilli appear as:
1. ABright orange rods on a green background
2. BYellow-orange rods on a dark background
3. CPurple rods on a pink background
4. DRed rods on a blue background
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Answer: B · Yellow-orange rods on a dark background
Auramine O and rhodamine bind the mycolic acids of acid-fast cells and stay in them after acid-alcohol decolorization. A counterstain such as potassium permanganate quenches background fluorescence, so the bacilli glow yellow to orange on a dark field. Smears can be scanned at lower magnification, which makes the method faster and more sensitive than carbol fuchsin stains. A positive fluorochrome smear can be confirmed by restaining with Ziehl-Neelsen or Kinyoun.
- A: Orange organisms on a green background describe the acridine orange stain.
- C: Purple and pink describe gram-positive and gram-negative cells on a Gram stain.
- D: Red rods on a blue background describe carbol fuchsin stains such as Kinyoun.
Question 5 · Medium
Why may gram-positive bacteria from old cultures, or from a patient on a cell wall-active antibiotic, stain pink?
1. ATheir damaged walls cannot hold the dye complex
2. BThey form spores that take up only safranin
3. CTheir capsules block entry of crystal violet
4. DThey gain a lipid-rich outer membrane as they age
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Answer: A · Their damaged walls cannot hold the dye complex
The Gram reaction depends on an intact peptidoglycan wall that traps the crystal violet-iodine complex. As cultures age, or when beta-lactams or vancomycin damage the wall, the complex leaks out during decolorization and the cells take up safranin. Some Clostridium and Bacillus species therefore often look gram-variable or gram-negative. Staining young (18 to 24 hour) growth and checking colony features and other tests avoids misreading.
- B: Spores resist staining and look clear; they do not decide the cell's Gram reaction.
- C: Capsules do not stop crystal violet from staining the cell.
- D: Gram-positive bacteria do not develop an outer membrane with age.
Question 6 · Hard
Three sputum smears from different patients are read as 1+ on Kinyoun stain. Findings: few bacilli per slide; background evenly blue; all stained in one jar with a 4+ smear from another patient; no patient on TB treatment; all cultures negative at 6 weeks. The most likely explanation is:
1. AMycobacteria too few to grow in the cultures
2. BBacilli carried between slides in the shared jar
3. CNocardia mistaken for acid-fast bacilli
4. DUnder-decolorization leaving red background dye
Answer and explanation in LearnFree account
Question 7 · Easy
How does the Kinyoun acid-fast method differ from the Ziehl-Neelsen method?
1. AUses auramine O as the primary stain
2. BDecolorizes with 1% sulfuric acid
3. CNeeds no heat to drive in the stain
4. DUses crystal violet as the primary stain
Answer and explanation in LearnFree account
Question 8 · Medium
The neutrophil nuclei in a Gram-stained sputum smear are deep purple. What does this show about the stain?
1. AThe safranin was left on too long
2. BThe iodine step was left out
3. CThe smear was under-decolorized
4. DThe smear was over-decolorized
Answer and explanation in LearnFree account
Question 9 · Easy
Gram-positive bacteria keep the crystal violet-iodine complex during decolorization mainly because they have:
1. AA polysaccharide capsule around the cell
2. BAn outer membrane rich in lipopolysaccharide
3. CA cell wall rich in long-chain mycolic acids
4. DA thick, cross-linked peptidoglycan layer
Answer and explanation in LearnFree account
Question 10 · Hard
Colonies from a 60-year-old man's leg-wound culture are thin rods. Results: Gram stain, gram-variable on repeated smears with correct controls; 3% KOH test, the suspension turns viscous and forms a string when the loop is lifted. How should the organism be handled?
1. ATreat it as a gram-negative rod
2. BTreat it as a gram-positive rod
3. CRepeat the Gram stain until clear
4. DReport it as gram-variable only
Answer and explanation in LearnFree account
Question 11 · Medium
A blood culture from a patient with advanced HIV signals positive. The Gram stain shows faint, beaded rods that barely take up crystal violet. Which stain should be done next?
1. AAcridine orange stain
2. BGiemsa stain
3. CCalcofluor white stain
4. DKinyoun acid-fast stain
Answer and explanation in LearnFree account
Question 12 · Medium
Why is methanol fixation often preferred over heat fixation for Gram smears of clinical specimens?
1. AIt replaces the need for an iodine mordant
2. BIt sterilizes the smear, including bacterial spores
3. CIt preserves host cells and bacterial shapes better
4. DIt protects gram-positive cells from over-decolorizing
Answer and explanation in LearnFree account
Question 13 · Medium
A 30-year-old man with HIV has watery diarrhea. A modified acid-fast stain of stool shows round pink-red bodies 4 to 6 µm across on a blue-green background. Which organism fits best?
1. AMicrosporidia
2. BCryptosporidium species
3. CCyclospora cayetanensis
4. DCystoisospora belli
Answer and explanation in LearnFree account
Question 14 · Easy
Which change to the Kinyoun procedure makes it the modified acid-fast stain used to detect Nocardia?
1. AReplacing carbol fuchsin with auramine O
2. BCounterstaining longer with methylene blue
3. CUsing 1% sulfuric acid as the decolorizer
4. DHeating the carbol fuchsin until it steams
Answer and explanation in LearnFree account
Question 15 · Hard
Gram stain control slide: Staphylococcus aureus pink; Escherichia coli pink; neutrophil nuclei pink. Reagent check: crystal violet in date; Gram's iodine pale yellow instead of dark brown; decolorizer 95% ethanol applied for 5 seconds. The most likely cause is:
1. ASafranin left out of the procedure
2. BUnder-decolorization of a thick smear
3. CIneffective iodine, so the dye complex did not form
4. DOver-decolorization by too long in ethanol
Answer and explanation in LearnFree account

Stains: Gram, acid-fast, modified acid-fast

In the Gram stain, Gram's iodine is applied after crystal violet in order to:

A Gram stain shows irregular purple granules of uneven size scattered over the background and on top of cells. The most likely cause is:

On an auramine-rhodamine smear read by fluorescence microscopy, acid-fast bacilli appear as:

Why may gram-positive bacteria from old cultures, or from a patient on a cell wall-active antibiotic, stain pink?

Three sputum smears from different patients are read as 1+ on Kinyoun stain. Findings: few bacilli per slide; background evenly blue; all stained in one jar with a 4+ smear from another patient; no patient on TB treatment; all cultures negative at 6 weeks. The most likely explanation is:

How does the Kinyoun acid-fast method differ from the Ziehl-Neelsen method?

The neutrophil nuclei in a Gram-stained sputum smear are deep purple. What does this show about the stain?

Gram-positive bacteria keep the crystal violet-iodine complex during decolorization mainly because they have:

Colonies from a 60-year-old man's leg-wound culture are thin rods. Results: Gram stain, gram-variable on repeated smears with correct controls; 3% KOH test, the suspension turns viscous and forms a string when the loop is lifted. How should the organism be handled?

A blood culture from a patient with advanced HIV signals positive. The Gram stain shows faint, beaded rods that barely take up crystal violet. Which stain should be done next?

Why is methanol fixation often preferred over heat fixation for Gram smears of clinical specimens?

A 30-year-old man with HIV has watery diarrhea. A modified acid-fast stain of stool shows round pink-red bodies 4 to 6 µm across on a blue-green background. Which organism fits best?

Which change to the Kinyoun procedure makes it the modified acid-fast stain used to detect Nocardia?

Gram stain control slide: Staphylococcus aureus pink; Escherichia coli pink; neutrophil nuclei pink. Reagent check: crystal violet in date; Gram's iodine pale yellow instead of dark brown; decolorizer 95% ethanol applied for 5 seconds. The most likely cause is: