Over 2 weeks, a TB laboratory reports scanty acid-fast bacilli (1 to 9 AFB per 100 oil-immersion fields) on Ziehl-Neelsen smears from seven new patients, but Xpert MTB/RIF on the same sputa is negative in all seven. This week the negative control slides, prepared from a known AFB-negative suspension, also show a few red rods, although the background and the other bacteria on every slide are evenly blue. The slides are new, they are stained flat on a rack and never dipped in shared jars, and the carbol fuchsin was freshly made and filtered. Since a plumbing repair, staff have rinsed the slides with tap water from a storage tank instead of distilled water. The most likely cause is:

Acid-fast saprophytes in the rinse water

Cross-contamination of sputa during processing

Bacilli carried between slides during staining

Too little decolorization of thick smears

A man has taken anti-TB treatment for 2 months. His sputum auramine smear still shows a few acid-fast bacilli, but the culture is negative at 8 weeks. What is the most likely explanation?

The smear is detecting nonviable bacilli

Auramine also stains non-acid-fast bacteria

The culture was read before growth could appear

The bacilli have become resistant to the drugs

In the Ziehl-Neelsen stain, the decolorizing agent is usually:

3% hydrochloric acid in ethanol

Acetone-alcohol, as used in the Gram stain

A Gram smear of wound pus is uneven. In thick areas, neutrophil nuclei and many rods are purple; in thin areas, the nuclei are pink and the same-shaped rods are pink. Gram-positive cocci in clusters are purple in both areas. How should the rods be reported?

As gram-negative rods, read from the thin areas

As gram-positive rods, read from the thick areas

As uninterpretable, with no organisms reported

As gram-variable rods with no further comment

Which pair of organisms is commonly used as the gram-positive and gram-negative controls when Gram stain reagents are checked?

Staphylococcus aureus and Escherichia coli

Enterococcus faecalis and Clostridium perfringens

Bacillus cereus and Mycobacterium smegmatis

Streptococcus pyogenes and Candida albicans

Microscopy and stains

14 DOH Abu Dhabi (HAAD) lab licensing practice questions on Microscopy and stains, part of Microbiology. The first 14 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
Over 2 weeks, a TB laboratory reports scanty acid-fast bacilli (1 to 9 AFB per 100 oil-immersion fields) on Ziehl-Neelsen smears from seven new patients, but Xpert MTB/RIF on the same sputa is negative in all seven. This week the negative control slides, prepared from a known AFB-negative suspension, also show a few red rods, although the background and the other bacteria on every slide are evenly blue. The slides are new, they are stained flat on a rack and never dipped in shared jars, and the carbol fuchsin was freshly made and filtered. Since a plumbing repair, staff have rinsed the slides with tap water from a storage tank instead of distilled water. The most likely cause is:
1. AAcid-fast saprophytes in the rinse water
2. BCross-contamination of sputa during processing
3. CBacilli carried between slides during staining
4. DToo little decolorization of thick smears
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Answer: A · Acid-fast saprophytes in the rinse water
Scanty positive smears in several patients, all negative by Xpert, point to acid-fast rods that did not come from the patients. The negative control slides were prepared separately from patient material and are also positive, so the source lies in the staining process, not in the specimens. The evenly blue background rules out poor decolorization, and new slides stained flat on a rack make transfer between slides unlikely. Tap water and storage tanks often hold environmental mycobacteria, which is why AFB staining procedures call for distilled or filtered water for rinsing and for reagents. The water source should be changed and the affected reports reviewed and corrected.
- B: Processing errors cannot put acid-fast rods on negative control slides prepared separately.
- C: Slides stained flat on a rack are not exposed to bacilli from other smears, as they are in a shared jar.
- D: Under-decolorized smears have a red-tinged background; here the background is evenly blue.
Question 2 · Medium
A man has taken anti-TB treatment for 2 months. His sputum auramine smear still shows a few acid-fast bacilli, but the culture is negative at 8 weeks. What is the most likely explanation?
1. AAuramine also stains non-acid-fast bacteria
2. BThe smear is detecting nonviable bacilli
3. CThe culture was read before growth could appear
4. DThe bacilli have become resistant to the drugs
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Answer: B · The smear is detecting nonviable bacilli
Microscopy shows bacilli whether they are alive or dead, while culture grows only living ones. During effective treatment, killed bacilli can still be seen for weeks, so a positive smear with a negative culture is expected and is not by itself a sign of failure. Culture is the more reliable guide to whether viable bacilli remain. Resistant bacilli would survive the drugs and grow in culture.
- A: Auramine is read for acid-fast morphology, and controls guard against false staining.
- C: Eight weeks is a full incubation period for mycobacterial culture.
- D: Resistant bacilli would still be alive and would grow in culture.
Question 3 · Easy
In the Ziehl-Neelsen stain, the decolorizing agent is usually:
1. AAcetone-alcohol, as used in the Gram stain
2. B3% hydrochloric acid in ethanol
3. C1% aqueous sulfuric acid
4. D95% ethanol alone
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Answer: B · 3% hydrochloric acid in ethanol
After carbol fuchsin is driven into the cell wall with heat, a strong decolorizer is applied; acid-fast organisms keep the red dye because their mycolic acids resist it. Acid-alcohol, 3% HCl in 95% ethanol, is standard, and some WHO procedures use 25% sulfuric acid instead. Methylene blue then counterstains the background and non-acid-fast cells. The weak 1% sulfuric acid is used only in the modified stain for partially acid-fast organisms such as Nocardia.
- A: Acetone-alcohol is the Gram stain decolorizer and is not used for acid-fast staining.
- C: 1% sulfuric acid is a weak decolorizer used for partially acid-fast organisms.
- D: Ethanol alone is too weak to separate acid-fast from non-acid-fast cells.
Question 4 · Hard
A Gram smear of wound pus is uneven. In thick areas, neutrophil nuclei and many rods are purple; in thin areas, the nuclei are pink and the same-shaped rods are pink. Gram-positive cocci in clusters are purple in both areas. How should the rods be reported?
1. AAs gram-positive rods, read from the thick areas
2. BAs gram-negative rods, read from the thin areas
3. CAs uninterpretable, with no organisms reported
4. DAs gram-variable rods with no further comment
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Answer: B · As gram-negative rods, read from the thin areas
Host cells act as a built-in decolorization control: neutrophil nuclei should be pink where decolorization is correct. In the thick areas the purple nuclei show under-decolorization, so purple rods there cannot be trusted. In the thin areas the nuclei are pink while the cocci stay purple, so the stain worked, and the pink rods are truly gram-negative. Smears should be spread thinly to avoid this problem.
- A: Purple nuclei show the thick areas are under-decolorized, so their colors are unreliable.
- C: The thin areas are correctly stained and can be reported.
- D: The rods are pink wherever decolorization was correct, so they are not gram-variable.
Question 5 · Medium
Which pair of organisms is commonly used as the gram-positive and gram-negative controls when Gram stain reagents are checked?
1. AStaphylococcus aureus and Escherichia coli
2. BEnterococcus faecalis and Clostridium perfringens
3. CBacillus cereus and Mycobacterium smegmatis
4. DStreptococcus pyogenes and Candida albicans
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Answer: A · Staphylococcus aureus and Escherichia coli
A control slide carrying a gram-positive coccus such as Staphylococcus aureus and a gram-negative rod such as Escherichia coli is stained with each new reagent lot and at set intervals. The cocci should be purple and the rods pink. If either shows the wrong color, patient smears are not reported until the cause, such as weak iodine or a decolorizing error, is fixed. Some laboratories make the slide from a mixed suspension so both appear in one field.
- B: Both are gram-positive, so the pair cannot check the gram-negative reaction.
- C: Mycobacterium smegmatis is an acid-fast control, and neither organism is gram-negative.
- D: Both are gram-positive, so a decolorizing error would not be detected.
Question 6 · Medium
A laboratory uses 0.8% basic fuchsin instead of safranin as the Gram counterstain. The main benefit is better staining of:
1. AAcid-fast bacilli such as mycobacteria
2. BGram-positive cocci such as streptococci
3. CYeasts such as Candida species
4. DFaint gram-negative rods such as Legionella
Answer and explanation in LearnFree account
Question 7 · Hard
In a Ziehl-Neelsen run, the positive control slide shows only blue rods and every patient smear is negative. A new staff member covered the slides with carbol fuchsin for 5 minutes at room temperature, then decolorized with acid-alcohol and counterstained with methylene blue. The most likely cause is that:
1. AThe methylene blue counterstain was left on too long
2. BThe smears were heat-fixed before staining began
3. CThe acid-alcohol was applied for too short a time
4. DThe carbol fuchsin was not heated to steaming
Answer and explanation in LearnFree account
Question 8 · Easy
CSF from a patient with advanced HIV and headache is examined with India ink. Cryptococcus neoformans is recognized as:
1. ABudding yeast surrounded by a clear halo
2. BYeast cells with a dark-staining capsule
3. CNarrow septate hyphae branching at 45°
4. DPseudohyphae with clusters of blastoconidia
Answer and explanation in LearnFree account
Question 9 · Easy
A main advantage of auramine-rhodamine staining over Ziehl-Neelsen staining for AFB screening is that the smears:
1. ACan be scanned faster at a lower magnification
2. BNeed no decolorizing step at all
3. CNo longer need a positive control slide in each run
4. DShow acid-fast bacilli as red rods
Answer and explanation in LearnFree account
Question 10 · Easy
Acid-fast bacteria hold carbol fuchsin against acid-alcohol because their cell walls are rich in:
1. ATeichoic acids
2. BLipopolysaccharide
3. CCapsular polysaccharide
4. DMycolic acids
Answer and explanation in LearnFree account
Question 11 · Medium
Compared with a cryptococcal antigen test on CSF, the India ink preparation:
1. ADetects early, low-burden infection better
2. BNeeds the organism to be grown in culture first
3. CIs the preferred test for monitoring treatment
4. DIs less sensitive and misses many cases
Answer and explanation in LearnFree account
Question 12 · Medium
A young man has scaly, pale patches on his upper back. A KOH mount of skin scrapings shows short, curved hyphae mixed with clusters of round yeast cells. Which organism is most likely?
1. ATrichophyton rubrum
2. BMalassezia species
3. CMicrosporum canis
4. DCandida albicans
Answer and explanation in LearnFree account
Question 13 · Hard
A Ziehl-Neelsen smear of sputum is read under oil immersion, and 180 acid-fast bacilli are counted in 50 fields. On the WHO/IUATLD grading scale, the result is reported as:
1. A1+
2. B3+
3. CScanty
4. D2+
Answer and explanation in LearnFree account
Question 14 · Easy
Gram-positive organisms appear pink on a Gram stain. What is the most likely technical cause?
1. ALeaving out the safranin counterstain
2. BUsing too thin a smear
3. COver-decolorization
4. DUnder-decolorization
Answer and explanation in LearnFree account

Microscopy and stains

A man has taken anti-TB treatment for 2 months. His sputum auramine smear still shows a few acid-fast bacilli, but the culture is negative at 8 weeks. What is the most likely explanation?

In the Ziehl-Neelsen stain, the decolorizing agent is usually:

A Gram smear of wound pus is uneven. In thick areas, neutrophil nuclei and many rods are purple; in thin areas, the nuclei are pink and the same-shaped rods are pink. Gram-positive cocci in clusters are purple in both areas. How should the rods be reported?

Which pair of organisms is commonly used as the gram-positive and gram-negative controls when Gram stain reagents are checked?

A laboratory uses 0.8% basic fuchsin instead of safranin as the Gram counterstain. The main benefit is better staining of:

CSF from a patient with advanced HIV and headache is examined with India ink. Cryptococcus neoformans is recognized as:

A main advantage of auramine-rhodamine staining over Ziehl-Neelsen staining for AFB screening is that the smears:

Acid-fast bacteria hold carbol fuchsin against acid-alcohol because their cell walls are rich in:

Compared with a cryptococcal antigen test on CSF, the India ink preparation:

A young man has scaly, pale patches on his upper back. A KOH mount of skin scrapings shows short, curved hyphae mixed with clusters of round yeast cells. Which organism is most likely?

A Ziehl-Neelsen smear of sputum is read under oil immersion, and 180 acid-fast bacilli are counted in 50 fields. On the WHO/IUATLD grading scale, the result is reported as:

Gram-positive organisms appear pink on a Gram stain. What is the most likely technical cause?