Microbial monitoring

• Surfaces should be cleaned, disinfected, and dry before swabbing.
• Sterile templates can help define the sampling area, especially useful when starting environmental swabbing.
• Microbiological tests are either:
  • Qualitative – presence/absence (used for pathogen detection)
  • Quantitative – number of bacterial cells e.g. cfu/cm² (used for indicator organisms)
• Quantitative tests are commonly used to assess cleaning and sanitation efficiency.
• Where microbiological testing is impractical or prohibitively expensive rapid test kits might be a suitable alternative for microbial monitoring

Microbial monitoring in food production areas is essential for verifying hygiene and preventing contamination. Swabbing is the primary method used to assess surface cleanliness and detect the presence of microorganisms on equipment, workspaces, and other contact surfaces. This approach provides a practical means of environmental sampling, supporting routine verification of cleaning procedures. In some cases, rapid detection kits offer a quicker, although less comprehensive, alternative to traditional microbiological testing.

Before choosing a testing method, consider the organism of interest and the purpose of testing.

I.  Qualitative testing

Reports whether a target organism is present or absent. Often used for detecting pathogens. Due to their rarity, pathogen tests rely on enrichment techniques, e.g. special growth media to enhance target organism growth while suppressing non-target microbes. Because multiplication rates are unknown, results are binary, i.e. presence/absence.

Pathogen testing typically requires larger sample areas, often using sponges rather than small swabs (1).

II. Quantitative testing

Provides numerical counts of bacterial cells and is commonly used to verify cleaning and disinfection effectiveness. It typically involves indicator organisms, such as aerobic mesophiles, faecal coliforms, or Enterobacteriaceae. Indicator tests help assess the hygienic status of surfaces. The Total Count of Aerobic Mesophiles (TCAM), also known as Total Viable Count (TVC), is widely used and cost-effective.

Table 1: Common indicator bacteria and their significance

Under Regulation (EC) 2073/2005 Article 5(2): "Food business operators manufacturing ready-to-eat foods, which may pose a Listeria monocytogenes risk for public health, shall sample the processing areas and equipment for Listeria monocytogenes as part of their sampling scheme." This may be a consideration for some fresh produce. Further, Regulation (EC) 852/2004 Chapter III Section 2(c) requires: "Adequate provision is to be made for the cleaning and, where necessary, disinfecting of working utensils and equipment.". Indicator testing may be used to validate the cleaning and disinfection protocol. 

While no specific numerical criteria for indicator organisms exist in the fresh produce sector, guidance values from other food sectors may assist in setting internal hygiene targets (table 2).

Table 2: Hygiene Benchmarks for Environmental Surfaces (Adapted from Griffiths, 2016 (2))

Swabbing should be conducted on flat, smooth, dry surfaces that have already been cleaned and disinfected. While no legally mandated surface area exists for environmental sampling, the Food Standards Agency recommends at least 20 cm².

To help standardise sampling, sterile templates are often used. These may be single use irradiated plastic, or reusable steel, sterilised by autoclave. Common template sizes include 50 cm² and 100 cm², although larger areas may be swabbed when testing for pathogens6. For instance, Listeria monocytogenes is commonly sampled over 1,000 cm² on food contact surfaces (1).
 

A universally accepted protocol for swabbing surfaces does not exist, though there are some standard methods. Wet-dry swabbing is well established as a method for the collection of environmental surface samples and widely used in the food processing industries. The following wet-dry swabbing method is widely accepted and was authorised by the Food Standards Agency as compatible with the 2002 EU Meat HACCP regulations (superseded by Regulation 2073/2005).

Required equipment:

Image: Swabs, latex gloves, alcohol wipes.

Sampler wears gloves to prevent contamination from the sampler’s skin and falsify the bacterial count. Gloves are disinfected with an alcohol wipe.

If using single use templates, it must be ensured that each template is only used once, if steel multiple use templates are utilised, they should ideally be autoclaved or gamma-irradiated, which might be cost-prohibitive and/or impractical for most food businesses. If reusing multiple-use templates in a sampling session, disinfection with fresh alcohol wipes between uses is required. Any cutting tools must also be sterilised.

After sterilisation and/or between samples place sampling tools on fresh alcohol wipes to avoid contamination. Ensure tools are visibly dry before use.

Use two sterile swabs per sample: one wet (pre-moistened with neutralising agent – see below), one dry. The wet swab provides a liquid phase for bacteria to move into; the dry swab then collects the bacterial solution. While swabbing the sterilised template should be firmly held in place and the swabs should be rolled between the fingers to rotate them while swabbing. 
 

Apply the wet swab within the template area, rubbing in three directions: horizontal, vertical, and diagonal.

The wet swab is held (not place down anywhere); then use the dry swab to absorb residual moisture, rubbing in the same three directions.

Cut the ends of both swabs into a labelled tube containing peptone salt solution (also called Maximum Recovery Diluent or MRD).

To avoid the potential effects of sanitising chemicals on the sampling surface which could kill collected bacteria and lead to inaccurate results, neutralisers should be used. One of the most effective is Dey Engley (D/E) broth (also called Dey Engley neutralising broth - DENbroth), a broad-spectrum neutraliser. The effectiveness of common neutralisers in shown below (table 3). 

Table 3: Effectiveness of neutralisers against disinfectants

Squeeze excess liquid from the swab into the tube. If the neutraliser supports bacterial growth, ensure re-cleaning of the swabbed area after sampling to prevent it becoming a fomite.

Store samples in an insulated cool box with frozen packs or crushed ice. Maintain temperature between 0°C and 4°C (cold but not frozen)

Keeping samples cold prevents growth, keeping samples from freezing prevents cold damage to the cells. Samples should reach the lab within 24 hours to ensure valid results.

Rapid detection kits can serve as a suitable alternative or complement to traditional microbiological testing for monitoring surface hygiene in food production areas. These methods are particularly useful when rapid results, operational flexibility, or cost savings are priorities. Two main types of rapid detection kits are commonly used: ATP detection kits and protein detection kits. ATP (adenosine triphosphate) is a molecule found in all living organisms, including microorganisms. ATP detection kits use a reaction that produces light in the presence of ATP, which is measured using a luminometer, which is a required instrument for this type of test. The amount of light emitted correlates with the level of biological material present, offering a proxy measurement for microbial contamination.

Protein detection kits, by contrast, measure residual protein on surfaces regardless of whether it originates from living or dead organisms. These kits typically rely on a colour-change reaction when protein is present, providing a quick visual indication of surface cleanliness. Depending on the cleanliness of the surface tested this can lead to false positives as the protein detection cannot discriminate the origin of the protein contamination.

Both types of kits have been shown to correlate with traditional microbiological indicators such as total viable counts (TVC), particularly when used before and after cleaning procedures. A comparative study by Moore and Griffith found that results from protein-based rapid tests aligned well with TVC results in various food production environments, supporting their value as reliable hygiene indicators (7). While these methods cannot identify specific pathogens, they are effective for assessing general sanitation standards and verifying the effectiveness of cleaning procedures.