A susceptibility result can shift before the organism changes. That is why KLIGLER Iron Agar Culture Media 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 30087 is discussed as a laboratory measurement component; implementation should follow the laboratory SOP and validated reference method.
Analytical scope and method role
KLIGLER IRON AGAR is a differential medium used in order to distinguish enterobacteria on the basis of their ability to ferment glucose and lactose and to produce sulfides.
Method principle
Proteose Peptone and meat extract are a source of amino acids and peptides. Yeast extract supplies amino acids and vitamin of group B. Sodium chloride maintains the osmotic balance of the medium. The combination of ferrous sulphate and sodium tiosulfate enables the detection of hydrogen sulphide production which is evidenced by a black color either throughout the butt or in a ring formation near the top of the butt. Lactose and glucose enable the differentiation of species of enteric bacilli due to color change of phenol red pH indicator in response to the acid produced during the fermentation of these sugars.
Formula and functional interpretation
(g/L) Proteose Peptone… … … … … … … … … … … … … … .. 20.0 Yeast Extract… ..… … … … … ..… … … … … … … … … ... 3.0 Meat Extract… … … … … … ..… … … … … … … … … … ... 3.0 Sodium Chloride.… … … … … … … … … … … … … … … 5.0 Ferrous Sulfate… … … … … … … … … … … … … … … … . 0.2 Sodium Thiosulfate… … … … … … … … … … … … … … .. 0.3 Lactose… … … … … … … … … … … … … … … … … … … . 10.0 Glucose… … … … … … … … … … … … … … … … … … … . 1.0 Phenol Red… … … … … … … … … … … … … … … … … ... 0.024 Agar… … … … … … … … … … … … … … … … … … … … … 11.0 Final pH= 7.4 ± 0.2
Review the nutritive, selective, differential, buffering, reducing, and indicator components against the expected target and background flora. Preparation errors can change recovery, inhibition, colour development, or endpoint visibility even when incubation is correct.
Preparation and execution: controlled method points
Procedure: Inoculate the medium with the specimen by stabbing the butt and streaking the slope using a sterile loop to ensure an adeguate dispersion of the organisms. Tubes are incubated at 36+/-1°C for 18-24 hours.
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
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
Typical reactions produced by members of Enterobacteriaceae are presented in the following table: MICROORGANISM SLANT BUTT GAS H2S Citrobacter Alkaline Acid + + or - Edwardsiella Alkaline Acid + + Escherichia coli Acid Acid + - Enterobacter Acid Acid + - Morganella Alkaline Acid ± - Proteus Alkaline or acid Acid + + Providencia Alkaline Acid ± - Salmonella Alkaline Acid + + Shigella Alkaline Acid
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
10-25°C away from light, until the expiry date on the label or until signs of deterioration or contamination are evident. WARNING and
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
For laboratory use. The product doesn’t contain dangerous substances according to directives 1999/45/CE and 2001/60/CE or for which exist recognized exposure limits.
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.
FDA BAM Chapter 5: Salmonella
The current FDA BAM Salmonella workflow separates pre-enrichment, selective enrichment, selective plating, and confirmation. That sequence is scientifically important: enrichment increases target representation in a mixed matrix, selective conditions manage competing flora, and confirmation prevents a colour or colony morphology from being treated as identity. A laboratory transferring the method should verify recovery and selectivity on its own matrix classes rather than assuming that a medium performs identically across foods, water, and environmental samples. Review the authoritative source.
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.
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 30087.
- FDA BAM Chapter 5: Salmonella (access checked 2026-07-20).
- EUCAST disk diffusion and quality control (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.