Rapid Detection and Enumeration based on ATP Bioluminescence

General ATP Bioluminescence

Rapid detection of microbial contamination is challenging and demands highly sensitive and reliable technologies. An alternative approach to monitoring bacterial contamination is to use the Adenosine Triphosphate (ATP) bioluminescence technology. ATP is generally accepted as an indicator of cell viability and is also a universal energy molecule stored in all microorganisms.

Methods based on ATP-bioluminescence have been developed and widely used in the food industry to monitor surface contamination and to detect contaminants in drinking water or beverages. The reaction involves ATP, luciferin, luciferase, magnesium and oxygen. Chemically, ATP transfers its energy to the luciferin and forms luciferyl adenylate. The luciferyl adenylate is then oxidized by the luciferase in the presence of magnesium ions to form oxyluciferin, carbon dioxide, adenosine monophosphate, pyrophosphates and the reaction emits light.

Applications

The Milliflex Rapid system can be successfully used in all applications where the sample is filterable and the target is an enumeration of the microorganisms, e.g., TAMC and TYMC. The following matrices have been tested or are already in use in the industry:

Pharmaceutical water
Bioburden of in-process or final products
- Antibiotics
- Syrups
- Cell culture clarification and purification intermediates
- Intralipid emulsions
Complex matrices like beverages

It has been demonstrated that even difficult to filter matrices containing a lot of endogenous ATP like mammalian cell cultures (e.g., CHO – cells), can be tested for microbiological contamination. As a strategy for these samples, a differential lysis of the cells is followed by an efficient rinsing of the membrane to remove the ATP that was not related to microbial contamination. Microorganisms that grow on the membrane are not affected by this treatment and can easily be detected by the system. Additionally, the Milliflex Rapid system has been shown to speed up the detection and enumeration of microorganisms when the sample can be delivered in a filterable format, e.g. swabs and dissolvable membranes.

Millipore has proven that the specific detection of P. aeruginosa by a DNA/RNA hybridization of probes linked to a chemiluminescence assay reveals excellent results, either in a single test format or as a confirmation after TAMC detection.

The typical time from the sample preparation to the final result is characterized by an overnight incubation step for environmental non-ATCC microorganisms leading to a time-to-detect at around 16-24 h.

Milliflex Rapid Technology

Figure 1: Workflow of the Milliflex Rapid procedure

The ATP bioluminescence protocol includes the following steps:

1) filter the sample through a Milliflex unit,

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 a Milliflex Rapid Autospray Station. The reaction between the ATP from the 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 (2D) exhibiting spots that represent colonies and in three dimensions (3D) with peaks that correlate with the ATP content of the colony (Figure 2) . The result is analyzed by a 21CFR part 11 - compatible software and directly displayed in CFUs on the screen.

Figure 2: Typical result of a Milliflex Rapid Analysis displayed in 2D (1) or 3D (2).
Here: mixed bacterial contamination in a cell culture.

Validation Strategy of Milliflex Rapid System

The validation of the Milliflex Rapid system at Millipore was based on the available literature at the time of development, e.g. PDA Technical Report #33 and drafts of the chapters USP <1223> and EP 5.1.6. The validation criteria accuracy, precision, specificity, limit of detection, limit of quantification, linearity, range, ruggedness and robustness have been qualified in-house and are available for customer use as a validation summary. Customers can decide to take results from these experiments or to verify them by additional testing.

Millipore provides a complete service package around this product, including IQ/OQ protocols, onsite validation services and method development. Additionally, repair and maintenance contracts and services are available to support the implementation at customer site.

The key point during validation of the method is to show the comparability of the results with routine samples on a statistical basis.