12.1 Microbroth Dilution, Disk Diffusion, and Gradient Diffusion
Key Takeaways
- A 0.5 McFarland standard is about 1–2×10^8 CFU/mL; too heavy an inoculum shrinks zones and raises MICs (false resistance), and too light does the opposite.
- Nonfastidious CLSI disk and dilution tests use Mueller-Hinton agar or cation-adjusted Mueller-Hinton broth at 35°C ±2°C in ambient air for 16–18 h; S. pneumoniae needs MHA with 5% sheep blood in CO2 for 20–24 h, and Haemophilus needs HTM in CO2.
- M02 yields zone diameters, M07 yields microbroth MICs, and M100 supplies breakpoints that are updated on a regular, usually annual, cycle — learn the method, not a frozen year-stamped table.
- Gradient diffusion (Etest) is an MIC method: read the ellipse where it intersects the strip and interpret with MIC breakpoints.
- Do not report S. aureus oxacillin disk results without cefoxitin-screen rules; QC conceptually uses E. coli ATCC 25922, S. aureus 25923 (disk) / 29213 (MIC), and P. aeruginosa 27853.
Antimicrobial susceptibility testing (AST) on the M(ASCP) exam is official outline II.K (guideline revised 2025-09-25). You are tested on how CLSI methods are inoculated, incubated, read, and quality-controlled — not on photocopying a single year’s breakpoint table. CLSI M02 is disk diffusion (Kirby-Bauer zone diameters in millimeters). CLSI M07 is dilution testing; broth microdilution (microbroth MIC) is the format you will meet on panels. CLSI M100 publishes interpretive breakpoints (susceptible, susceptible-dose-dependent, intermediate, resistant) and QC ranges. Those M100 tables are revised on a regular, usually annual, cycle. Learn how a zone or MIC is generated and that you look up the current table. Do not memorize a frozen millimeter list as if it were still active M100. Selecting which antimicrobials are appropriate to report by species and body site (cascade-by-site lists) is SM-only (II.K.6) and is not M-tested reporting curriculum. M candidates still must know when a method is valid and when a resistance marker rewrites a result.
Inoculum: 0.5 McFarland
Every standardized method starts with a 0.5 McFarland turbidity standard, a barium-sulfate suspension optically equivalent to about 1–2×10^8 CFU/mL. Pick 3–5 morphologically similar colonies from an overnight (18–24 hour) plate — not mixed colony types and not a single starved pinpoint colony — emulsify in saline or broth, and match the standard against a Wickerham card or, better, a calibrated densitometer. Remix the barium standard; a settled standard looks too light and produces a heavy bacterial suspension.
Inoculum error is bidirectional and highly testable:
- Too heavy (extra colonies, unmatched turbidity, delay that lets the tube grow): zones shrink and MICs rise → false resistance.
- Too light (over-diluted, unmixed McFarland, one tiny colony in a large volume): zones enlarge and MICs fall → false susceptibility.
For disk diffusion you swab the 0.5 McFarland onto the agar as a lawn within about 15 minutes of adjusting turbidity. For reference broth microdilution that same 0.5 McFarland is further diluted so the final well density is about 5×10^5 CFU/mL. Pipetting neat 0.5 McFarland into a MIC well is a 100-fold overload and a classic cause of falsely elevated oxacillin, vancomycin, or carbapenem MICs. Direct-colony versus log-phase broth inoculum must still land on the same turbidity target; the SOP, not habit, wins.
Mueller-Hinton agar and broth as reagents
Nonfastidious organisms are tested on unsupplemented Mueller-Hinton agar (disk) or cation-adjusted Mueller-Hinton broth (CAMHB, dilution). The medium is as much a reagent as the disk. Pour depth is about 4 mm. pH is 7.2–7.4. Calcium and magnesium concentrations are specified because they change aminoglycoside and some other activity against Pseudomonas. Thymidine must be low because excess thymidine lets organisms bypass trimethoprim–sulfamethoxazole and fill in the zone.
| Medium variable | Distortion | AST consequence |
|---|---|---|
| Agar too thin (<~4 mm) | Antibiotic diffuses farther | Zones too large (false S) |
| Agar too thick | Shallower gradient | Zones too small (false R) |
| pH too acidic | Aminoglycosides, macrolides, quinolones lose activity | False resistance |
| Excess Ca2+/Mg2+ | Aminoglycosides vs P. aeruginosa look weaker | False resistance |
| Low Ca2+/Mg2+ | Aminoglycosides look stronger | False susceptibility |
| Excess thymidine | Folate-pathway bypass | TMP-SMX zones fill (false R) |
| Disks crowded or at the edge | Overlapping gradients | Unreadable zones |
Incubate nonfastidious disk plates 16–18 hours at 35°C ±2°C in ambient air. Do not use CO2 for routine enteric or Pseudomonas disk tests: CO2 acidifies the agar surface, which shrinks aminoglycoside, macrolide, and quinolone zones (false resistance) while enlarging tetracycline zones (false susceptibility). Do not stack plates so the center never reaches temperature. Measure the zone of complete inhibition from the back of the plate against a dark background, to the nearest millimeter. Ignore faint Proteus swarm beyond the inner true zone. For TMP-SMX, read at about 80% inhibition because slight trailing is expected. Imipenem and some clavulanate disks are labile — store with desiccant and do not leave cartridges on a warm bench.
Fastidious exceptions: pneumococcus and Haemophilus
Streptococcus pneumoniae will not produce a usable lawn on unsupplemented MHA. Use Mueller-Hinton agar with 5% sheep blood, incubate in CO2 for 20–24 hours, and measure zones from the surface (you cannot read through blood agar). An oxacillin 1 µg disk is a penicillin screen for pneumococci, not a stand-alone MIC: a large oxacillin zone predicts penicillin susceptibility; a reduced zone requires a penicillin MIC (and often a ceftriaxone MIC) before you report. That screen is method theory — look up the current M100 cutoff rather than treating a blog number as this year’s table.
Haemophilus spp. require Haemophilus Test Medium (HTM): Mueller-Hinton base plus yeast extract, hematin (X factor), and NAD (V factor). Incubate HTM disks in CO2. A nitrocefin (Cefinase) test on the colony is a separate beta-lactamase assay, covered in the next section; it does not replace HTM ampicillin disk or MIC when a full AST is required. Chocolate agar is a growth plate, not the CLSI disk medium for Haemophilus.
Disk diffusion, microbroth MIC, and gradient diffusion
Disk diffusion (M02): after the lawn dries for a few minutes (not so long that the inoculum dies), apply specified disks, press so the entire face contacts agar, invert, and incubate as above. Drug diffuses in a roughly circular gradient. Zone diameter is compared with the current M100 column for that organism group. Disk testing is flexible but is invalid for some organism–drug pairs. Vancomycin disk diffusion does not reliably detect vancomycin-intermediate S. aureus (VISA); use an MIC. Staphylococcal oxacillin and vancomycin wells, and enterococcal vancomycin tests, often need a full 24-hour read — do not issue a 16-hour “S” on those combinations.
Microbroth dilution (M07): trays contain two-fold dilutions in CAMHB, plus CLSI-required supplements (2% NaCl in staphylococcal oxacillin wells; blood supplements for streptococci). After overnight incubation, the MIC is the lowest concentration without visible growth. Skip wells (growth, no growth, growth again) make that drug uninterpretable — repeat; do not interpolate. Trailing with bacteriostatic agents is read at the SOP endpoint, not at the last pinpoint button. A growth-control well must be cloudy and a sterility well must be clear.
Gradient diffusion (Etest or equivalent): a strip carries a dried exponential gradient labeled in µg/mL. Place it on a standardized lawn. After incubation an ellipse of inhibition forms. Read the MIC where the ellipse intersects the strip; if the intersection falls between marks, follow the product instruction (usually round up to the next two-fold value). Gradient MIC is still an MIC, interpreted with M100 MIC breakpoints, not with the disk-zone column. It is useful for fastidious organisms, drugs missing from the commercial panel, and confirmation of an unusual MIC.
QC ATCC strains and the oxacillin disk rule
Recognize the conceptual QC set:
- Escherichia coli ATCC 25922 — Gram-negative disk and MIC QC.
- Staphylococcus aureus ATCC 25923 — disk diffusion QC.
- Staphylococcus aureus ATCC 29213 — dilution/MIC QC. Do not swap 25923 and 29213.
- Pseudomonas aeruginosa ATCC 27853 — Pseudomonas, aminoglycoside, and many beta-lactam QC challenges.
QC results must fall in the current M100 range. Out-of-range QC invalidates patient AST until the incubator, atmosphere, depth, inoculum, or reagent is corrected.
Do not report S. aureus oxacillin susceptibility from an oxacillin disk without cefoxitin-screen rules. mecA-mediated MRSA is induced more reliably by cefoxitin than by oxacillin. CLSI uses a cefoxitin disk or cefoxitin MIC (or a validated mecA/PBP2a assay) as the surrogate that predicts oxacillin resistance. An oxacillin disk alone can look falsely susceptible. When the cefoxitin screen or mecA is positive, report oxacillin-resistant/MRSA; do not override with a large oxacillin disk. That is a method rule, not the SM-only body-site reporting list.
A laboratory reports oxacillin susceptibility for Staphylococcus aureus from an oxacillin disk zone alone, without a cefoxitin screen or mecA/PBP2a result. Which statement is most accurate for CLSI-based M(ASCP) method rules?
What organism density is a 0.5 McFarland standard intended to match when preparing CLSI disk diffusion and the starting suspension for dilution AST?
Which setup matches CLSI disk diffusion for Streptococcus pneumoniae?