Diamidex: 032201: Legionella pneumophila in water by fluorescence tagging

Summary
Analytes
Revision
Data and Sites

Official Method Name

Enumeration of Legionella pneumophila in sanitary waters and cooling tower waters

Current Revision

Original

Media

WATER

Instrumentation

Fluorescence

Method Subcategory

Microbiological

Method Source

Diamidex

Citation

Passot, F., Peslier, S., Benzinger, Jr., M.J., Blackburn, J., Thompson, W., Bastin, B., Dumont, A., Dukan, S., 2023, "Validation of MICA Legionella for Enumeration of Legionella pneumophila in Sanitary Waters and Cooling Tower Waters: AOAC Performance Tested Method^SM 032201", Journal of AOAC INTERNATIONAL, 106(3): 725–736.

Brief Method Summary

The original water sample is concentrated by membrane filtration. The membrane is then laid over a drop of culture supplement on a standard selective Legionella agar plate (GVPC). This culture supplement contains a specific molecule that will be internalized by growing L. pneumophila and integrated in their O-antigen on the surface of the cells. After 48h of bacterial growth, the membrane is transferred onto a drop of tagging solution containing a fluorescent molecule that will bind by click chemistry specifically onto the labelled L. pneumophila. This specific fluorescent tagging allows the CFU to be detected at the microcolony stage using the MICA microcolony counter. The MICA Legionella AI analyzer then uses multiple parameters to specifically identify L. pneumophila microcolonies and gives a result as a concentration of L. pneumophila in the original sample.

Scope and Application

This method detects and enumerates Legionella pneumophila in domestic and cooling tower waters.

Applicable Concentration Range

1 - 2,000,000 CFU/L

Interferences

The sensitivity of MICA Legionella is 99.7% and the specificity is 96.2%.

Quality Control Requirements

Sample Handling

As recommended in ISO 19458:2006 (Sampling for microbiological analysis)

Maximum Holding Time

2 days

Relative Cost

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Legionella pneumophila(ITIS-962877) Legionella pneumophila	1.000 CFU/L	106% Rec (SL)	0.05 RSD (SL)	3	6	1000.00 CFU/L

Precision Descriptor Notes:	Method developer studies were conducted in the laboratories of Diamidex, and included the inclusivity/exclusivity study, matrix studies for all claimed matrixes, product consistency and stability studies, and robustness testing. The results of MICA Legionella were compared with that of the standard reference method ISO 11731:2017 on two different matrixes: domestic hot water and cooling tower water. MICA Legionella and ISO 11731:2017 analyses were both started on the day of the inoculation. Five test portions of each contamination level and of the uncontaminated matrixes were tested with both methods. The independent laboratory study was conducted by Q Laboratories (MicroVal expert lab, accredited ISO 17 025) and included a matrix study for cooling tower water. The matrix was artificially contaminated with Legionella pneumophila serogroup 1 ATCC 33152, originally isolated from a human, at a range of target concentrations. Accuracy and precision were taken from the medium artificial contamination density in domestic hot water (approximately 10,000 CFU/L).
Detection Level Note:	Detection limit is 1 CFU/L (for 1 liter filtrated). The MICA counter detects all microcolonies present on the membrane.

Precision Descriptor Notes:

Method developer studies were conducted in the laboratories of Diamidex, and included the inclusivity/exclusivity study, matrix studies for all claimed matrixes, product consistency and stability studies, and robustness testing. The results of MICA Legionella were compared with that of the standard reference method ISO 11731:2017 on two different matrixes: domestic hot water and cooling tower water. MICA Legionella and ISO 11731:2017 analyses were both started on the day of the inoculation. Five test portions of each contamination level and of the uncontaminated matrixes were tested with both methods. The independent laboratory study was conducted by Q Laboratories (MicroVal expert lab, accredited ISO 17 025) and included a matrix study for cooling tower water. The matrix was artificially contaminated with Legionella pneumophila serogroup 1 ATCC 33152, originally isolated from a human, at a range of target concentrations. Accuracy and precision were taken from the medium artificial contamination density in domestic hot water (approximately 10,000 CFU/L).

Detection Level Note:

Detection limit is 1 CFU/L (for 1 liter filtrated). The MICA counter detects all microcolonies present on the membrane.

Revision	PDF/Link
Original

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Diamidex: 032201: Legionella pneumophila in water by fluorescence tagging

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