A susceptibility result can shift before the organism changes. That is why ESBL (Chromos. Induc. AmpC) disc kit (acc. to EUCAST) for confirmation of must be considered together with inoculum, atmosphere, incubation, and endpoint reading. The discussion below connects its documented role with preparation variables, controls, interpretation, and the limits of inference. Catalogue 99006 is discussed as a laboratory measurement component; implementation should follow the laboratory SOP and validated reference method.
Analytical scope and method role
Extended-spectrum beta-lactamases (ESBLs) are enzymes hydrolyzing most penicillins and cephalosporins, including oxyimino- beta-lactam compunds but not cephamycins and carbapenems. Most ESBLs belong to the Ambler class A of beta-lactamases and are inhibited by beta-lactamases inhibitors: clavulanic acid, sulbatam and tazobactam. ESBL production has been observed mostly in Enterobacteriaceae, particularly Escherichia coli and Klebsiella pnuemoniae, but all other clinically-relevant Enterobacteriaceae species are also common ESBL-producers. In many areas, ESBL detection and characterization is recommended or mandatory for infection control purpose. AmpC beta-lactamases are enzymes encoded on the chromosome of many Enterobacteriaceae and a few other organisms.
Method principle
Enterobacteriaceae suspected to be producers of both ESBL and AmpC enzymes may be confirmed by using cefepime to evaluate the inhibition of ESBL activity by Clavulanic acid. Combination Disc Test (CDT) For each test, discs containing cefepime alone and in combination with clavulanic acid are applied. The inhibition zone around the cefepime disc combined with clavulanic acid is compared with the zone around the disc with the cefepime alone. The test is positive if the inhibition zone diameter is ≥ 5 mm larger with clavulanic acid than without. GATHERING AND KEEPING SAMPLES The colonies that are to be subjected to the susceptibility test are taken up by culture media that have been previously swabbed with the sample under examination.
Preparation and execution: controlled method points
Procedure: 1.Using a fresh, pure culture prepare a suspension of the test organism equivalent to 0.5 McFarland. 2.Using a sterile cotton swab, spread the adjusted suspension over the entire area of a Mueller Hinton agar plate. 3.Apply the discs onto the inoculated plate, ensuring sufficient space between individual discs to allow for proper measurement of inhibition zones. 4.Incubate at 35±1°C for 16-20 hours. EVALUATING THE RESULTS At the end of the incubation period, measure the inhibition halos and interpret as indicated in the following table. Interpretative Table. CDT method for confirmation of ESBL in Enterobacteriaceae producing chromosomal AmpC. ESBL (Chromos. Induc. AmpC) disc kit (acc. to EUCAST) Disc tests for confirmation of ESBLs in organisms with chromosomally encoded inducible AmpC.
Translate the method into controlled worksheet fields: catalogue and lot, preparation mass or volume, water quality, pH where applicable, heating or sterilization, supplements, dispense volume, preparation date, expiry assignment, incubation atmosphere and temperature, reading window, equipment, and analyst. Record departures as deviations instead of silently normalizing them.
Quality control: what the control result protects
of discs, MHA plates, inoculum and
Challenge both sides of performance. Include a target reaction demonstrating recovery and, when relevant, a non-target or alternate reaction challenging inhibition or differentiation. Record expected and observed responses, acceptance criteria, strain and passage, lot, incubation, and disposition. A failed control blocks result interpretation until the cause is resolved.
Interpretation and confirmation pathway
Record the raw observation before the conclusion: morphology or colour, growth pattern, endpoint, reaction intensity, reading time, and control response. Presumptive reactions remain presumptive. Route atypical, weak, mixed, or delayed reactions through the confirmation step in the current method.
Storage, stability, and pre-use inspection
Store the unopened blister at -20°C to +8°C till the expiry date. Allow unopened cartridge to come to room temperature before removing it from the blister for minimising condensation on the discs. Leftover discs from an opened cartridge should be stored at 2-8°C for no more than 7 days. Return unused discs to the refrigerator as soon as the application of the discs has been completed. Dispose of expire discs.
Receipt and storage records should reconstruct temperature exposure, lot rotation, expiry, container integrity, and time outside controlled storage. Inspect colour, clarity, fill volume, gel integrity, contamination, dehydration, and precipitate before use.
Limitations and boundaries of use
The disc cannot be classified as being hazardous according to current legislation but fall within the specific field of application where a safety data sheet must be supplied because they can cause phenomena of sensitization in sensitive subjects if they come into contact with the skin. The discs are disposable products. They are only for diagnostic in vitro use and are intended for professional use. They must be used in the laboratory by properly trained operators using approved aseptic and safety methods for pathogenic agents.
Applicability depends on matrix, target organism, interfering flora, inoculum, validated method, and regulatory setting. Do not transfer conditions or acceptance criteria from a different matrix without verification.
Troubleshooting from evidence, not guesswork
- Poor recovery: check preparation, pH, overheating, supplement addition, inoculum, atmosphere, storage, and control performance.
- Loss of selectivity: review formulation, sterilization, expiry, inoculum burden, enrichment sequence, and incubation time.
- Weak or atypical reaction: confirm reading time and atmosphere, compare controls, document mixed growth, and follow confirmation requirements.
- Run-to-run drift: trend lot, analyst, equipment, water, preparation batch, controls, and environmental conditions.
External method context
The product should be understood as one measurement component in an antimicrobial susceptibility method. The analytical chain includes isolate purity, standardized inoculum, medium composition, antimicrobial concentration, incubation atmosphere and time, endpoint reading, and the interpretive table selected by the laboratory. A shift in any upstream variable can move an MIC or zone without a change in organism biology, which is why QC distributions and trend review are more informative than a single pass/fail control.
External laboratory-quality guidance also supports a lifecycle view: verify the method when introduced, bridge new lots against an accepted lot, trend control results, investigate nonconformities, and retain enough raw observations to reconstruct the decision. This is the practical difference between reproducing a preparation instruction and demonstrating that the resulting measurement is fit for its intended use.
EUCAST disk diffusion and quality control
EUCAST requires standardized methodology, current breakpoint tables, and routine quality control; the laboratory remains responsible for the final susceptibility report. For broth dilution, inoculum density, cation content, pH, incubation atmosphere, endpoint definition, and two-fold dilution design influence MIC reproducibility. Breakpoints are interpretive rules applied after a technically valid MIC or zone measurement; they are not properties of the product alone. Review the authoritative source.
FDA Bacteriological Analytical Manual
FDA BAM methods organize food microbiology around validated sampling, preparation, enrichment, isolation, and confirmation steps, with method applicability tied to the matrix and target organism. Sampling uncertainty, homogenization efficiency, inhibitory ingredients, dilution design, and countable range can dominate the result before the culture medium is ever inoculated; those variables belong in method verification and uncertainty review. Review the authoritative source.
WHO Laboratory Quality Management System handbook
WHO laboratory quality guidance treats document control, equipment, purchasing and inventory, process control, records, occurrence management, and continual improvement as connected parts of reliable testing. Review the authoritative source.
Technical references
- Manufacturer product documentation for catalogue 99006.
- EUCAST disk diffusion and quality control (access checked 2026-07-20).
- FDA Bacteriological Analytical Manual (access checked 2026-07-20).
- WHO Laboratory Quality Management System handbook (access checked 2026-07-20).
Bench-use checklist
- Verify product identity, catalogue number, lot, expiry, and storage history.
- Document preparation and incubation exactly as controlled in the SOP.
- Define controls and acceptance criteria before testing.
- Record raw observations, control response, reading time, and confirmation decision.
- Escalate failed controls, atypical reactions, and departures through the quality system.