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Setting up susceptibility tests

16 min

  • Recognize agar depth, cation, or thymidine errors in an AST setup
  • Distinguish broth dilution from agar and gradient-diffusion AST formats
  • Read the inhibition-zone diameter for a valid disk-diffusion test
  • Check that AST control strains are in range before releasing patient results

Read the full reference

Try first

Try first

Mueller-Hinton agar plates from a hand-poured batch measure about 6 mm deep. The method calls for 4 mm. How will zones read on these plates?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

The medium and the inoculum

Mueller-Hinton medium is the standard for routine susceptibility testing. Its lots perform consistently, and it holds little thymidine and few excess cations.1,2 Each part of the setup moves the endpoint when it drifts:

  • Depth. Agar is poured 4 mm deep. Deeper agar makes an isolate look more resistant, and shallower agar makes it look more susceptible.
  • pH. The final pH is 7.2 to 7.4.
  • Cations. Excess calcium and magnesium make Pseudomonas aeruginosa falsely resistant to aminoglycosides. Broth dilution uses cation-adjusted Mueller-Hinton broth.
  • Thymidine. Thymidine and thymine antagonize trimethoprim and sulfonamides.1,2

The inoculum starts as a 0.5 McFarland suspension of fresh colonies, about 1 to 2 × 108 CFU/mL. Broth microdilution dilutes it to a final 5 × 105 CFU/mL in the well. Plates incubate at 35 °C ± 2 °C. Disk diffusion is read at 16 to 18 hours and dilution at 16 to 20 hours. Staphylococcal oxacillin and vancomycin MICs and enterococcal vancomycin tests take a full 24 hours.3

Four formats

FormatEndpointWhere it fits
Broth microdilutionMICThe reference method for most nonfastidious organisms and the basis of commercial panels
Agar dilutionMICMany isolates against one drug on one plate; the reference method for anaerobes and gonococci
Gradient diffusionMIC where the ellipse meets the strip's scaleOnly cleared organism-drug combinations; never colistin or polymyxin B
Disk diffusionZone diameterRapidly growing aerobes and the fastidious organisms with published disk criteria

A gradient reading between two doubling dilutions is rounded up to the next dilution before it is compared with a breakpoint.3 Disk-diffusion result = inhibition-zone diameter in millimeters, read by the specified method and interpreted with the matching organism-drug zone criteria. The diameter is a separate measurement from an MIC. A 150-mm plate holds no more than 12 disks and a 100-mm plate no more than 5, at least 24 mm apart center to center.1

Controls come first

The Clinical Laboratory Improvement Amendments (CLIA) require a check of each batch of medium and each lot or shipment of drug before or with its first use. Control strains run each day of testing, and every control result is in range before patient results are reported.4 Testing less often than daily needs a written individualized quality control plan.5 When a control falls out of range:

  1. Stop reporting the affected drug and method.
  2. Find the cause.
  3. Review patient results reported since the last in-range control.
  4. Document the corrective action before reporting resumes.3,4
References
  1. Clinical and Laboratory Standards Institute. M02, Disk Diffusion 14th ed. and M07, Dilution 12th ed. CLSI; 2024.
  2. Centers for Disease Control and Prevention. Laboratory assessment of antibiotic resistance testing capacity: Mueller-Hinton medium specifications. Accessed September 27, 2026. https://stacks.cdc.gov/view/cdc/103871
  3. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 36th ed. CLSI supplement M100. CLSI; 2026. Accessed September 27, 2026. https://clsi.org/shop/standards/m100/
  4. Code of Federal Regulations. 42 CFR §493.1261: routine susceptibility testing controls. Accessed September 27, 2026. https://www.law.cornell.edu/cfr/text/42/493.1261
  5. Centers for Medicare and Medicaid Services. FAQs for Individualized Quality Control Plan (IQCP). Accessed September 27, 2026. https://www.cms.gov/regulations-and-guidance/legislation/clia/downloads/faqs-iqcp.pdf

Watch one

Tuesday's disk diffusion controls are read at 07:30. The plates come from a Mueller-Hinton agar lot opened this morning.

  • Escherichia coli ATCC 25922: every drug in range.
  • P. aeruginosa ATCC 27853: gentamicin zone 13 mm. The laboratory's posted range for this strain and disk is 17 to 23 mm. Every other drug is in range.
  • Three patient P. aeruginosa isolates on the same lot read gentamicin resistant.

What happens to the patient gentamicin results?

  1. Compare every control with its posted range. Only gentamicin on P. aeruginosa ATCC 27853 is out, and it is low.

    Each control covers one strain, drug and method, so the first job is to find exactly which one failed.

  2. Match the failure to patient work. The three P. aeruginosa gentamicin zones came from the same disks and the same agar lot.

    The patient results that share the failed drug, method and medium are the ones the failure could be misreading.

  3. Look at the pattern. A new lot with a low aminoglycoside zone on P. aeruginosa points first at the medium's cations.

    A small aminoglycoside zone on P. aeruginosa is the classic sign of excess calcium and magnesium in the medium.

  4. Report no patient gentamicin result from this lot. Drugs whose controls are in range follow the laboratory's procedure for release.

    An out-of-range control means the method may be calling susceptible isolates resistant.

  5. Review gentamicin results reported since the last in-range control.

    Earlier reports may carry the same error if the problem began before today.

  6. Retest the controls and the three isolates on another lot, document the corrective action, then report.

    Patient results return only after the fix is proven on the control strain.

The three gentamicin results stay unreported. The controls and isolates are retested on another agar lot, and the corrective action is documented before any gentamicin result is released.

Your turn

Problem 1 of 3

A physician requests a colistin MIC on an Acinetobacter baumannii isolate. Which format gives a valid result?

Gradient strips do not reproduce colistin's activity reliably. They are not valid for either polymyxin.

Colistin is tested by MIC methods only, and broth microdilution is the reference method for it.

Disk diffusion gives no MIC, and it is not a valid method for either polymyxin.

Hint
  1. Colistin's category depends on a measured MIC, so a zone alone will not do.
  2. Ask which MIC formats reproduce the polymyxins' activity reliably.

Review Dilution and gradient-diffusion methods

Problem 2 of 3

Put the disk diffusion setup in order.

  1. Suspend fresh colonies to a 0.5 McFarland standard
  2. Measure each zone diameter in millimeters
  3. Swab Mueller-Hinton agar for a confluent lawn
  4. Place the disks at least 24 mm apart
  5. Incubate at 35 °C for 16 to 18 hours
Hint
  1. The drug can only act on a standardized lawn.
  2. The measurement comes after the timed incubation.
Show the answer

Suspend fresh colonies to a 0.5 McFarland standard, Swab Mueller-Hinton agar for a confluent lawn, Place the disks at least 24 mm apart, Incubate at 35 °C for 16 to 18 hours, Measure each zone diameter in millimeters

The standardized suspension makes a lawn of known density, the disks go onto that lawn with room for each zone, and the zones are measured only after the timed incubation. A lawn from an unstandardized suspension changes every zone on the plate.

Review Disk diffusion

Problem 3 of 3

A new staff member made broth microdilution trays with plain Mueller-Hinton broth from the media room. The broth was not cation adjusted. Which results are most at risk?

The medium's cation content changes how some drugs act. Aminoglycoside and tetracycline MICs shift with calcium and magnesium.

Read an endpoint from a plate with the wrong depth or medium

Agar deeper than 4 mm makes an isolate look more resistant, excess calcium and magnesium make P. aeruginosa falsely resistant to aminoglycosides, and thymidine antagonizes trimethoprim and sulfonamides. An endpoint read from such a setup can place the isolate in the wrong category.

Thymidine interferes with folate drugs. Cations are the problem in this broth.

Vancomycin MICs for staphylococci depend on a full 24-hour read. The cations in this broth act on aminoglycosides and tetracyclines.

Aminoglycoside activity against P. aeruginosa changes with calcium and magnesium. Unadjusted broth moves those MICs, so the trays are remade with cation-adjusted broth.

Review Principles, media, inoculum, and incubation

Use it

  • An Enterococcus faecalis from a urine culture is set up by disk diffusion on a 100-mm Mueller-Hinton plate.
  • The plate carries 7 disks, including vancomycin.
  • The vancomycin disks come from a shipment opened today. No control has been tested on them.
  • At 18 hours a technologist reads a sharp vancomycin zone edge under the plate reader's reflected light and asks to release it.
Decision 1 of 3

How is the vancomycin zone read?

An enterococcal vancomycin zone read early and by reflected light can miss the haze of resistant growth inside it.

Ignored haze inside a vancomycin zone

Enterococcal vancomycin zones are read at a full 24 hours with transmitted light. A faint haze inside the zone, which direct light does not show, signals resistance. Reading only the clear edge reports a resistant isolate as susceptible.

Enterococcal vancomycin zones are read at 24 hours. Transmitted light shows a faint haze that signals resistance.

The 16-to-18-hour read suits most rapidly growing organisms. Enterococcal vancomycin needs the full 24 hours.

Review Disk diffusion

Decision 2 of 3

What about the plate itself?

Disks too close together let zones run into each other, and the diameters can no longer be measured.

The drug has already diffused from those disks. Lifting them leaves their drug in the agar.

A 100-mm plate holds no more than 5 disks. With 7, zones can overlap, so the test is set up again with the disks spread correctly.

Review Disk diffusion

Decision 3 of 3

What must happen before any result from the new vancomycin disks is reported?

Each new lot or shipment is checked with control strains before or with its first use. Patient results wait for in-range controls.

Yesterday's controls tested the old shipment. The new disks have no control result yet.

Released affected AST results despite unacceptable controls

Control strains must be in range before patient results are reported. An out-of-range control means the affected drug, medium, or method may be misreading patient isolates too, so results since the last in-range control are reviewed before any release.

Controls come before patient reporting. A weekly schedule also needs a written quality control plan.

Released affected AST results despite unacceptable controls

Control strains must be in range before patient results are reported. An out-of-range control means the affected drug, medium, or method may be misreading patient isolates too, so results since the last in-range control are reviewed before any release.

Review Quality control and verification

The clue that settles this case is the 18-hour read. An enterococcal vancomycin zone means nothing until the full 24 hours, read with transmitted light. The repeat plate, set up with 5 disks and checked with controls on the new shipment, gives a zone that can be reported.

Keep

Sources checked