A critical evaluation of sampling methods used for assessing microorganisms on surfaces

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Abstract

Methods used to evaluate the effectiveness of cleaning and disinfection regimes, or of putative antimicrobial surfaces, rely on an estimate of the amount of viable microbial cells remaining on a surface after treatment. In essence, microbial cells are applied to the surface, and the number remaining after a specified time/treatment is assessed by variety of methods. This paper provides a critical commentary on these methods.

The most common method relies on removal of the cells from the surface, by swabbing or agitation, plating onto culture media, and counting the number of colonies obtained. However, the surface should always be subsequently examined for residual cells: low numbers of colonies are deemed indicative of effective cleaning (i.e. few cells on the surface), but they could also indicate that cells have not been removed from the surface. Swabbing efficiency can be affected by moisture at the surface, presence of organic material, surface topography and presence of antimicrobial compounds.

It is important to be aware of the limitations of a given method for assessing the presence of microorganisms on a surface, as well as of the intended antimicrobial property of the surface or agent applied to the surface.

Research highlights

▶ Microorganisms not always removed from surface by swabbing. ▶ More than one method recommended for assessing surface contamination. ▶ Cells attached on surfaces should be visualised microscopically, and/or in situ viability assessed.

Introduction

With high numbers of microorganisms potentially present on surfaces, accurate methods for enumeration are essential. Numerous sampling techniques are available to microbiologists, including swabs, agar contact plates, wipes, tapes, hygiene monitors, dust and bulk sampling as well as microscopy of the surface. Swabbing is a widely used sampling method, but it lacks the standardisation required to provide the level of reproducibility required. The efficiency of swabbing is reliant on the efficiency of the individual carrying out the procedure in three areas: the removal of bacteria from the surface; the removal of bacteria from the swab, and cultivation of bacteria (Moore and Griffith, 2007). In addition, the properties of the surface (topography, wettability, porosity, etc.), and the presence of organic material on the surface can affect the efficiency of swabbing. It should perhaps also be routine to check the surface after swabbing for residual microorganisms.

Alternative or supplementary methods are many, but all have their limitations. Contact plates (agar plates pressed directly onto the test surface) are more successful if selective culture media are used for particular indicator microorganisms on a surface: if surfaces are rough, or wet, then the sampling is inaccurate, or resultant growth on the agar may be confluent (Egington et al., 1995).

Indicators for surface hygiene include ATP bioluminescence sampling (Holah et al., 1989), which requires swabbing, and ultra-violet light irradiation, which enables a simple visual assessment of gross soiling. Neither method discriminates between soil and microorganisms, but the presence of microorganisms raises ATP readings considerably (Whitehead et al., 2008).

It is highly likely that – in the food engineering plant – microorganisms will be present on the surface alongside organic material. This material can affect the efficiency of cleaning and disinfection protocols, and can also provide nutrient, or protection for microorganisms (Verran et al., 2008). However, studies on the effect of organic soil on methods for detecting microorganisms are few. In our laboratories, the use of a differential fluorescent stain accompanied with image analysis has enabled separate assessment of surface coverage (Whitehead et al., 2009), but there have been few comparisons between microscopic and culture methodologies.

This paper investigates some of the variables that affect the efficiency of swabbing, and compares this efficiency with that of alternative or complementary methods. Initially, Staphylococcus aureus was used, since this bacterium is common on the skin and would typically be found on surfaces with minimal additional material (other than sweat); subsequently Listeria monocytogenes was used, mixed with food soil, to provide a scenario more relevant to the food processing environment. Both are Gram positive bacteria, possessing a thick cell wall that may assist survival in adverse conditions.

Section snippets

Swabbing from smooth (glass) surfaces using microbial cell suspensions

Microscope slides provide a transparent, smooth, hard surface, thus facilitating simple methodologies for assessing surface contamination in vitro, and for evaluating the effectiveness of swabbing.

Degreased slides were inoculated with 0.1 ml of 10-fold dilutions of an 18 h S. aureus Oxford strain (NCTC 6571) nutrient broth culture in sterile phosphate buffered saline. In parallel, viable counts were made of the culture, to enable the number of cells inoculated onto the surfaces to be calculated.

Results and discussion

Swabbing has long been used to remove cells from surfaces, and thence as an indirect indication of cells on the surface. Using glass, a smooth, hard transparent surface, swabbing proved efficient (Fig. 1a), and light microscopy revealed that few cells remained on the surface. Recovery was around 30%, reducing as the inoculum dried (Fig. 1b). It is not surprising that cells dried onto a surface are harder to remove, but it is important to recognise that this may well be the case in an industrial

Conclusion

Most methods that are used to indicate the presence of microorganisms on a surface are inevitably limited in some way. Thus the effectiveness of recovery of cells from a surface by swabbing is affected by the efficiency of swabbing, whether the cells are dying/drying on the surface, the topography of the surface, and the presence of other material on the surface. It is important to recognise the limitations of methods when interpreting results, to avoid potentially costly deductive errors.

Acknowledgements

Much of the work presented in this paper was carried out as part of the EU FP6 PathogenCombat project.

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