Bacterial morphology, microscopy and staining

33 Kerala MSc MLT Entrance practice questions on Bacterial morphology, microscopy and staining, part of Microbiology. The first 25 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Medium

   In Hiss's capsule stain, the smear is rinsed with 20% copper sulphate rather than water. Why?

   1. ACopper sulphate stains the bacterial cell dark blue
   2. BWater would decolorize the crystal violet in the cells
   3. CThe capsule dissolves in water and would wash away
   4. DCopper sulphate fixes the capsule to the slide

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   Answer: C · The capsule dissolves in water and would wash away

   Most capsules are hydrated polysaccharide and are easily washed off with water. Copper sulphate removes excess crystal violet without destroying the capsule. It also tints the capsule pale blue around the deep purple cells. Heat fixation is avoided or kept gentle for the same reason, since it shrinks the capsule.

   • A: The cells take their deep purple from the crystal violet.
   • B: Water does not decolorize crystal violet from cells.
   • D: Copper sulphate does not act as a fixative.

2. Question 2 · Easy

   The volutin (metachromatic) granules of Corynebacterium diphtheriae are stores of:

   1. AGlycogen
   2. BSulphur
   3. CLipid
   4. DPolymetaphosphate

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   Answer: D · Polymetaphosphate

   Volutin granules are made of polymerised inorganic phosphate, an energy and phosphate reserve. They stain red-purple with polychrome dyes such as Albert's and Neisser's stains, while the cytoplasm stains a different colour. This colour change is called metachromasia. The granules lie at the poles of the bacillus and help in the presumptive diagnosis of diphtheria.

   • A: Glycogen granules are carbon stores, not metachromatic.
   • B: Sulphur granules occur in some environmental bacteria.
   • C: Lipid granules stain with Sudan black, not metachromatically.

3. Question 3 · Medium

   In the Ziehl-Neelsen stain for Mycobacterium leprae, 5% sulphuric acid is used as the decolorizer instead of 20%, because M. leprae is:

   1. AMore acid-fast than M. tuberculosis
   2. BLess acid-fast than M. tuberculosis
   3. CUnable to take up carbol fuchsin
   4. DDamaged by the heat used in staining

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   Answer: B · Less acid-fast than M. tuberculosis

   Mycobacterium leprae holds carbol fuchsin less firmly than M. tuberculosis. A strong 20% acid would decolorize it and give false negatives. Slit-skin smears are therefore decolorized with 5% sulphuric acid. Nocardia, which is weaker still, needs an even milder acid such as 0.5-1%.

   • A: A more acid-fast organism would tolerate the stronger acid.
   • C: It does take up carbol fuchsin; it holds it less firmly.
   • D: Heating is used for M. leprae smears as usual.

4. Question 4 · Hard

   With the oil-immersion objective, 25 ocular micrometer divisions coincide with 3 stage micrometer divisions, each stage division being 10 µm. A bacillus spans 4 ocular divisions. Its length is:

   1. A1.2 µm
   2. B48 µm
   3. C3.3 µm
   4. D4.8 µm

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   Answer: D · 4.8 µm

   The 3 stage divisions measure 30 µm, so each ocular division equals 30 / 25 = 1.2 µm at this objective. The bacillus spans 4 divisions, so it is 4 x 1.2 = 4.8 µm long. Inverting the ratio gives 3.3 µm, and forgetting to multiply by 4 gives 1.2 µm. Calibration must be repeated for each objective, because the value changes with magnification.

   • A: 1.2 µm is the value of one division, not the bacillus.
   • B: 48 µm treats each ocular division as one stage division (10 µm).
   • C: 3.3 µm comes from inverting the calibration ratio (25/30).

5. Question 5 · Hard

   A dairy farmer whose cow died suddenly has a painless black eschar on his forearm. A smear of vesicle fluid stained with polychrome methylene blue shows blue, square-ended rods in short chains surrounded by purple amorphous material. The purple material is the:

   1. AVolutin of Corynebacterium diphtheriae
   2. BSpore of Clostridium tetani
   3. CCapsule of Bacillus anthracis
   4. DSlime of Pseudomonas aeruginosa

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   Answer: C · Capsule of Bacillus anthracis

   This is the McFadyean reaction: polychrome methylene blue stains the polypeptide (D-glutamic acid) capsule of B. anthracis purple around blue bacilli. Square-ended rods in short chains, a sudden animal death and a painless eschar fit cutaneous anthrax. The capsule is made in the body, so it shows in clinical smears but often not in culture. Specimens are handled in a biosafety cabinet.

   • A: Volutin granules are inside the cell, at its poles.
   • B: Clostridium tetani spores are terminal and round, not a surrounding coat.
   • D: Pseudomonas aeruginosa is a slender gram-negative rod, not a chained square-ended rod.

6. Question 6 · Easy

   The heat resistance of bacterial endospores is linked mainly to their core content of:

   1. APoly-beta-hydroxybutyrate
   2. BTeichoic acid
   3. CMycolic acid
   4. DCalcium dipicolinate

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7. Question 7 · Hard

   A student stains a Bacillus smear by the Schaeffer-Fulton method, leaving malachite green on for 5 minutes at room temperature, then water and safranin. The bacilli are red, with clear oval areas inside them. What went wrong?

   1. AThe culture was too young to contain spores
   2. BThe safranin counterstain was left on too long
   3. CThe malachite green was not steamed into the spores
   4. DThe smear was washed too long with water

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8. Question 8 · Medium

   Bacterial flagella are about 20 nm thick, yet they can be seen with a light microscope after Leifson's stain. How?

   1. ABasic fuchsin binds only to flagellin and turns it black
   2. BThe stain makes the flagella fluoresce under UV light
   3. CA tannic acid mordant builds up a dye deposit on them
   4. DThe stain fixes the flagella in their extended position

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9. Question 9 · Medium

   In auramine-phenol staining for acid-fast bacilli, potassium permanganate is applied after the acid-alcohol to:

   1. AFix the bacilli to the slide
   2. BRemove the auramine from the bacilli
   3. CMake the bacilli fluoresce green
   4. DQuench background fluorescence

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10. Question 10 · Easy

    Wall-deficient variants that arise when bacteria grow with penicillin, can multiply, and may revert to the walled form when the drug is removed are called:

    1. AMesosomes
    2. BMycoplasmas
    3. CL-forms
    4. DProtoplasts

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11. Question 11 · Medium

    Phase-contrast microscopy makes living, unstained cells visible by:

    1. AUsing a beam of electrons of much shorter wavelength than light
    2. BConverting differences in refractive index into differences in brightness
    3. CAllowing only light scattered by the object to reach the objective
    4. DExciting natural fluorescence of cell components with ultraviolet light

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12. Question 12 · Medium

    A wound smear shows Gram-positive bacilli with round terminal spores wider than the cell, giving a drumstick appearance. This suggests:

    1. AClostridium tetani
    2. BBacillus cereus
    3. CBacillus anthracis
    4. DClostridium perfringens

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13. Question 13 · Easy

    In the Gram stain, the reagent applied after crystal violet that forms an insoluble dye complex inside the cell is:

    1. AAcetone-alcohol
    2. BDilute carbol fuchsin
    3. CGram's iodine
    4. DSafranin

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14. Question 14 · Hard

    In one week, an NTEP designated microscopy centre finds scanty AFB in several sputum smears from patients whose repeat smears and cultures are negative. A clean new slide with no specimen, stained with the same reagents and rinse water, also shows a few acid-fast rods. What is the most likely explanation?

    1. AUnder-decolorization of the patients' smears
    2. BOld methylene blue counterstain hiding the bacilli
    3. CCross-transfer of bacilli between slides on the rack
    4. DEnvironmental mycobacteria in the rinse water or reagents

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15. Question 15 · Hard

    During a Gram stain practical, a student stains smears of Staphylococcus aureus and Escherichia coli. Both appear pink. The student applied crystal violet for 1 minute, iodine for 1 minute, then acetone-alcohol for 60 seconds, then safranin. What most likely caused the error?

    1. ASafranin was applied for too short a time
    2. BThe iodine was left on for too long
    3. CThe smears were made too thick
    4. DThe decolorizer was left on for too long

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16. Question 16 · Easy

    Teichoic acids are a characteristic component of the cell wall of:

    1. AMycoplasma species
    2. BGram-negative bacteria
    3. CGram-positive bacteria
    4. DChlamydia species

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17. Question 17 · Hard

    A sputum smear stained by ZN is examined under oil immersion. Across 50 fields, the technologist counts 4 to 7 acid-fast bacilli in each field. Using the NTEP grading scale, how should the smear be reported?

    1. APositive, 2+
    2. BPositive, 1+
    3. CPositive, 3+
    4. DScanty, with the count recorded

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18. Question 18 · Medium

    A hanging drop of fresh rice-water stool shows curved bacilli moving with rapid darting motility. This movement is produced by a:

    1. ASingle polar flagellum
    2. BTuft of flagella at both poles
    3. CPair of periplasmic axial filaments
    4. DPeritrichous flagellar coat

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19. Question 19 · Hard

    A student examines a hanging drop of an unknown broth culture. The bacteria vibrate in place but do not change their position across the field. A drop of the same culture after heating to kill the cells shows exactly the same movement. What should be concluded?

    1. AThe organism is non-motile and the movement is Brownian
    2. BThe culture is too young for flagella to have formed
    3. CThe organism is motile with a single polar flagellum
    4. DThe organism shows gliding motility on the coverslip

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20. Question 20 · Hard

    A 72-hour culture of a Bacillus species on nutrient agar gives a Gram stain in which many rods are pink and some are purple. An 18-hour subculture of the same isolate stains uniformly purple. How should this be interpreted?

    1. AGram-variable staining due to ageing cell walls in the old culture
    2. BL-form conversion caused by a penicillin-containing medium
    3. CA mixed culture of a Gram-positive and a Gram-negative bacillus
    4. DUnder-decolorization of the 18-hour smear

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21. Question 21 · Easy

    In the Ziehl-Neelsen stain for sputum, the counterstain that colours the background and non-acid-fast cells is usually:

    1. AMethylene blue
    2. BMalachite green in alcohol
    3. CCrystal violet
    4. DSafranin

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22. Question 22 · Hard

    Auramine-stained sputum smears were stained on Friday, left on the bench under daylight, and read on Tuesday. Several known positive smears now show very few faint bacilli. What is the most likely reason, and the corrective action?

    1. AFluorescence fades with light and time; read within 24 hours, store in the dark
    2. BThe permanganate quench step was too long; shorten it to 10 seconds
    3. CThe smears were under-fixed; heat fix them again before reading
    4. DThe smears dried out; add a drop of water before reading

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23. Question 23 · Medium

    In negative staining with nigrosin or India ink, bacteria appear as clear bodies on a dark background because:

    1. AThe anionic dye is repelled by the negative cell surface
    2. BThe dye is positively charged and binds only to the glass slide
    3. CThe cell wall lipids dissolve the dye and wash it out of the cell
    4. DHeat fixation removes the dye from the cells but not the background

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24. Question 24 · Medium

    Unlike the polysaccharide capsules of most bacteria, the capsule of Bacillus anthracis is made of:

    1. AMycolic acid
    2. BLipoteichoic acid
    3. CHyaluronic acid
    4. DPoly-D-glutamic acid

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25. Question 25 · Easy

    Treponema pallidum is too thin to be seen in a Gram-stained smear of chancre fluid. Which staining method makes it visible as dark brown to black spirals?

    1. AFontana's silver impregnation
    2. BSchaeffer-Fulton stain
    3. CAlbert's stain
    4. DHiss's capsule stain

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