Antimicrobial Susceptibility Testing

Why AST

Species ID tells expected pattern; AST tells actual susceptibility of this isolate. Result drives treatment, not textbook reputation.

Disk Diffusion (Kirby-Bauer)

Standard qualitative method. Standardized suspension (0.5 McFarland) spread on Mueller-Hinton agar. Antibiotic disks placed → diffuse outward, concentration gradient. Zone of inhibition diameter measured (mm) → compared to breakpoint table (CLSI) → Sensitive/Intermediate/Resistant.

Zone size = interaction of diffusion + susceptibility, not pure potency. Never compare raw mm across different drugs.

MIC (Minimum Inhibitory Concentration)

Lowest concentration preventing visible growth (overnight). More quantitative than disk diffusion. Used for serious infections/borderline susceptibility/narrow therapeutic window drugs.

Methods:

• Broth dilution / broth microdilution — doubling dilution series in broth/microtiter wells, lowest clear well = MIC
• Agar dilution — same principle, series of agar plates with fixed concentrations
• E-test — plastic strip, continuous gradient, teardrop inhibition zone, MIC read directly off strip scale

MBC (Minimum Bactericidal Concentration)

Lowest concentration killing ≥99.9% of inoculum (not just inhibiting). Distinguishes cidal from static drugs.

Method: subculture from clear MIC wells onto antibiotic-free medium — no growth = truly bactericidal concentration.

Reserved for: infective endocarditis, severely immunocompromised patients. Not routine.

Specific Resistance Detection Tests

D-test (double disk approximation): detects inducible clindamycin resistance. Erythromycin disk near clindamycin disk → flattened D-shaped zone (not full circle) = positive. Prevents false report of clindamycin susceptibility.

ESBL confirmatory test: cephalosporin disk alone vs + clavulanic acid, side by side. Bigger zone with clavulanate (synergy) = ESBL positive.

Cefoxitin screening for MRSA: more reliable surrogate than direct oxacillin testing. Cefoxitin resistance predicts mecA-mediated resistance.

Automated Systems

VITEK and similar — combine ID + AST. Standardized inoculum → card/panel with graded antibiotic wells → turbidity/fluorescence read at intervals → MIC + category within hours.

Faster than manual, but manual disk diffusion/E-test still used for uncovered organisms/drugs and where automation unavailable.

Reading/Reporting

Raw zone/MIC value → compared to breakpoint table (CLSI/EUCAST) → categorical report: Sensitive/Intermediate/Resistant. This category, not the raw number, guides prescribing.

Why Test Susceptibility at All

Knowing an organism’s species tells a clinician what it is usually susceptible to; it does not tell them what this particular isolate, from this particular patient, actually is susceptible to right now, given how freely resistance moves between bacteria. Antimicrobial susceptibility testing (AST) answers that question directly, and its result is what should actually drive antibiotic selection — not the organism’s textbook reputation.

Disk Diffusion (Kirby-Bauer Method)

This remains the standard qualitative method in most diagnostic laboratories, valued for being cheap, simple, and flexible enough to test many drugs against one organism on a single plate.

A standardized bacterial suspension (adjusted to a reference turbidity, the 0.5 McFarland standard) is spread evenly across a Mueller-Hinton agar plate, and paper disks impregnated with a fixed concentration of each antibiotic to be tested are placed on the surface. As the plate incubates, the antibiotic diffuses outward from each disk in a concentration gradient — highest near the disk, falling with distance — and bacterial growth is inhibited wherever that local concentration exceeds what the organism can tolerate. The result is a clear zone of inhibition around each disk, whose diameter is measured in millimetres and compared against standardized interpretive breakpoints (published by bodies such as CLSI) to classify the isolate as Sensitive, Intermediate, or Resistant to that drug.

The zone diameter is not itself the potency of the drug — it reflects the interaction between the drug’s diffusion characteristics and the organism’s susceptibility, which is exactly why it must be read against a validated breakpoint table for that specific drug-organism combination, never compared as a raw number across different antibiotics.

Minimum Inhibitory Concentration (MIC)

Where disk diffusion gives a categorical answer, MIC testing gives a number: the lowest concentration of an antibiotic that visibly prevents growth of the organism after overnight incubation. This is the more quantitative gold standard and matters most where the precise potency needed is clinically important — serious infections, borderline susceptibility, or drugs with a narrow therapeutic window.

• Broth dilution — a doubling series of antibiotic concentrations is prepared in broth (or, more practically today, in the wells of a microtiter plate for broth microdilution), each well inoculated with a standard bacterial suspension; after incubation, the lowest concentration showing no visible turbidity is the MIC.
• Agar dilution — the same principle carried out in a series of agar plates, each containing a different fixed antibiotic concentration, with multiple organisms inoculated per plate (useful for testing many isolates against one drug efficiently, though less practical for testing one isolate against many drugs).
• E-test (epsilometer test) — a plastic strip carrying a continuous, predefined antibiotic gradient along its length is laid on an inoculated agar plate; a teardrop-shaped zone of inhibition forms around it, and the MIC is read directly from the strip’s calibrated scale at the point where the inhibition ellipse intersects it. This combines the convenience of a disk-based format with the quantitative precision of a dilution method, at higher cost per test.

Minimum Bactericidal Concentration (MBC)

The MBC is the lowest antibiotic concentration that actually kills at least 99.9% of the original inoculum, rather than merely inhibiting visible growth — a distinction that matters because MIC alone cannot tell a bactericidal drug from a bacteriostatic one. It is determined by subculturing from the clear (no-growth) wells of an MIC test onto antibiotic-free medium: wells that show no growth on subculture represent concentrations that were truly bactericidal, not just inhibitory. MBC testing is reserved for specific clinical situations where cidal activity genuinely changes management — infective endocarditis and infections in severely immunocompromised patients being the classic examples — rather than run routinely.

Detecting Specific Resistance Mechanisms

Beyond simply reporting Sensitive/Resistant, several targeted phenotypic tests exist to flag a specific, clinically important resistance mechanism directly:

• D-test (double disk approximation) — detects inducible clindamycin resistance in a Staphylococcus or Streptococcus isolate that appears clindamycin-susceptible but erythromycin-resistant on standard testing. Placing an erythromycin disk near a clindamycin disk produces a flattened, D-shaped zone of inhibition around the clindamycin disk (rather than a full circle) if inducible resistance is present — reporting the isolate as clindamycin-susceptible without this test risks clinical failure on a drug that looks safe on paper.
• ESBL confirmatory test — places a cephalosporin disk (ceftazidime or cefotaxime) alone and the same disk combined with clavulanic acid side by side; a substantially larger zone around the combination disk (synergy) confirms ESBL production, since clavulanic acid inhibits the ESBL enzyme and restores the cephalosporin’s activity.
• Cefoxitin screening for MRSA — because oxacillin/methicillin resistance in S. aureus can be difficult to detect reliably by direct oxacillin testing, a cefoxitin disk is used as a more reliable surrogate marker; resistance to cefoxitin predicts the presence of the mecA-mediated resistance mechanism even in strains where oxacillin itself gives an ambiguous result.

Automated Susceptibility Testing Systems

Automated instruments (such as VITEK and similar platforms) combine organism identification and susceptibility testing in one workflow: a standardized inoculum is loaded into a card or panel containing miniaturized wells of graded antibiotic concentrations, and the system reads growth (by turbidity or fluorescence) at intervals, calculating an MIC and interpretive category for a whole panel of drugs within hours rather than the overnight-plus turnaround of manual methods. These systems have become standard in high-volume laboratories, though manual disk diffusion and E-test retain a role for organisms or drugs the automated panels do not cover well, and for laboratories without the capital investment automation requires.

Reading and Reporting

Whichever method is used, the raw measurement (zone diameter or MIC value) only becomes clinically useful once compared against a validated breakpoint table specific to that organism-drug pair, issued and periodically revised by standard-setting bodies (CLSI in much of the world, EUCAST in Europe). The final report to a clinician is the categorical result — Sensitive, Intermediate, or Resistant — because that is what actually guides prescribing, not the underlying millimetre or microgram-per-millilitre value itself.

Hand-drawing notes only (no rendered diagrams)

D-test (inducible clindamycin resistance). Draw a Mueller-Hinton plate with two disks placed close together: erythromycin and clindamycin. Around the erythromycin disk, draw a small zone of inhibition (or none, since resistant). Around the clindamycin disk, draw a zone that is flattened/truncated on the side facing the erythromycin disk — a “D” shape rather than a full circle — labelling this flattening as the positive result (inducible resistance present). Contrast with a full circular zone around clindamycin (negative — true susceptibility) as a second small panel. Common exam mistake: describing the flattening as occurring on the side of the clindamycin disk away from erythromycin — it is the side facing the inducing erythromycin disk that flattens.

E-test strip. A single rectangular strip laid on an agar plate with a printed numeric gradient scale along its length, surrounded by a teardrop/ellipse-shaped zone of inhibition — wide near the high-concentration end of the strip, narrowing to a point where it crosses the strip at the MIC value. Label the point where the ellipse edge intersects the strip as the MIC reading.

Not rendered

Disk diffusion zone-diameter interpretation and MIC dilution series are already captured clearly as procedural text and would not gain clarity from a rendered figure — no qualifying diagram beyond the two hand-drawn items above.

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