**Use of ATP Bioluminescence for Rapid Detection and Enumeration of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System**

Renaud Chollet and Sébastien Ribault *Merck-Millipore France* 

#### **1. Introduction**

98 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

Skrabanek L, Murcia M, Bouvier M et al. (2007) Requirements and ontology for a G proteincoupled receptor oligomerization knowledge base. *BMC Bioinformatics*, 8: 177. Terrillon S, Bouvier M. (2004) Roles of G-protein-coupled receptor dimerization. *EMBO Rep*,

Urizar E, Yano H, Kolster R, Gales C, Lambert N, Javitch JA. (2000) CODA-RET reveals

Veatch W, Stryer L. (1977) The dimeric nature of the gramicidin A transmembrane channel:

Vrecl M, Drinovec L, Elling C, Heding A. (2006) Opsin oligomerization in a heterologous cell

White JH, Wise A, Main MJ et al. (1998) Heterodimerization is required for the formation of

Xu Y, Kanauchi A, von Arnim AG, Piston DW, Johnson CH. (2003) Bioluminescence

Xu Y, Piston DW, Johnson CH. (1999) A bioluminescence resonance energy transfer (BRET)

Zacharias DA, Baird GS, Tsien RY. (2000) Recent advances in technology for measuring and

system. *Journal of Receptors and Signal Transduction*, 26: 505-526.

a functional GABA(B) receptor. *Nature*, 396: 679-682.

manipulating cell signals. *Curr Opin Neurobiol*, 10: 416-421.

*Methods Enzymol*, 360: 289-301.

21-30.

5: 30-34.

113: 89-102.

96: 151-156.

630.

recruitment to G protein-coupled receptor heteromers. *Assay Drug Dev Technol*, 9:

functional selectivity as a result of GPCR heteromerization. *Nat Chem Biol*, 7: 624-

conductance and fluorescence energy transfer studies of hybrid channels. *J Mol Biol,*

resonance energy transfer: monitoring protein-protein interactions in living cells.

system: application to interacting circadian clock proteins. *Proc Natl Acad Sci U S A*,

Rapid microbial detection becomes increasingly essential to many companies in pharmaceutical, clinical and in food and beverage areas. Faster microbiological methods are required to contribute to a better control of raw materials as well as finished products. Rapid microbiological methods can also provide a better reactivity throughout the manufacturing process. Implementing rapid technologies would allow companies for cost saving and would speed up products release. Despite clear advantages, traditional methods are still widely used. Current methods require incubation of products in liquid or solid culture media for routinely 2 to 7 days before getting the contamination result. This necessary long incubation time is mainly due to the fact that stressed microorganisms found in complex matrices require several days to grow to visible colonies to be detected. Moreover, this incubation period can be increased up to 14 days in specific application like sterility testing for the release of pharmaceutical compounds. Although these techniques show advantages like simplicity, the use of inexpensive materials and their acceptability to the regulatory authorities, the major drawback is the length of time taken to get microbiological results. Thus, face to the growing demand for rapid detection methods, various alternative technologies have been developed. In the field of rapid microorganisms detection, ATPbioluminescence based on luciferine/luciferase reaction has shown great interest. Indeed, adenosine triphosphate (ATP) is found in all living organisms and is an excellent marker for viability and cellular contamination. Detection of ATP through ATP-luminescence technology is therefore a method of choice to replace traditional method and significantly shorten time to detection without loosing reliability.

This chapter will address the ATP-bioluminescence principle as a sensitive and rapid detection technology in the Milliflex Rapid Microbiology Detection and Enumeration System (RMDS). This system combines membrane filtration principle, detection of microorganisms by ATP-bioluminescence and light capture triggered by a Charged Coupled Device camera (CCD) followed by software analysis.

Use of ATP Bioluminescence for Rapid Detection and Enumeration

**2.3 ATP-Bioluminescence applications** 

et al., 1992; Yan et al., 2011).

**3.1 System description** 

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 101

N-terminal domain affects bioluminescence color by modulating slightly the polarity of the active site environment (Hosseinkhani, 2011; Shapiro et al., 2005). This interesting feature

With the isolation, cloning and purification of various luciferases from many bioluminescence-producing organisms (bacteria, beetles, marines organisms, etc), bioluminescent assays have been developed and widely used in microbiology to detect bacterial contamination by measuring presence of ATP and in molecular and cellular biology with luciferase as reporter gene to monitor gene expression, protein-protein interaction, etc (Francis et al., 2000; Roda et al, 2004; Thorne et al., 2010). The average intracellular ATP content in various microorganisms has been quantified and ATP has been shown to be a reliable biomarker of the presence of living organisms (Kodata et al., 1996; Thore et al., 1975; Venkateswaran et al., 2003). To be able to specifically detect living organisms by ATP-bioluminescence, the first step is to extract ATP from cells. This step is critical and impacts directly the reliability of the detection (Selan et al., 1992). Chemical solution or physical extraction methods were used in liquid samples (Selan et al., 1992; Siro et al., 1982). Some false negative results were described in few studies (Conn et al., 1975; Kolbeck et al., 1985). Additional studies investigated the cause of false negative results and demonstrated that ATP extraction was not efficient. Indeed, extensive sonication of bacterial samples for instance caused a significant increase of Relative Light Unit (RLU) measured (Selan et al., 1992). Taking into account this limitation, ATP-bioluminescent assay has already proved to provide good detection properties in many areas. Bioluminescent assay is broadly used to monitor air and surface cleanliness and product quality mainly in food industries and in less extent in pharmaceutical industries (Aycicek et al., 2006; Bautisda et al., 1995; Davidson et al., 1999; Dostalek & Branyik, 2005; Girotti et al., 1997; Hawronskyj & Holah, 1999). Studies shows that the level of contamination assessed though surface swabbing, ATP extraction and bioluminescent assay correlate well for 80 % of the samples tested with traditional plate method (Poulis et al., 1993). Availability of sensitive luminometers as well as many commercial ATP-bioluminescent kits has allowed the development of various protocols and applications in industrial microbiology. Currently, ATP- bioluminescence is an accepted and common technology used to monitor contamination in areas such as food and beverage, ecology, cosmetic, and clinical (Andreotti & Berthold, 1999; Chen & Godwin, 2006; Davidson et al., 1999; Deininger & Lee, 2001; Frundzhyan & Ugarova, 2007; Miller et al., 1992; Nielsen & Van Dellen, 1989; Selan

opens the way to wide applications in biotechnology (Branchini et al., 2005).

**3. Milliflex rapid microbiological detection and enumeration system** 

RMDS offers a way to detect and quantify living microorganisms grown on a membrane. By combining ATP-bioluminescence and sensitive detection system, the microbial detection is obtained more rapidly than traditional method. In order to detect a colony or a micro-colony on a membrane by ATP-bioluminescence, the first step is to release ATP from cells. This critical step is achieved by nebulizing automatically an ATP-releasing solution onto the
