Listeria monocytogenes is a foodborne bacterium that can cause a serious infection called listeriosis. Unlike many other foodborne bacteria, it can survive and grow even at refrigeration temperatures. Although listeriosis is relatively rare, it can cause severe illness in pregnant women, newborns, older adults, and people with weak immune systems. Infection during pregnancy can also lead to miscarriage, stillbirth, or serious illness in the newborn.

The bacterium can contaminate a variety of foods, including ready-to-eat (RTE) meats, soft cheeses, unpasteurized dairy products, smoked seafood, prepared salads, and fresh produce. RTE foods require particular attention because they are usually consumed without further cooking, which means there may be no additional step to destroy the bacteria before consumption. If the food supports bacterial growth, L. monocytogenes can multiply during refrigerated storage. This makes proper temperature control, hygienic food handling, and appropriate shelf-life management essential for reducing the risk of contamination.

For food manufacturers and testing laboratories, detecting and measuring L. monocytogenes are important steps in assessing food safety and verifying hygiene controls. Detection determines whether the bacterium is present in a specified test portion, while enumeration estimates the number of bacteria present in a given quantity of food. The choice between these approaches depends on the purpose of testing and the applicable microbiological requirements.

The ISO 11290 series provide standardized methods for both types of analysis. ISO 11290-1 describes the method for detecting Listeria spp., including L. monocytogenes, while ISO 11290-2 describes the method for enumerating these bacteria. Understanding the differences between the two methods helps laboratories select the appropriate testing procedure, interpret results correctly, and support effective food safety management.

The recent amendment, ISO 11290-1:2017/Amd 1:2026, introduces provisions for sample storage before analysis and revises the control strains used for performance testing of culture media and reagents in both the detection and enumeration methods.

Understanding ISO 11290-1 and ISO 11290-2

The ISO 11290 series provide two complementary methods for testing Listeria monocytogenes and other Listeria species in food. Although both methods support food safety assessment, they answer different questions: Is the organism present? or quantity of organism present.

ISO 11290-1: Detection of Listeria spp.

ISO 11290-1 describes a qualitative method that determines whether Listeria spp., including L. monocytogenes, are detected in a specified test portion. A 25 g test portion is commonly used, although the required sample size depends on the applicable procedure and product category.

The method uses enrichment to help recover the organism when it is present in low numbers or has been stressed by food processing, storage, or other environmental conditions. The final result indicates whether the target organism was detected in the test portion; it does not quantify the bacterial concentration.

ISO 11290-2: Enumeration of Listeria spp.

ISO 11290-2 describes a quantitative method used to estimate the concentration of Listeria spp. or L. monocytogenes in a food sample. Results are generally expressed as colony-forming units per gram (CFU/g) or per millilitre (CFU/mL), as applicable.

Enumeration is useful when a numerical microbiological criterion applies, when manufacturers need to assess contamination levels, or when evaluating whether the organism may multiply during a product's shelf life. The method's ability to quantify the organism depends on the concentration present and the analytical conditions.

Which method should a food testing laboratory use?

The choice depends on the product, the purpose of testing, and the applicable regulatory or customer requirements. For ready-to-eat (RTE) foods, detection may be required to assess the presence of L. monocytogenes in a defined test portion, while enumeration may be needed to determine whether its concentration complies with a specified limit.

For example, a limit of 100 CFU/g applies to certain RTE foods under specific regulatory conditions when compliance throughout shelf life can be demonstrated. These criteria are not universal and must be interpreted according to the relevant jurisdiction and food category.

Key takeaway: ISO 11290-1 establishes whether the organism is detected in the specified test portion, whereas ISO 11290-2 estimates its concentration. Laboratories should select and report the method appropriate to the testing objective and applicable requirements.

Why does detection need two enrichment stages?

Listeria monocytogenes may be present in low numbers in food samples alongside a large population of competing microorganisms. The two-stage enrichment process used in ISO 11290-1 helps recover the target organism while progressively suppressing background flora that could interfere with its detection.

Stage 1: Half-Fraser broth:

The primary suspension prepared in buffered peptone water is transferred to Half-Fraser broth and typically incubated at 30 °C for 24 h, in accordance with the applicable method. Half-Fraser broth contains lower concentrations of selective agents than full-strength Fraser broth, helping promote the recovery of Listeria cells that may have been injured during food processing or storage while inhibiting competing microorganisms.

Stage 2: Fraser broth

A portion of the primary enrichment is then transferred to Fraser broth, typically incubated at 37°C for 24 hours. The higher concentrations of selective agents provide stronger inhibition of competing microorganisms, helping facilitate the subsequent isolation of Listeria. The exact procedure and incubation conditions should follow the applicable edition of ISO 11290-1.

What Does Blackening of the Broth Indicate?

Both Half-Fraser and Fraser broths contain esculin and ferric ammonium citrate. When Listeria hydrolyses esculin into esculetin and glucose, the esculetin reacts with ferric ions to form a dark-coloured complex, resulting in the characteristic darkening or blackening of the medium.

However, blackening is an indicator of a possible positive result, not confirmation of L. monocytogenes. Other microorganisms may also hydrolyse esculin, so further isolation and confirmation are necessary.

Importantly, a clear Fraser broth must not automatically be considered negative or discarded. The prescribed plating procedure should be followed even when blackening is absent, because relying on broth colour alone could lead to missed detections. Laboratories should follow the relevant ISO procedure for transferring, plating, and confirming presumptive colonies.

ISO 11290-1 specifies the use of a chromogenic agar medium, such as Ottaviani and Agosti (ALOA), alongside a second selective agar medium, typically Oxford or PALCAM, in accordance with the standard and the laboratory’s validated procedure.

Why Are Two Plating Media Required?

Using two selective agar media improves the chances of recovering Listeria when strains respond differently to selective agents or show atypical colony characteristics. Because the media use different selective systems, a strain that is inhibited or poorly recovered on one medium may grow more readily on the other.

Using only one medium may reduce the likelihood of detecting certain strains. Following the prescribed two-medium approach therefore supports reliable recovery and helps minimize the risk of false-negative results.

How Has Chromogenic Agar Improved Listeria Identification?

On traditional selective media, such as Oxford and PALCAM agar, L. monocytogenes and L. innocua can produce similar-looking colonies, often with grey-green or grey colonies and dark halos. As a result, presumptive colonies may require additional testing to distinguish the two species.

Chromogenic media, such as ALOA-type agar, improves differentiation through characteristic colony colour and halo formation. Typically, L. monocytogenes produces blue-green colonies with an opaque halo, while L. innocua generally produces blue-green colonies without the halo. These features help laboratories identify presumptive L. monocytogenes colonies more efficiently.

However, chromogenic appearance alone does not confirm the species. Appropriate confirmation remains necessary according to the applicable method.

Why Are Plates Examined at Both 24 and 48 Hours?

Plates are examined after the initial incubation period, typically around 24 hours, and re-examined after further incubation up to 48 hours, as specified by the method.

Some colonies may develop slowly and may not display their characteristic appearance during the first reading. On chromogenic agar, colour may appear before a fully developed halo becomes visible. The second reading can therefore help identify colonies that were not clearly distinguishable earlier.

Conversely, prolonged incubation may allow colonies to enlarge or competing organisms to grow, potentially making individual colonies harder to distinguish. Examining the plates at both specified time points helps improve recovery and recognition of presumptive colonies.

Key takeaway:

Using the prescribed combination of selective media, carefully interpreting colony characteristics, and examining plates at the specified incubation times are essential for reliable Listeria detection and enumeration. Laboratories should follow the applicable editions of ISO 11290-1 and ISO 11290-2, including relevant amendments. They should also consider the provisions of ISO 11290-1:2017/Amd 1:2026 regarding sample storage before analysis and the revised control strains used to assess the performance of culture media and reagents, while adhering to the manufacturer's instructions for the media used.

How does enumeration differ?

Unlike ISO 11290-1, which uses selective enrichment to detect Listeria in a defined test portion, ISO 11290-2 focuses on estimating the concentration of the organism in a sample. Enumeration generally involves direct plating onto a suitable selective agar medium, with a prescribed resuscitation step where applicable to aid the recovery of sub lethally injured cells before exposure to selective agents. The method also specifies the use of an additional control strain from another Listeria species for performance testing, as required by the applicable standard and its amendments.

This difference affects the sensitivity of the two methods. Detection can identify the organism when present at low levels because enrichment allows it to multiply before isolation. Enumeration, by contrast, depends on recovering and counting colonies from the portion plated and may not detect concentrations below its practical detection limit.

Consequently, a sample may be positive by ISO 11290-1 but yield no countable colonies by ISO 11290-2. These results are not necessarily contradictory: detection assesses presence in a defined test portion, whereas enumeration estimates the concentration within the method's measurable range.

How Is Listeria monocytogenes Confirmed?

Colonies that appear typical on selective agar are considered presumptive and require further testing to confirm their identity. Under ISO 11290-1, presumptive colonies are purified and subjected to the prescribed identification and confirmation procedures. A non-selective medium, such as tryptone soya yeast extract agar (TSYEA), may be used for purification.

Confirmation may include the following characteristics, depending on the applicable procedure:

• The mandatory confirmatory tests include haemolysis and carbohydrate fermentation. The other tests like microscopic aspect, catalase, motility and CAMP test are optional.
• Catalase reaction and Gram stain: Listeria typically shows a positive catalase reaction and appears as Gram-positive short rods.
• Motility: At approximately 20–25°C, L. monocytogenes typically exhibits characteristic tumbling motility. In semi-solid motility medium, it may produce an umbrella-shaped growth pattern. Motility is generally reduced or absent at 37°C.
• Haemolysis: L. monocytogenes typically produces narrow, sometimes faint beta-haemolysis on sheep blood agar.

Note: Some rare strains of Listeria monocytogenes may not exhibit haemolysis or produce a positive CAMP test reaction under certain conditions.

• CAMP test: A characteristic positive reaction may be observed with Staphylococcus aureus under the specified test conditions.
• Carbohydrate utilisation: The typical reaction pattern is rhamnose positive and xylose negative.

The interpretation of these tests should follow the current standard and the laboratory’s validated confirmation procedure. No single characteristic should be used alone to establish species identity. Further confirmation of Listeria spp. includes mandatory tests for microscopic appearance, which typically reveals short, slender rods or coccobacilli, and catalase activity. Other tests, such as the Voges–Proskauer (VP) test and motility testing, may be performed as optional tests, depending on the applicable standard and confirmation procedure.

Why Are Rhamnose and Xylose Reactions Important?

Carbohydrate utilization helps differentiate L. monocytogenes from other haemolytic Listeria species, including L. ivanovii. The typical pattern for L. monocytogenes is rhamnose positive and xylose negative, whereas L. ivanovii typically shows the opposite pattern.

This distinction is useful because some species may share other characteristics, such as haemolysis, that make them difficult to distinguish based on colony appearance alone. Carbohydrate reactions therefore contribute to the overall identification process. They should, however, be interpreted alongside the other prescribed confirmation tests rather than treated as a standalone result.

Why Can Haemolysis Be Difficult to Interpret?

The beta-haemolysis produced by L. monocytogenes may be narrow and faint, making it difficult to distinguish from a negative reaction. Reliable interpretation depends on suitable blood agar, appropriate incubation conditions, and careful examination of the plates.

For clearer assessment, laboratories should use fresh sheep blood agar where specified by the procedure and examine the plate under suitable transmitted light. Appropriate positive and negative controls can help establish whether the observed reaction is valid. Over-incubation, unsuitable plate conditions, or inadequate lighting may make weak haemolysis difficult to recognise.

Because haemolysis contributes to presumptive identification, an uncertain reaction should be assessed according to the standardised procedure and considered alongside the other confirmation results.

Key takeaway: Confirmation of L. monocytogenes relies on a combination of morphological, biochemical, and other prescribed identification characteristics. Careful interpretation of each result helps reduce the risk of misidentification.

Which Media and Reagents Are Required for ISO 11290 Testing?

The media and reagents required for Listeria monocytogenes detection and confirmation depend on the applicable part of ISO 11290 and the laboratory's validated procedure. The main requirements typically include:

• Half-Fraser broth with selective supplement: Primary enrichment in ISO 11290-1.
• Fraser broth with selective supplement: Secondary enrichment in ISO 11290-1.
• Chromogenic Listeria agar: Such as ALOA-type agar, for presumptive isolation and differentiation.
• Oxford or PALCAM agar with selective supplement: A second selective plating medium, as specified by the method.
• Tryptone soya yeast extract agar (TSYEA): For purification of presumptive colonies.
• Sheep blood agar: For assessing haemolysis on Blood Agar Plates (M2096I).
• Motility medium: For evaluating characteristic motility by inoculating isolated colony in a non-selective nutrient liquid medium.
• Carbohydrate utilisation media or equivalent validated tests: For the prescribed biochemical identification reactions.
• Staphylococcus aureus culture: The CAMP test may be performed when required by the confirmation procedure, particularly when the haemolysis test results are difficult to interpret. Haemolytic strains of Staphylococcus aureus and Rhodococcus equi are used to help identify and differentiate haemolytic Listeria species by demonstrating synergistic enhancement of red blood cell (RBC) lysis on sheep blood agar.

How Is Culture-Medium Performance Verified?

Culture media should be prepared, tested, and performance-verified in accordance with ISO 11133, where applicable. Verification includes assessing whether the medium supports the growth of target organisms (productivity), produces the expected diagnostic reactions, and inhibits specified non-target organisms (selectivity).

Suitable reference strains are selected according to the medium and its intended use. For example, L. monocytogenes can be used to assess productivity and characteristic reactions on appropriate media, while L. innocua may help assess differentiation on chromogenic agar. An organism such as Enterococcus faecalis may be used to assess selectivity where specified by the relevant medium-performance protocol.

The required strains, acceptance criteria, and performance checks vary by medium. Laboratories should consult the applicable edition of ISO 11133 and the manufacturer's instructions rather than applying one set of organisms or criteria to every medium.

Where Can Listeria Testing Results Go Wrong?

Understanding common testing errors helps laboratories troubleshoot unexpected results and maintain the reliability of Listeria monocytogenes analysis.