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

### **3.1 System description**

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

Use of ATP Bioluminescence for Rapid Detection and Enumeration

**3.2 RMDS ATP-Bioluminescence protocol** 

Fig. 4.

after incubation.

**microorganisms** 

evaluated.

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 103

Fig. 3. Example of image given by RMDS software. Picture show the image of the membrane with spots (A) or peaks in 3 dimensions (B) representing exactly the place on the membrane where light is emitted. The result in colony forming unit is directly given by the system.

A B

1. filter the sample through a Milliflex funnel; 2. incubate the sample onto media; 3. separate the membrane from the media and let the membrane dry inside a laminar flow hood; 4. spray the ATP-releasing reagent and bioluminescence reagent onto the membrane by means of the Milliflex Rapid Autospray Station. The reaction between the ATP from microorganisms and the luciferase enzyme produces light; 5. place the membrane onto the detection tower and initiate detection and enumeration. Photons are detected by the system via a photon counting imaging tube coupled to a CCD camera. The photons generated by the ATP bioluminescence reaction are captured, and the integrated picture is displayed on the computer monitor; 6. after data treatment, a picture of the membrane is provided in two dimensions (2-D) exhibiting spots that represent colonies and in three dimensions (3-D) with peaks that correlate with the ATP content of the colony. The result is directly displayed in colony-forming unit (cfus)on the software screen. The successive steps are summarized in

1. filter the sample through a Milliflex funnel; 2. incubate the sample and visually count cfus

**3.3 Evaluation of Luciferin/Luciferase relative concentrations for optimal detection of** 

The relative concentrations of the 2 key components of the detection reagents were

The RMDS ATP-bioluminescence protocol includes the following steps:

The standard protocol, performed in parallel, includes the following steps:

membrane. ATP extraction is made on microcolonies grown on membrane which represents an advantage compared to chemical or physical extraction in liquid. Once ATP is released from lysed cells, it becomes accessible to bioluminescent reaction. A second solution is then automatically nebulized onto the same membrane. This solution brings to lysed cells all components, except ATP, involved in the Luciferin/Luciferase bioluminescence chemical reaction. A spray station is used to uniformly apply small volumes of reagents onto the membrane. As soon as bioluminescent reagents are sprayed onto the membrane, the bioluminescence reaction starts and photons are emitted. The membrane is then transferred manually from the spray station to the detection system. The Milliflex Rapid detection system combines the use of a highly sensitive CCD camera to monitor light emitted from microorganisms and an image analysis software to analyze the signal and give the number of microorganisms counted. The figure 2 shows the detection tower components and their function.

Fig. 2. Milliflex detection tower components: RMDS collects, amplifies, and registers on a CCD camera the light activity of bioluminescent reaction. Photons emitted by microorganisms go through the tapered fiber in order the light to be concentrated and becomes compatible with the size diameter of the CCD camera. In the intensifier, photons hit a photocathode and each photon is converted into cloud of electrons. Then electrons hit a phosphorous screen and are converted back into photons. The CCD camera records light every 30 times per second.

Data collected by the CCD camera are analyzed and treated by software to build an image of the membrane loaded on the top of the detection tower. The image indicates the place where light is emitted. As the signal is collected over a short period (integration time), spots size on the picture represents the light intensity accumulated or emitted by microorganisms (Fig.3).

Fig. 3. Example of image given by RMDS software. Picture show the image of the membrane with spots (A) or peaks in 3 dimensions (B) representing exactly the place on the membrane where light is emitted. The result in colony forming unit is directly given by the system.

## **3.2 RMDS ATP-Bioluminescence protocol**

102 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

membrane. ATP extraction is made on microcolonies grown on membrane which represents an advantage compared to chemical or physical extraction in liquid. Once ATP is released from lysed cells, it becomes accessible to bioluminescent reaction. A second solution is then automatically nebulized onto the same membrane. This solution brings to lysed cells all components, except ATP, involved in the Luciferin/Luciferase bioluminescence chemical reaction. A spray station is used to uniformly apply small volumes of reagents onto the membrane. As soon as bioluminescent reagents are sprayed onto the membrane, the bioluminescence reaction starts and photons are emitted. The membrane is then transferred manually from the spray station to the detection system. The Milliflex Rapid detection system combines the use of a highly sensitive CCD camera to monitor light emitted from microorganisms and an image analysis software to analyze the signal and give the number of microorganisms counted. The figure 2 shows the detection tower components and their

Fig. 2. Milliflex detection tower components: RMDS collects, amplifies, and registers on a

Data collected by the CCD camera are analyzed and treated by software to build an image of the membrane loaded on the top of the detection tower. The image indicates the place where light is emitted. As the signal is collected over a short period (integration time), spots size on the picture represents the light intensity accumulated or emitted by microorganisms (Fig.3).

microorganisms go through the tapered fiber in order the light to be concentrated and becomes compatible with the size diameter of the CCD camera. In the intensifier, photons hit a photocathode and each photon is converted into cloud of electrons. Then electrons hit a phosphorous screen and are converted back into photons. The CCD camera records light

CCD camera the light activity of bioluminescent reaction. Photons emitted by

function.

every 30 times per second.

The RMDS ATP-bioluminescence protocol includes the following steps:

1. filter the sample through a Milliflex funnel; 2. incubate the sample onto media; 3. separate the membrane from the media and let the membrane dry inside a laminar flow hood; 4. spray the ATP-releasing reagent and bioluminescence reagent onto the membrane by means of the Milliflex Rapid Autospray Station. The reaction between the ATP from microorganisms and the luciferase enzyme produces light; 5. place the membrane onto the detection tower and initiate detection and enumeration. Photons are detected by the system via a photon counting imaging tube coupled to a CCD camera. The photons generated by the ATP bioluminescence reaction are captured, and the integrated picture is displayed on the computer monitor; 6. after data treatment, a picture of the membrane is provided in two dimensions (2-D) exhibiting spots that represent colonies and in three dimensions (3-D) with peaks that correlate with the ATP content of the colony. The result is directly displayed in colony-forming unit (cfus)on the software screen. The successive steps are summarized in Fig. 4.

The standard protocol, performed in parallel, includes the following steps:

1. filter the sample through a Milliflex funnel; 2. incubate the sample and visually count cfus after incubation.

#### **3.3 Evaluation of Luciferin/Luciferase relative concentrations for optimal detection of microorganisms**

The relative concentrations of the 2 key components of the detection reagents were evaluated.

Use of ATP Bioluminescence for Rapid Detection and Enumeration

Formulation 1

Formulation 2

Formulation 3

Formulation 4

Formulation 5

**3.4 ATP background removal** 

of Contaminants: The Milliflex Rapid Microbiology Detection and Enumeration System 105

*E. coli S. aureus* 

Table 2. RMDS results obtained with the 5 formulations of Luciferin/Luciferase tested

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

Fig. 4. RMDS ATP-bioluminescence protocol

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 concentrations as described in table 1.


Table 1. Formulations relative concentrations of Luciferin/Luciferase tested

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 increase signal on background ratio.

Table 2. RMDS results obtained with the 5 formulations of Luciferin/Luciferase tested
