Daily disk diffusion QC with Staphylococcus aureus ATCC 25923 on Mueller-Hinton agar gives these zones, with the acceptable ranges in brackets: gentamicin 16 mm (19-27); erythromycin 19 mm (22-30); tetracycline 33 mm (24-30); cefoxitin 25 mm (23-29); trimethoprim-sulfamethoxazole 27 mm (24-32). The inoculum was a fresh 0.5 McFarland suspension, and the agar depth was 4 mm. A new staff member had incubated the plates for 18 hours at 35 °C in the 5% CO2 incubator used for chocolate agar. What is the most likely cause of the out-of-range results?

Excess calcium and magnesium in the agar

A 26-year-old woman with uncomplicated cystitis is to be treated with oral cephalexin. Her urine grows more than 10^5 CFU/mL of Escherichia coli, and the laboratory tests cefazolin, which CLSI allows as a surrogate for oral cephalosporins. The cefazolin MIC is 8 µg/mL. The CLSI cefazolin breakpoints for Enterobacterales (µg/mL) are: systemic infections, S ≤2, I 4, R ≥8; uncomplicated urinary tract infection, as a surrogate for oral agents such as cephalexin, S ≤16, R ≥32. For this patient, the cefazolin result should be reported as:

Susceptible, by the urinary surrogate breakpoint

Not reportable without a cephalexin MIC

Resistant, by the systemic breakpoint

Intermediate, one dilution from susceptible

The disk diffusion test shown was set up on a 90-mm Mueller-Hinton plate for an Escherichia coli isolate. Why can its zones not be measured reliably?

Too many disks were placed on one plate

The inoculum was heavier than 0.5 McFarland

The plate was incubated in 5% CO2

The disks had lost potency in storage

Susceptibility testing: methods, theory and interpretation

21 ASCP MLS practice questions on Susceptibility testing: methods, theory and interpretation, part of Microbiology. The first 21 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
Daily disk diffusion QC with Staphylococcus aureus ATCC 25923 on Mueller-Hinton agar gives these zones, with the acceptable ranges in brackets: gentamicin 16 mm (19-27); erythromycin 19 mm (22-30); tetracycline 33 mm (24-30); cefoxitin 25 mm (23-29); trimethoprim-sulfamethoxazole 27 mm (24-32). The inoculum was a fresh 0.5 McFarland suspension, and the agar depth was 4 mm. A new staff member had incubated the plates for 18 hours at 35 °C in the 5% CO2 incubator used for chocolate agar. What is the most likely cause of the out-of-range results?
1. ALow agar surface pH from CO2
2. BLoss of potency of the disks
3. CExcess thymidine in the agar
4. DExcess calcium and magnesium in the agar
Show answerHide answer
Answer: A · Low agar surface pH from CO2
Non-fastidious organisms on Mueller-Hinton agar must be incubated in ambient air, because CO2 dissolves in the agar and lowers its pH. Aminoglycosides and macrolides lose activity at acid pH, so the gentamicin and erythromycin zones shrink, while tetracycline becomes more active and its zone grows. A pattern of some zones too small and one too large points to pH rather than to a general problem such as inoculum or disk potency. Excess divalent cations would shrink the tetracycline zone too, and thymidine would affect trimethoprim-sulfamethoxazole, which is in range. Patient results from this run are held until QC repeated in ambient air is in range.
- B: Weak disks give small zones and cannot explain an oversized tetracycline zone.
- C: Excess thymidine affects trimethoprim-sulfamethoxazole, which is in range here.
- D: Excess cations shrink aminoglycoside and tetracycline zones; they do not enlarge the tetracycline zone.
Question 2 · Hard
A 26-year-old woman with uncomplicated cystitis is to be treated with oral cephalexin. Her urine grows more than 10^5 CFU/mL of Escherichia coli, and the laboratory tests cefazolin, which CLSI allows as a surrogate for oral cephalosporins. The cefazolin MIC is 8 µg/mL. The CLSI cefazolin breakpoints for Enterobacterales (µg/mL) are: systemic infections, S ≤2, I 4, R ≥8; uncomplicated urinary tract infection, as a surrogate for oral agents such as cephalexin, S ≤16, R ≥32. For this patient, the cefazolin result should be reported as:
1. ANot reportable without a cephalexin MIC
2. BResistant, by the systemic breakpoint
3. CIntermediate, one dilution from susceptible
4. DSusceptible, by the urinary surrogate breakpoint
Show answerHide answer
Answer: D · Susceptible, by the urinary surrogate breakpoint
CLSI gives cefazolin two sets of Enterobacterales breakpoints. The systemic breakpoints predict the activity of intravenous cefazolin in infections such as bacteremia, where an MIC of 8 µg/mL is resistant. For uncomplicated UTI caused by E. coli, Klebsiella pneumoniae or Proteus mirabilis, cefazolin is also a surrogate for oral cephalosporins, which reach high levels in urine, and an MIC of 16 µg/mL or less predicts susceptibility. This patient has uncomplicated cystitis and will take cephalexin, so the surrogate breakpoint applies and the result is susceptible. Laboratories label the surrogate result clearly so that it is not applied to a systemic infection.
- A: Cefazolin is tested so that a separate cephalexin MIC is not needed.
- B: The systemic breakpoint applies to intravenous cefazolin, not to oral therapy for cystitis.
- C: The surrogate breakpoints have no intermediate category, and 8 µg/mL is susceptible.
Question 3 · Hard
An 18-month-old with meningitis has Streptococcus pneumoniae in the CSF. The penicillin gradient strip, on Mueller-Hinton agar with 5% sheep blood in CO2, is shown. CLSI penicillin breakpoints (µg/mL): meningitis S ≤0.06, R ≥0.12; non-meningitis S ≤2, I 4, R ≥8. How should penicillin be reported?
Figure: MedExams
1. ASusceptible; MIC 0.06 µg/mL
2. BResistant; MIC 0.12 µg/mL
3. CIntermediate; MIC 0.094 µg/mL
4. DSusceptible; MIC 0.12 µg/mL
Show answerHide answer
Answer: B · Resistant; MIC 0.12 µg/mL
The ellipse meets the strip at the 0.094 µg/mL mark. A gradient strip reading that falls between two-fold dilutions is rounded up to the next two-fold value before it is interpreted, so the MIC is 0.12 µg/mL. For a CSF isolate the meningitis breakpoints apply, and 0.12 µg/mL is resistant, because penicillin does not reach a high enough level in the CSF. The non-meningitis breakpoints would call the same isolate susceptible, which is why the specimen source must be known before reporting.
- A: Strip readings are rounded up, never down, to the next two-fold value.
- C: 0.094 µg/mL must be rounded up to 0.12, and the meningitis breakpoints have no intermediate category.
- D: Non-meningitis breakpoints do not apply to an isolate from CSF.
Question 4 · Easy
The gradient diffusion strip shown tests vancomycin against a Staphylococcus aureus isolate from blood. Which MIC should be recorded?
Figure: MedExams
1. A1 µg/mL
2. B4 µg/mL
3. C0.5 µg/mL
4. D2 µg/mL
Show answerHide answer
Answer: A · 1 µg/mL
A gradient strip releases a continuous gradient of drug into the agar, and the MIC is read where the edge of the ellipse of inhibition meets the strip. Here the ellipse meets the strip at the 1 µg/mL mark. Concentrations above that point inhibit growth and those below it do not. For vancomycin, read at complete inhibition of all growth, including haze and microcolonies, so that heteroresistant subpopulations are not missed.
- B: 4 µg/mL lies well inside the ellipse, far above the intersection.
- C: 0.5 µg/mL lies below the intersection, where growth reaches the strip.
- D: 2 µg/mL lies above the intersection, inside the ellipse.
Question 5 · Medium
The disk diffusion test shown was set up on a 90-mm Mueller-Hinton plate for an Escherichia coli isolate. Why can its zones not be measured reliably?
Figure: MedExams
1. AThe inoculum was heavier than 0.5 McFarland
2. BThe plate was incubated in 5% CO2
3. CToo many disks were placed on one plate
4. DThe disks had lost potency in storage
Show answerHide answer
Answer: C · Too many disks were placed on one plate
The plate holds nine disks, and neighbouring zones run into each other, so most zone edges cannot be measured. CLSI allows no more than 5 disks on a 100-mm plate (12 on a 150-mm plate) and wants disk centres at least 24 mm apart. Where zones overlap, the drugs can also interact at the borders. Repeat the test with fewer disks per plate or on a 150-mm plate.
- A: A heavy inoculum makes zones smaller; it does not make them merge.
- B: CO2 lowers the agar pH and alters some zones, but it cannot make zones overlap.
- D: Lost potency gives smaller zones, not crowded, merging ones.
Question 6 · Medium
The Mueller-Hinton plate shown was inoculated from a suspension that was not checked against a McFarland standard. Compared with a correct test, its zones are most likely:
Figure: MedExams
1. AToo large, from an inoculum that was too light
2. BToo small, from an inoculum that was too heavy
3. CToo small, from agar that was poured too deep
4. DUnchanged, as inoculum density does not matter
Answer and explanation in LearnFree account
Question 7 · Medium
The trimethoprim-sulfamethoxazole wells of a broth microdilution panel for a urine Escherichia coli are shown. The MIC to report for the patient isolate is:
Figure: MedExams
1. A0.5/9.5 µg/mL
2. B8/152 µg/mL
3. C1/19 µg/mL
4. D16/304 µg/mL
Answer and explanation in LearnFree account
Question 8 · Hard
In daily disk diffusion QC, Pseudomonas aeruginosa ATCC 27853 gives gentamicin and tobramycin zones below the acceptable range, while all other drugs are in range. The most likely cause is:
1. AInoculum lighter than the 0.5 McFarland standard
2. BExcess thymidine in the agar
3. CExcess calcium and magnesium in the agar
4. DAgar poured thinner than 4 mm
Answer and explanation in LearnFree account
Question 9 · Hard
For broth dilution, a 0.5 McFarland suspension (1.5 x 10^8 CFU/mL) is diluted 1:150 in broth, and then 0.5 mL of this is added to 0.5 mL of broth containing antibiotic. The final inoculum in the tube is:
1. A1 x 10^6 CFU/mL
2. B7.5 x 10^5 CFU/mL
3. C2.5 x 10^5 CFU/mL
4. D5 x 10^5 CFU/mL
Answer and explanation in LearnFree account
Question 10 · Medium
CLSI gives Streptococcus pneumoniae separate penicillin breakpoints for meningitis and non-meningitis isolates. Why?
1. AMeningitis strains usually carry different capsule types
2. BCSF isolates grow too slowly for standard tests
3. CPenicillin reaches lower levels in CSF than in blood
4. DCSF strains often produce beta-lactamase
Answer and explanation in LearnFree account
Question 11 · Hard
A 2-year-old with meningitis has Streptococcus pneumoniae in the CSF. The oxacillin 1-µg disk zone is 14 mm (20 mm or more predicts penicillin susceptibility). What should the laboratory do?
1. AReport penicillin resistant
2. BReport penicillin susceptible
3. CRepeat oxacillin on plain Mueller-Hinton agar
4. DDetermine the penicillin MIC
Answer and explanation in LearnFree account
Question 12 · Hard
A diabetic foot wound grows Pseudomonas aeruginosa. MICs are shown with the CLSI breakpoints (µg/mL): cefepime 16 (S ≤8, R ≥32); meropenem 2 (S ≤2, R ≥8); ciprofloxacin 1 (S ≤0.5, R ≥2); tobramycin 4 (S ≤1, R ≥4). Which agent is reported susceptible?
1. AMeropenem
2. BCefepime
3. CTobramycin
4. DCiprofloxacin
Answer and explanation in LearnFree account
Question 13 · Medium
Mueller-Hinton plates for disk diffusion were poured to a depth of 2 mm instead of 4 mm. The zones of inhibition will be:
1. AToo small, giving false resistance
2. BUnchanged if the inoculum is correct
3. CToo large, giving false susceptibility
4. DSmaller for beta-lactams only
Answer and explanation in LearnFree account
Question 14 · Hard
A patient with MRSA bacteremia is not improving on vancomycin. Disk diffusion gives a vancomycin zone of 17 mm. What is the best next step?
1. AReport susceptible because the zone is 15 mm or more
2. BRepeat disk diffusion with a heavier inoculum
3. CDetermine the vancomycin MIC
4. DReport resistant because therapy has failed
Answer and explanation in LearnFree account
Question 15 · Hard
A broth microdilution row for one drug reads (µg/mL): 0.5 growth, 1 growth, 2 no growth, 4 growth, 8 no growth, 16 no growth. The growth control grew and the sterility control is clear. What MIC should be recorded?
1. A16 µg/mL
2. B8 µg/mL
3. C2 µg/mL
4. D4 µg/mL
Answer and explanation in LearnFree account
Question 16 · Easy
A bacterial suspension matched to a 0.5 McFarland standard contains about how many organisms?
1. A1.5 x 10^8 CFU/mL
2. B1.5 x 10^6 CFU/mL
3. C3 x 10^8 CFU/mL
4. D5 x 10^5 CFU/mL
Answer and explanation in LearnFree account
Question 17 · Easy
In a broth microdilution panel, the MIC is read as the:
1. ALowest concentration that kills 99.9% of cells
2. BHighest concentration that still shows growth
3. CLowest concentration with no visible growth
4. DMidpoint between the last growth and first clear well
Answer and explanation in LearnFree account
Question 18 · Medium
On a trimethoprim-sulfamethoxazole disk diffusion plate, a faint haze of growth is seen inside the zone. How should the zone be measured?
1. AReport resistant because growth is in the zone
2. BRepeat the test with a lighter inoculum
3. CMeasure to the edge of the faint haze
4. DIgnore the haze and read the obvious margin
Answer and explanation in LearnFree account
Question 19 · Medium
Excess thymidine in a lot of Mueller-Hinton medium mainly gives falsely resistant results for:
1. APiperacillin-tazobactam
2. BGentamicin and tobramycin
3. CTrimethoprim-sulfamethoxazole
4. DTetracycline and minocycline
Answer and explanation in LearnFree account
Question 20 · Medium
An Enterobacterales isolate is reported 'SDD' (susceptible-dose dependent) to cefepime. This means that:
1. AThe drug works only where it is physiologically concentrated
2. BAn ESBL is present, so cefepime must not be used
3. CHigher or more frequent dosing is needed for success
4. DThe MIC is in a technical buffer zone and needs repeating
Answer and explanation in LearnFree account
Question 21 · Medium
In Kirby-Bauer disk diffusion testing, which conditions must be standardized?
1. AAnaerobic incubation for all organisms
2. B0.5 McFarland inoculum, Mueller-Hinton agar of set depth
3. CSheep blood agar plates for all organisms
4. DReading the zones after 6 hours of incubation
Answer and explanation in LearnFree account

Susceptibility testing: methods, theory and interpretation

The gradient diffusion strip shown tests vancomycin against a Staphylococcus aureus isolate from blood. Which MIC should be recorded?

The disk diffusion test shown was set up on a 90-mm Mueller-Hinton plate for an Escherichia coli isolate. Why can its zones not be measured reliably?

The Mueller-Hinton plate shown was inoculated from a suspension that was not checked against a McFarland standard. Compared with a correct test, its zones are most likely:

The trimethoprim-sulfamethoxazole wells of a broth microdilution panel for a urine Escherichia coli are shown. The MIC to report for the patient isolate is:

In daily disk diffusion QC, Pseudomonas aeruginosa ATCC 27853 gives gentamicin and tobramycin zones below the acceptable range, while all other drugs are in range. The most likely cause is:

For broth dilution, a 0.5 McFarland suspension (1.5 x 10^8 CFU/mL) is diluted 1:150 in broth, and then 0.5 mL of this is added to 0.5 mL of broth containing antibiotic. The final inoculum in the tube is:

CLSI gives Streptococcus pneumoniae separate penicillin breakpoints for meningitis and non-meningitis isolates. Why?

A 2-year-old with meningitis has Streptococcus pneumoniae in the CSF. The oxacillin 1-µg disk zone is 14 mm (20 mm or more predicts penicillin susceptibility). What should the laboratory do?

A diabetic foot wound grows Pseudomonas aeruginosa. MICs are shown with the CLSI breakpoints (µg/mL): cefepime 16 (S ≤8, R ≥32); meropenem 2 (S ≤2, R ≥8); ciprofloxacin 1 (S ≤0.5, R ≥2); tobramycin 4 (S ≤1, R ≥4). Which agent is reported susceptible?

Mueller-Hinton plates for disk diffusion were poured to a depth of 2 mm instead of 4 mm. The zones of inhibition will be:

A patient with MRSA bacteremia is not improving on vancomycin. Disk diffusion gives a vancomycin zone of 17 mm. What is the best next step?

A broth microdilution row for one drug reads (µg/mL): 0.5 growth, 1 growth, 2 no growth, 4 growth, 8 no growth, 16 no growth. The growth control grew and the sterility control is clear. What MIC should be recorded?

A bacterial suspension matched to a 0.5 McFarland standard contains about how many organisms?

In a broth microdilution panel, the MIC is read as the:

On a trimethoprim-sulfamethoxazole disk diffusion plate, a faint haze of growth is seen inside the zone. How should the zone be measured?

Excess thymidine in a lot of Mueller-Hinton medium mainly gives falsely resistant results for:

An Enterobacterales isolate is reported 'SDD' (susceptible-dose dependent) to cefepime. This means that:

In Kirby-Bauer disk diffusion testing, which conditions must be standardized?