Overview
Once a bacterium causing an infection has been identified, the clinical laboratory must determine which antibiotics will be effective against it. This process is called antibiotic susceptibility testing (AST), and it directly guides the treating physician's choice of therapy.
This lesson covers the key terminology (selectivity, therapeutic index, bactericidal vs bacteriostatic), and the major methods used — disk diffusion, breakpoint testing, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), resistance mechanism detection, and automated systems.
Learning Objectives
After this lesson you will be able to…- Describe various terminologies related to antibiotic susceptibility testing.
- Describe the principle for antibiotic susceptibility testing.
- Describe the procedure for performing antibiotic susceptibility testing.
- Describe different methods used for antibiotic susceptibility testing.
- Differentiate between MIC and MBC and explain their clinical relevance.
Clinical Story
Why This MattersA blood culture from a critically ill ICU patient grows a Gram negative rod. The physician needs to know within hours which antibiotics will work — a disk diffusion sensitivity test set up alongside the identification will determine whether the patient survives on empirical therapy or needs an urgent change to a more targeted, potentially life-saving antibiotic.
Core Concepts
Selectivity — clinically effective antimicrobial agents show selective toxicity toward bacteria rather than the host; this distinguishes antibiotics from disinfectants.
Therapeutic Index — the ratio of the toxic dose (to host) to the effective therapeutic dose. A higher therapeutic index means a safer, better antibiotic.
Bactericidal vs Bacteriostatic — bactericidal antibiotics kill susceptible bacteria; bacteriostatic antibiotics reversibly inhibit bacterial growth, relying on host defenses to eradicate the organism. Bactericidal drugs are preferred for infections of the endocardium or meninges where host defenses are weak.
A filter paper disk pre-impregnated with a defined amount of antibiotic is placed on an agar plate inoculated with the test bacterium. As the plate incubates (18–24 h), the antibiotic diffuses out along a concentration gradient. If the bacterium is sensitive, a clear zone of inhibition forms around the disk. Zone diameter depends on disk potency, degree of susceptibility, physicochemical properties of the drug, agar depth, and inoculum density.
Two approaches exist: the comparative disk test (Stokes' method), which compares zones between a test organism and a control organism of known sensitivity on the same plate, and standardized disk testing, which uses carefully standardized inocula and reports results as sensitive or resistant based on defined cut-off points (e.g. <18 mm = resistant).
A defined "breakpoint" concentration of antibiotic is incorporated uniformly into the agar. Using multipoint inoculators, many bacterial strains (>30) can be tested simultaneously against a range of antibiotics. Bacteria that grow at the breakpoint concentration are resistant; those that don't grow are sensitive. A control plate without antibiotic confirms viability.
Minimum Inhibitory Concentration (MIC) — the lowest antibiotic concentration that inhibits visible bacterial growth, determined by macro/micro broth dilution or E-test strips.
Minimum Bactericidal Concentration (MBC) — the lowest concentration that kills (99.9% reduction) the bacteria, determined by subculturing MIC tubes with no visible growth onto antibiotic-free agar. Highly bactericidal drugs have MIC ≈ MBC; bacteriostatic drugs have MBC much higher than MIC. A strain with high MBC but low MIC to a normally bactericidal drug (e.g. penicillin) shows "tolerance".
Rapid tests can directly detect resistance mechanisms — e.g. a rapid "stick" test for beta-lactamase production (predicts ampicillin/amoxicillin resistance), latex agglutination or PCR for the mecA gene (MRSA), and PCR for rpoB gene mutations (rifampicin-resistant M. tuberculosis). Automated systems use liquid cultures and turbidity/CO₂ measurement to shorten incubation and reporting time significantly.
Laboratory Principle
In disk diffusion, antibiotic diffuses radially outward from the disk through the agar, creating a concentration gradient — highest near the disk, lowest at the periphery. Bacterial growth is inhibited wherever the local antibiotic concentration exceeds the organism's MIC, producing a visible clear zone whose diameter correlates inversely with the organism's resistance. In dilution methods, a doubling series of antibiotic concentrations is tested directly against a standardized bacterial inoculum, and the lowest concentration preventing visible turbidity (growth) is the MIC.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Antibiotic-impregnated disks | Standardized potency per drug | Disk diffusion sensitivity testing | Refrigerated / frozen, protect from moisture |
| E-test strips | Manufactured concentration gradient | Direct MIC determination on agar | Refrigerated (2–8°C) |
| Antibiotic stock solutions | Doubling dilution series | Broth macro/microdilution MIC | Freshly prepared or frozen aliquots |
| Nitrocefin sticks | Rapid beta-lactamase detection | Detect beta-lactamase production | Room temperature, protect from light |
Step-by-Step Procedure
Prepare a bacterial suspension from a pure culture matched to a standard turbidity (e.g. 0.5 McFarland) and spread evenly across the surface of the agar plate to achieve semiconfluent growth.
Place up to six antibiotic-impregnated disks on the inoculated agar surface, spaced to avoid overlapping zones. For comparative (Stokes') testing, inoculate a control strain of known sensitivity in the centre of the plate.
Incubate the plate at 35–37°C for 18–24 hours to allow the antibiotic to diffuse and bacterial growth/inhibition to develop.
Measure the diameter of each clear zone in millimetres. Compare to standardized cut-off tables (e.g. <18 mm = resistant) or, for Stokes' method, compare directly to the control organism's zone (>3 mm reduction compared to control = resistant).
For precise cases (e.g. endocarditis, meningitis), set up a broth macro/microdilution series or an E-test strip. Read the MIC as the lowest concentration with no visible turbidity; subculture MIC tubes to antibiotic-free agar to determine the MBC.
Flow Diagram
Quality Control
Every batch of antibiotic disks and every plate lot must be checked against reference control strains of known susceptibility (e.g. E. coli ATCC 25922) to confirm zone diameters fall within the expected QC range before reporting patient results.
Laboratories should participate in a recognized external proficiency scheme, receiving blinded challenge organisms periodically to verify accuracy and reproducibility of susceptibility results across the laboratory network.
Reference Values
Typical Cut-offs⚠️ Reference cut-offs are drug- and organism-specific and follow standardized guidelines (e.g. CLSI/EUCAST). Always apply your laboratory's current breakpoint tables.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Large zone of inhibition | Organism is sensitive to the tested antibiotic | Report as sensitive; physician may use this drug |
| No zone / growth up to the disk | Organism is resistant | Physician selects an alternative agent |
| Positive beta-lactamase stick test | Predicts resistance to ampicillin/amoxicillin | Avoid unprotected penicillins; consider beta-lactamase inhibitor combinations |
| MRSA (mecA positive) | Resistant to all beta-lactams including methicillin/cloxacillin | Consider vancomycin or other MRSA-active agents; institute infection control |
| High MBC, low MIC (tolerance) | Bacteriostatic effect despite normally bactericidal drug | May require prolonged therapy or combination treatment, especially in endocarditis |
Common Errors & How to Avoid Them
Cause: Placing more than the recommended maximum (usually six) disks on a standard plate causes overlapping zones and inaccurate measurements.
Prevention: Space disks according to standard guidelines; use a second plate if more antibiotics need testing.
Cause: A non-standardized inoculum (not matched to 0.5 McFarland or equivalent) produces falsely large or small zones.
Prevention: Always standardize the inoculum turbidity before plating; aim for semiconfluent growth.
Cause: MBC tests are technically difficult to standardize and results can be misinterpreted as more clinically significant than they are.
Prevention: Reserve MBC testing for specific difficult cases and interpret alongside MIC and clinical context, not in isolation.
Laboratory Tips from the Bench
Always include a control plate with no antibiotic when running breakpoint sensitivity tests — if the organism fails to grow on the control plate, the whole test is invalid regardless of the antibiotic plates.
Report a laboratory "resistant" result to the clinician as a guide, not an absolute — a patient already responding clinically to therapy does not always need a change in antibiotic.
Remember: MIC = lowest concentration that INHIBITS (no visible growth); MBC = lowest concentration that KILLS (99.9% reduction, confirmed by subculture). "Inhibit before you kill."
Important Notes
In vitro sensitivity results are only a guide. An organism reported with sensitivity results does not always require treatment — for example, catheter urine specimens are treated only if the patient is symptomatic.
Direct sensitivity tests (set up straight from a specimen or liquid broth) provide faster results but are harder to standardize, may involve mixed inocula, and the antibiotic panel chosen may not match the organism eventually isolated.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with dysuria, frequency and low-grade fever for two days. History of three UTIs in the past year, each treated with a different antibiotic.
Disk diffusion shows ampicillin resistance (likely beta-lactamase production) but sensitivity to nitrofurantoin and ciprofloxacin. The recurrent history and multiple prior antibiotic exposures raise concern for evolving resistance patterns.
- →Zone diameter cut-offs are drug- and organism-specific and must be checked against standardized tables.
- →Recurrent infections warrant repeat susceptibility testing since resistance patterns can change over time.
- →Nitrofurantoin remains a good empirical choice for uncomplicated lower UTIs due to low resistance rates.
Frequently Asked Questions
A standard agar plate has limited surface area. Placing more than about six disks causes the diffusion zones to overlap, making it impossible to accurately measure individual zone diameters and interpret results.
MIC testing is reserved for situations requiring a precise, quantitative assessment of susceptibility — such as pneumococcal meningitis or streptococcal endocarditis — where the exact drug concentration needed matters for dosing decisions.
No. In vitro results are a guide, not an absolute rule. Clinical response depends on immune status, drug pharmacokinetics, site of infection, and biofilm presence, so a resistant report does not automatically mean treatment failure, and vice versa.
Quick Revision
10-Minute ReviewKey Takeaways
- Antibiotic susceptibility testing guides safe and effective antibiotic therapy.
- Disk diffusion and breakpoint testing are the two classic agar-based methods.
- MIC and MBC provide quantitative measures of susceptibility for difficult cases.
- Resistance mechanism detection (beta-lactamase, mecA, rpoB) offers rapid, targeted results.
- Automated systems speed up reporting, especially for slow-growing organisms.
- Laboratory results must always be interpreted alongside the patient's clinical picture.
Competency Checklist
Track Your MasteryReferences
- Collee JG, Fraser AG, Marmion BP, Simmons A. Mackie & McCartney Practical Medical Microbiology. 14th ed. Churchill Livingstone.
- Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing.
- National Institute of Open Schooling (NIOS). Microbiology — Antibiotic Susceptibility Testing, Module Notes.