A SYBR Green real-time PCR for the Staphylococcus aureus nuc gene, with melt-curve analysis, is run on a staphylococcal colony. From the derivative melt curves shown, the colony result is best reported as:

nuc not detected; the colony peak is primer-dimer

nuc variant; confirm it by sequencing

Amplicon contamination; repeat the run

nuc detected; the colony is S. aureus

Automated identification systems such as VITEK 2 identify an isolate by:

Reading colour or turbidity changes in biochemical wells

Detecting CO2 produced in a culture bottle

Amplifying species-specific DNA targets

Matching the protein mass spectrum to a reference library

A patient with culture-negative endocarditis has valve surgery. Broad-range 16S rRNA PCR with Sanger sequencing on the valve gives a trace with overlapping double peaks along its whole length. The most likely explanation:

More than one bacterial species is present

The organism is not in the database

A 70-year-old with pneumonia has an alpha-hemolytic blood isolate that MALDI-TOF reports as Streptococcus pneumoniae. Why do many laboratories still confirm it with bile solubility or optochin?

Its spectrum is very close to that of S. mitis/oralis

Pneumococcal capsules block ionization

MALDI-TOF cannot identify catalase-negative cocci

The matrix degrades pneumococcal proteins

A febrile patient's blood culture shows gram-positive cocci in clusters. A multiplex panel that reports mecA alone, with no S. aureus-specific junction target, detects S. aureus, S. epidermidis and mecA. What is the correct conclusion?

mecA cannot yet be assigned to either species

The S. aureus is methicillin-resistant

The S. aureus result is a false positive

The S. epidermidis is methicillin-susceptible

Identification: automated, MALDI-TOF, multiplex molecular, 16S sequencing

11 ASCP MLS practice questions on Identification: automated, MALDI-TOF, multiplex molecular, 16S sequencing, part of Microbiology. The first 11 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 · Medium
A SYBR Green real-time PCR for the Staphylococcus aureus nuc gene, with melt-curve analysis, is run on a staphylococcal colony. From the derivative melt curves shown, the colony result is best reported as:
Figure: MedExams
1. Anuc variant; confirm it by sequencing
2. BAmplicon contamination; repeat the run
3. Cnuc not detected; the colony peak is primer-dimer
4. Dnuc detected; the colony is S. aureus
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Answer: C · nuc not detected; the colony peak is primer-dimer
SYBR Green binds any double-stranded DNA, so melt analysis is needed to show what was amplified. The positive control gives the specific nuc product, which melts at about 85.5 °C. The colony's only peak is at about 77 °C, the same low temperature as the small peak in the no-template control, which is typical of short primer-dimers. The target was not amplified, so the colony is not S. aureus (for example, a coagulase-negative staphylococcus).
- A: A sequence variant shifts the melting point by a degree or two and would not appear in the no-template control.
- B: Contaminating amplicon would give a peak at the target's melting point in the no-template control.
- D: A specific product would melt near the positive control's 85.5 °C, not at 77 °C.
Question 2 · Easy
Automated identification systems such as VITEK 2 identify an isolate by:
1. AReading colour or turbidity changes in biochemical wells
2. BDetecting CO2 produced in a culture bottle
3. CAmplifying species-specific DNA targets
4. DMatching the protein mass spectrum to a reference library
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Answer: A · Reading colour or turbidity changes in biochemical wells
A standardized suspension is loaded into a card or panel of miniaturized biochemical and substrate tests. An optical reader records colour, turbidity or fluorescence changes over several hours. The pattern becomes a biocode that is compared with a database and reported with a probability. Results depend on a pure culture and the correct inoculum density.
- B: CO2 detection is how continuous-monitoring blood culture systems flag growth.
- C: Amplifying DNA targets describes molecular panels.
- D: Matching protein spectra is how MALDI-TOF works.
Question 3 · Medium
A patient with culture-negative endocarditis has valve surgery. Broad-range 16S rRNA PCR with Sanger sequencing on the valve gives a trace with overlapping double peaks along its whole length. The most likely explanation:
1. AThe organism is not in the database
2. BThe valve contains a yeast
3. CPCR inhibitors were present
4. DMore than one bacterial species is present
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Answer: D · More than one bacterial species is present
Sanger sequencing reads one template cleanly. When two or more different 16S sequences are amplified together, their bases overlap and the trace shows mixed peaks that cannot be read. This happens with polymicrobial infection or contamination during collection or processing. Next-generation amplicon sequencing can separate mixed sequences.
- A: A missing database entry gives a clean sequence with no close match.
- B: A yeast lacks the bacterial 16S target and would give no product.
- C: Inhibitors cause a failed reaction, not a mixed trace.
Question 4 · Medium
A 70-year-old with pneumonia has an alpha-hemolytic blood isolate that MALDI-TOF reports as Streptococcus pneumoniae. Why do many laboratories still confirm it with bile solubility or optochin?
1. AIts spectrum is very close to that of S. mitis/oralis
2. BPneumococcal capsules block ionization
3. CMALDI-TOF cannot identify catalase-negative cocci
4. DThe matrix degrades pneumococcal proteins
Show answerHide answer
Answer: A · Its spectrum is very close to that of S. mitis/oralis
S. pneumoniae and the viridans species S. mitis and S. oralis have nearly identical ribosomal protein patterns. Some library versions call mitis group strains pneumococci, or the reverse. A quick bile solubility or optochin test separates them. The distinction matters because pneumococcal bacteremia and a viridans contaminant are managed very differently.
- B: The capsule does not stop MALDI-TOF from producing a spectrum.
- C: MALDI-TOF identifies most streptococci and enterococci well.
- D: The matrix helps ionize proteins; it does not destroy them.
Question 5 · Hard
A febrile patient's blood culture shows gram-positive cocci in clusters. A multiplex panel that reports mecA alone, with no S. aureus-specific junction target, detects S. aureus, S. epidermidis and mecA. What is the correct conclusion?
1. AThe S. aureus is methicillin-resistant
2. BThe S. aureus result is a false positive
3. CmecA cannot yet be assigned to either species
4. DThe S. epidermidis is methicillin-susceptible
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Answer: C · mecA cannot yet be assigned to either species
The panel detects genes in the whole bottle, not in single cells. mecA is common in S. epidermidis, so it may come from the coagulase-negative staphylococcus, the S. aureus, or both. S. aureus is reported with methicillin status pending, and cefoxitin testing on isolated colonies settles it. Panels that add a mecA-SCCmec junction (MREJ) target can link the resistance to S. aureus.
- A: The mecA may belong only to the S. epidermidis.
- B: A mixed culture does not make the S. aureus result false.
- D: The mecA may come from the S. epidermidis, so its status is open too.
Question 6 · Easy
In a MALDI-TOF instrument, the ions travelling down the flight tube are separated according to their:
1. AMass-to-charge ratio
2. BHydrophobicity
3. CElectrophoretic mobility
4. DLight absorbance
Answer and explanation in LearnFree account
Question 7 · Hard
A 6-year-old with bloody diarrhea has a stool isolate that is lactose negative, non-motile, lysine decarboxylase negative and makes no gas from glucose. MALDI-TOF reports Escherichia coli with a high score. What should the laboratory do next?
1. AReport E. coli, because the score is high
2. BReport Shigella without further testing
3. CRepeat MALDI-TOF after formic acid extraction
4. DExclude Shigella with biochemicals and antisera
Answer and explanation in LearnFree account
Question 8 · Easy
MALDI-TOF mass spectrometry identifies a bacterial colony mainly from the peak pattern of its:
1. ACapsular polysaccharides
2. BRibosomal proteins
3. C16S rRNA gene fragments
4. DCell wall fatty acids
Answer and explanation in LearnFree account
Question 9 · Medium
A multiplex PCR panel on a positive blood culture from a patient in septic shock detects Klebsiella pneumoniae group and none of its resistance gene targets. Why is phenotypic susceptibility testing still needed?
1. APanel results are not valid for gram-negative rods
2. BOther resistance mechanisms are not detected
3. CThe panel detects only nonviable organisms
4. DThe panel cannot identify Klebsiella to species
Answer and explanation in LearnFree account
Question 10 · Easy
Why is the 16S rRNA gene the usual target for sequence-based identification of bacteria?
1. AIt is carried on easily extracted plasmids
2. BIt codes for species-specific surface antigens
3. CIt is found only in pathogenic species
4. DIt has conserved and variable regions
Answer and explanation in LearnFree account
Question 11 · Medium
A yeast from a urine culture gives no reliable identification when the colony is smeared directly on the MALDI-TOF target. What is the best next step?
1. ARe-spot using a much thicker layer of the colony
2. BReport the closest match in the library
3. CSend the isolate for 16S rRNA sequencing
4. DRepeat with an on-target formic acid extraction
Answer and explanation in LearnFree account

Identification: automated, MALDI-TOF, multiplex molecular, 16S sequencing

A SYBR Green real-time PCR for the Staphylococcus aureus nuc gene, with melt-curve analysis, is run on a staphylococcal colony. From the derivative melt curves shown, the colony result is best reported as:

Automated identification systems such as VITEK 2 identify an isolate by:

A patient with culture-negative endocarditis has valve surgery. Broad-range 16S rRNA PCR with Sanger sequencing on the valve gives a trace with overlapping double peaks along its whole length. The most likely explanation:

A 70-year-old with pneumonia has an alpha-hemolytic blood isolate that MALDI-TOF reports as Streptococcus pneumoniae. Why do many laboratories still confirm it with bile solubility or optochin?

A febrile patient's blood culture shows gram-positive cocci in clusters. A multiplex panel that reports mecA alone, with no S. aureus-specific junction target, detects S. aureus, S. epidermidis and mecA. What is the correct conclusion?

In a MALDI-TOF instrument, the ions travelling down the flight tube are separated according to their:

A 6-year-old with bloody diarrhea has a stool isolate that is lactose negative, non-motile, lysine decarboxylase negative and makes no gas from glucose. MALDI-TOF reports Escherichia coli with a high score. What should the laboratory do next?

MALDI-TOF mass spectrometry identifies a bacterial colony mainly from the peak pattern of its:

A multiplex PCR panel on a positive blood culture from a patient in septic shock detects Klebsiella pneumoniae group and none of its resistance gene targets. Why is phenotypic susceptibility testing still needed?

Why is the 16S rRNA gene the usual target for sequence-based identification of bacteria?

A yeast from a urine culture gives no reliable identification when the colony is smeared directly on the MALDI-TOF target. What is the best next step?