Field Epidemiology Manual Wiki

Methods for assessing environmental cleanliness

Last modified at 4/21/2016 7:19 PM by Vladimir Prikazsky

1. Introduction.

1.1 Methods for assessing environmental cleanliness.

Assessing environmental cleanliness is a key point in the management of infectious risk in healthcare settings. Its objective is to evaluate the effectiveness of the interventions aimed at reducing the microbial contamination on surfaces, being an useful means to support quality improvement activities.

Methods for assessing environmental cleanliness can be divided into two types: methods evaluating only the process (Visual inspection, Fluorescent marker); and methods that evaluate the outcome, measuring directly the microbial contamination or measuring the Adenosine triphosphate (ATP), therefore demonstrating the effectiveness of cleaning and disinfection procedures.

  • Visual inspection is based on direct observation of the environmental surfaces. It is easy to implement and can be used for large areas to assess all surfaces for gross deficiencies; however, it does not assess bioburden and is subjective so that its accuracy is variable. Visual inspection directly addresses also patient perception of cleanliness which is an increasingly important element of patient satisfaction.
  • Fluorescent marker is based on the use of transparent gel, visible only using a UV lamp, which dries quickly on surfaces after application and is easily removed with light cleaning. The gel is applied to surfaces before cleaning, and checked after cleaning, providing an immediate feed-back on performance. This method, as the visual inspection, does not assess bioburden; however can provide a more standardised approach to process evaluation compared to visual inspection.
  • Microbiological evaluation of surfaces provides the most accurate indication of infection risk; it allows the isolation, identification and typing of the viable microorganisms. Microbial sampling of surfaces can be performed by using a contact device (RODAC plates, dip slides, nitrocellulose membranes) or by application of a swabbing technique (swabs, sponges, wipes).
  • The Adenosine triphosphate (ATP) bioluminescencemethod relies on the measurement of ATP, as a marker for organic material. ATP is quantified by measuring the light produced through its reaction with the firefly luciferase using a luminometer. Light output from the reaction is proportional to the amount of ATP present and is measured in relative light units, or RLUs.


  1. Guidelines for environmental infection control in health-care facilities. recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Atlanta: U.S. Department of Health and Human Services; 2003.

  2. Carling P. Methods for assessing the adequacy of practice and improving room disinfection. Am J Infect Control. 2013 May;41(5 Suppl): S20-5.

  3. Mitchell, B, Wilson, F, Ware C, Brown S, McGregor, A, Dancer, S. Evaluating environmental cleanliness in hospitals and other healthcare settings. What are the most effective and efficient methods to use?. Hobart: Department of Health and Human Services; 2012.


Microbial evaluation of surfaces

Adenosine triphosphate (ATP) bioluminescence method

1. Microbiological evaluation of surfaces.

Microbiological evaluation of surfaces may be performed by contact devices (RODAC plates, replicate organism detection and counting, dipslides, nitrocellulose membranes) or by application of a swabbing technique (swabs, sponges, wipes). Each method has advantages and disadvantages, therefore, it is essential to acquire the basic knowledge to make appropriate choices. Standards for microbial contamination of surfaces in healthcare settings have been proposed; however, there is a lack of generally accepted methods and microbiological standards.

Contact devices allow biological particles to be transferred from the surface to be sampled by direct contact to the nutrient medium, if RODAC plates or dip slides are used, or to the nitrocellulose membrane, which is then transferred to a Petri dish containing the culture medium. The resulting colonies give a mirror-image “map” of the original viable units. The contact device is pressed on to the surface to be sampled and incubated directly after sampling. In order to standardize weight and time, for RODAC plates, the RODAC-Weight sampler is available.

The results are usually expressed as cfu/cm2 or cfu/dm2, but can also be expressed as cfu/plate.

Contact devices are simple to use and can be standardized. They are not suitable for heavily contaminated surfaces because colony overgrowth makes enumeration difficult; only a limited area can be sampled and generally they are mainly suitable for flat surfaces; as nitrocellulose membranes or dip slides are flexible they may be used for curved surfaces.

The use of swabbing techniques is particularly convenient for sampling large, irregular or recessed surfaces not accessible to contact devices and are useful in case of heavily contaminated surfaces. The swab is pre-moistened with a sterile rinse medium and then stroked in close parallel sweeps over the defined sampling area, while being slowly rotated. It is then placed in a specified amount of rinse liquid which is assayed for viable units. A template may be used to quantify accurately the area to be sampled. Sponges and wipes are particulary suitable for sampling large areas.

Nitrocellulose membranes, as settle plates, exposed for a known period, can be used In order to evaluate the rate at which microorganisms are falling on a specific surface.

A disadvantage of microbial cultures is the time required to obtain the results, due to the need of incubating the culture media. The use of molecular methods can help in reducing the time, resulting in the quicker implementation of infection prevention and control activities.

Microbiological evaluation of surfaces in a healthcare setting is mainly indicated to demonstrate effectiveness of cleaning and disinfection procedures, as part of an outbreak investigation, to identify potentially hazardous environmental conditions, for research, for education purposes, for quality assurance purposes. The process should include a written protocol, analysis and interpretation of results and expected actions based on the results obtained.

2. Adenosine triphosphate (ATP) bioluminescence method.

The Adenosine triphosphate (ATP) bioluminescence method relies on the measurement of ATP, which is the basic energy component of all cells and is present in all microorganisms and organic residues; therefore, detection of this molecule would indicate that organic material is still present on that substrate.

The surface to be tested is sampled by using a specialized swab, which is then placed in a portable handheld luminometer that uses the firefly enzyme and substrate luciferase and luciferin, respectively, to catalyse a reaction with ATP. When ATP reacts with luciferin in the presence of luciferase, it produces a release of energy in the form of light, called bioluminescence. Light output from the reaction is proportional to the amount of ATP present which can be measured with a luminometer and expressed as relative light units (RLUs). The results can be delivered within about 20 seconds. Benchmark values have been proposed (from 100 RLU, very clean surfaces; 500 RLU clean surfaces); however, difficulties emerge considering that a great variability in results obtained by using different luminometers has been observed.

ATP results cannot be considered as indicators of microbial contamination, considering also that total amount of ATP, both microbial (including dead bacteria) and non-microbial (e.g. organic debris) is quantified. A scarse correlation between microbial contamination and RLU has been demonstrated in healthcare settings.


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  4. Pitzurra M, Savino A, Pasquarella C. Microbial Environmental Monitoring. Ann Ig 1997;9:439-54.

  5. Standard di Sicurezza e di Igiene del Lavoro nel Reparto operatorio. Roma; Istituto Superiore per la Prevenzione e la Sicurezza sul Lavoro: 2009.

Original contribution from:

Cesira Pasquarella, Department of Biomedical, Biotechnological and Translational, Sciences, University of Parma.