**3.4 ATP background removal**

104 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

The protocol used is described in the previous paragraph "RMDS ATP bioluminescence protocol". Only the reagent used for detection varies for the 2 components relative

growth medium Imaging

Results in CFUs 90s

Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5

The signal and background were determined using membranes incubated during 6h at 32.5°C on Tryptic Soy Agar inoculated with *Escherichia coli* or *Staphylococcus aureus* (table 2). Formulation 1 gave a signal so strong that the detection system was almost saturated. This saturation did not allow the accurate detection of bacteria on the membrane. The same issue occurred to a weaker extent using formulation 2. On the other hand, while the detection of *S. aureus* was accurate using formulation 5, the signal was too weak to allow all colonies of *E. coli* to be counted. Formulations 3 and 4 were both able to generate a good signal associated with low background. We can conclude from these results that the luciferin and luciferase concentration can be increased to optimize the signal but also that the balance between the 2 components is key. Signal will be increased while increasing concentrations but background as well. Formulation 3 which benefits from the best signal on background ratio has been used during the rest of the studies presented here. It is noticeable that depending on the application, the type of sample tested and the resulting background, this luciferase to luciferin balance can be adjusted to better match the detection criteria and

Luciferase 3x 1.5x 1x 1.5x 1x

Luciferin 1x 1x 1x 0.5x 0.5x

Table 1. Formulations relative concentrations of Luciferin/Luciferase tested

Incubation on

Spraying Membrane

Fig. 4. RMDS ATP-bioluminescence protocol

concentrations as described in table 1.

Sample

Filtration via Milliflex

increase signal on background ratio.

One advantage to use an ATP bioluminescent assay to detect microorganisms is that ATP is present in all living organisms and is an excellent and sensitive biomarker of contamination. However this advantage can become an issue when non microbial or extracellular ATP is detected, generating bioluminescent background and preventing a reliable detection. Extracellular ATP is usually found either in culture media or in products containing eukaryotic cells. In both cases, the presence of unwanted ATP generates an overestimation of the contamination and impacts negatively the sensitivity of the ATP-bioluminescent assay. Two approaches are commonly used to remove extracellular ATP: enzymatic treatment to cleave ATP and lysis treatment to selectively lyse non bacterial cells. Methods including a treatment with ATP dephosphorylating enzymes such as apyrase or adenosine

Use of ATP Bioluminescence for Rapid Detection and Enumeration

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 107

A

B

Fig. 6. A) RMDS analysis of 1mL of Chinese Hamster Ovary cells at 106cell/mL. Eukaryotic ATP content generates a high bioluminescent background. B) RMDS analysis of a sample containing Chinese Hamster Ovary cells at 106cell/mL contaminated with *E. coli* pretreated with a mammalian cells lysis solution and with apyrase. The sample pretreatment induces

Water is a key raw material utilized in the manufacturing of products within the food and beverage, healthcare, microelectronics and pharmaceutical industries. Within each industry, different regulatory requirements exist for microbial contamination in the water used for the manufacturing of a product for a specific application. The microorganisms found in these water systems are mainly stressed, slow-growing strains characterized by long incubation times before growth can be detected using traditional microbiology methods such as membrane filtration or pour plates. The time it takes before contamination can be detected in water can cause delays in product release, and extend the storage time of products. Using a rapid Bioluminescence based detection method allows manufacturers to identify microbial

ATP background removal allowing contaminant detection.

**3.5.1 Use of Bioluminescence for microorganisms detection in water** 

**3.5 RMDS applications** 

phosphatase, have been described and used to remove efficiently ATP (Askgaard et al., 1995; Thore et al., 1975). Combination of apyrase and adenosine phosphate deaminase showed a good reduction of extracellular ATP and was applied to successfully detect E. coli and *S. aureus* in media broth and biological specimens (Sakakibara et al., 1997). When the objective of the assay is to detect and quantify bacterial contamination from a mixed population containing eukaryotic cells and bacteria, a differential lysis can be applied to selectively remove eukaryotic cells from the sample. This approach was used to separate bacterial ATP from biological fluids by lysing somatic cells with detergent as Triton X 100 at low concentration and combining this step with an enzymatic degradation of ATP released from lysed cells (Chapelle et al., 1978). RMDS protocol is based on sample filtration through membrane which naturally helps to eliminate extracellular ATP. If background ATP remains after filtration, rinsing the membrane with physiological serum or sterile water contributes to removal of residual ATP and allows bacterial detection. The figure 5 shows the impact of adding rinsing steps to reduce background on beverage products.

Fig. 5. Example of 2D and 3D views given by RMDS software for flavored water analysis with and without rinsing with sterile water. Picture A shows light spots corresponding to ATP present naturally in the filtered sample. Picture B shows the impact of rinsing water to remove background.

A protocol was developed to use RMDS to detect and quantify bacterial contamination from a mixture of mammalian cells and bacteria. The filtration of mammalian cells and bioluminescence detection through RMDS protocol shows (see Fig.6A) a high amount of light produced by mammalian cells preventing any bacterial detection. The sample treatment with a combination of a mammalian cells lysis solution and with apyrase contributes to efficiently remove the bioluminescent background and the figure 6B demonstrates that light spots remain detectable. These spots correspond to light emitted by bacteria in the mixture. Results obtained show that ATP-bioluminescent assay could be a powerful tool to microbiologically and quickly monitor eukaryotic cell cultures.

Fig. 6. A) RMDS analysis of 1mL of Chinese Hamster Ovary cells at 106cell/mL. Eukaryotic ATP content generates a high bioluminescent background. B) RMDS analysis of a sample containing Chinese Hamster Ovary cells at 106cell/mL contaminated with *E. coli* pretreated with a mammalian cells lysis solution and with apyrase. The sample pretreatment induces ATP background removal allowing contaminant detection.
