Antimicrobial Susceptibility Testing (AST): Basics Explained

Antimicrobial susceptibility testing (AST) measures how a bacterial isolate responds to antibiotics in the laboratory, so clinicians can choose a treatment likely to work. Results are reported as susceptible, intermediate or susceptible at increased exposure, or resistant, based on zone diameters or minimum inhibitory concentrations (MICs) compared against published breakpoints.

AST is one of the most consequential reports a microbiology laboratory issues. An error in inoculum, medium or breakpoint table can push a patient onto an antibiotic that fails, or onto a broader agent than needed, which adds to the pressure driving antimicrobial resistance.

Because AST is so standardised, most errors come from small departures from the method. This guide covers the main methods, how interpretation works and where laboratories usually go wrong.

The main AST methods

Disc diffusion (Kirby-Bauer)

A standardised suspension of the isolate is swabbed evenly across a Mueller-Hinton agar plate, and paper discs containing fixed amounts of antibiotic are placed on the surface. The antibiotic diffuses outwards, and after incubation the diameter of the zone of inhibition is measured in millimetres and interpreted against breakpoints.

Disc diffusion is flexible and inexpensive, which is why it remains widely used in India and the GCC. It gives a category, not an MIC.

Broth microdilution MIC

Broth microdilution tests the isolate against twofold dilutions of each antibiotic in liquid medium, usually in microtitre plates. The MIC is the lowest concentration that prevents visible growth. It is the international reference method, described in ISO 20776-1, and is the method other approaches are compared with.

Gradient strips

A plastic strip carrying a continuous antibiotic gradient is placed on an inoculated plate. After incubation, the MIC is read where the elliptical zone of inhibition meets the strip scale. Strips are useful for single agents or fastidious organisms, but cost more per test.

Automated systems

Commercial automated systems inoculate cards or panels and read growth optically, often combined with identification. They improve throughput and standardisation, but each has validated organism and drug limits, and some results need confirmation by a reference method.

Breakpoints and the S/I/R categories

Breakpoints are the zone diameters or MICs that separate susceptible from resistant isolates. The two main sources are CLSI, through its M100 performance standards, and EUCAST, through its breakpoint tables. Both are updated regularly, so laboratories must record which version they use and review changes at each update.

Many laboratories in India use CLSI, while EUCAST is used widely in Europe and by an increasing number of laboratories elsewhere. Follow your national guidance and accreditation requirements, and do not mix breakpoint systems for the same report.

• S (susceptible): a high likelihood of therapeutic success at a standard dosing regimen.
• I: in CLSI, intermediate. In EUCAST, since 2019, susceptible, increased exposure, meaning the agent is likely to work if exposure is increased, by dose or by concentration at the site of infection.
• R (resistant): a high likelihood of therapeutic failure even with increased exposure.

CLSI also uses susceptible-dose dependent (SDD) for some combinations, and EUCAST defines an area of technical uncertainty (ATU) where results need extra care. Read the guidance notes in your chosen tables, not only the numbers.

Getting the inoculum and medium right

Standardisation is the core of AST. Prepare a suspension from several well-isolated colonies of a pure overnight culture and adjust it to a 0.5 McFarland standard, using a densitometer or a visual comparison against a standard.

Use Mueller-Hinton agar poured to the depth the method specifies, commonly 4 mm, and supplemented as required for fastidious organisms. EUCAST describes a 15-15-15 rule for disc diffusion: use the suspension within 15 minutes, apply discs within 15 minutes of inoculation and incubate within 15 minutes of applying discs.

Store discs as the manufacturer directs and let containers reach room temperature before opening, so condensation does not degrade the antibiotic. Incubation conditions and times are set by the method for each organism group.

Quality control strains

Test reference strains with known expected ranges alongside patient isolates. Common examples include Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 for disc diffusion, S. aureus ATCC 29213 for MIC methods and Pseudomonas aeruginosa ATCC 27853.

The QC frequency, strain selection and acceptable ranges come from your chosen standard. When a QC result is out of range, investigate before reporting patient results tested in the same run. Results should be monitored through your internal quality control system.

Worked example: a urinary E. coli at a Kochi lab

A diagnostic laboratory in Kochi receives a midstream urine from a woman with symptoms of cystitis. Culture yields significant growth of a single lactose-fermenting Gram-negative bacillus, identified as E. coli. The laboratory uses disc diffusion interpreted with the CLSI M100 edition listed in its procedure.

The technologist checks a purity plate and confirms that the day's QC with E. coli ATCC 25922 is within range. Zones are measured with callipers against a dark background, and the results show resistance to ceftriaxone and ciprofloxacin, with susceptibility to nitrofurantoin and fosfomycin.

Reduced zones for third-generation cephalosporins trigger the laboratory's ESBL screening procedure, and a phenotypic confirmation test with clavulanate supports ESBL production. The microbiologist applies the laboratory's selective reporting rules, highlighting the oral agents appropriate for uncomplicated cystitis and adding a comment about ESBL production for infection control.

Before release, the report is checked for unusual phenotypes and for consistency with expected resistance patterns. The isolate is stored in case the clinician requests further testing.

Common mistakes

• Using a suspension that is too heavy, which shrinks zones and over-calls resistance, or too light, which does the opposite.
• Pouring Mueller-Hinton agar at the wrong depth.
• Testing mixed cultures without a purity plate.
• Opening cold disc containers straight from the fridge.
• Applying an outdated breakpoint table or mixing CLSI and EUCAST.
• Reporting patient results after a QC strain falls out of range.
• Reporting every agent tested, rather than following selective reporting rules.

Key takeaway: Accurate AST depends on a pure 0.5 McFarland inoculum, correctly prepared Mueller-Hinton agar, in-range QC strains and a clearly identified, current breakpoint table. Treat each of these as a controlled step, not a habit.

Frequently Asked Questions

What does MIC mean in microbiology?

MIC stands for minimum inhibitory concentration, the lowest concentration of an antibiotic that prevents visible growth of a bacterial isolate under standard test conditions. It is measured by broth microdilution, agar dilution, gradient strips or automated systems, then compared with clinical breakpoints to categorise the isolate as susceptible, susceptible at increased exposure or intermediate, or resistant.

What is the difference between CLSI and EUCAST?

CLSI and EUCAST are two organisations that publish breakpoints and AST methods. They use different breakpoints for some organism-drug combinations, define the I category differently and recommend some different procedures. Laboratories should choose one system, follow the current version consistently and confirm which system their national guidance or accreditation body expects.

What does 'susceptible, increased exposure' mean?

Since 2019, EUCAST defines I as susceptible, increased exposure. It means the antibiotic is likely to work if exposure at the site of infection is increased, for example through a higher dose or because the drug concentrates there. It is not a borderline or uncertain result, and clinicians may need guidance to interpret it correctly.

Why is a 0.5 McFarland standard used?

The 0.5 McFarland standard gives a reproducible bacterial density, so results can be compared with published breakpoints. If the inoculum is too heavy, zones shrink and resistance may be over-called. If it is too light, zones grow and resistance may be missed. Check turbidity with a densitometer or against a standard under good lighting.

Next steps

To strengthen quality and competence across your microbiology service, explore our ISO 15189 Advanced: Medical Laboratory Quality and Competence course. For the infection control side of resistant organisms, see Infection Prevention and Control Essentials.

For related guides, read antimicrobial resistance and One Health, Gram staining procedure and interpretation and internal quality control in medical labs. Unsure where to start? Speak to a counsellor.