Attachment 07 - NIH Legionella.1714658485927.docx

Attachment 07: NIH Legionella (ISO 17031 protocol )

Summary This document specifies culture methods for the isolation of Legionella and estimation of their numbers in water samples. Samples collected are incubated, presumptive colonies are confirmed as legionella after morphologically characteristic colonies on the selective culture media are identified by a subculture to demonstrate their growth requirement for L-cysteine and iron (III). Not all legionella species are culturable. Therefore, this method may not recover all species of Legionella.

Sample Collection and Storage:

The Contractor shall collect the samples for legionella analysis.

The Contractor shall provide representative characteristics and account for all vertical, horizontal, and temporal variation. Sampling points where conditions are unstable shall be avoided, and the heterogeneity of the hydraulic system shall be taken into consideration. For routine sampling, sterile bottles shall be used. The volume of the bottles shall be 500 mL or adequate for analysis of all requested parameters. Bottles shall be made of glass or various plastics (polypropylene, polystyrene, polyethylene, polycarbonate). Glass bottles can be re-used, but polyethylene shall be disposed. Adhesion to surfaces can lower the detection of microorganisms. Closures shall be a ground glass or plastic stopper for glass bottles, a plastic press-on lid for plastic bottles or jars, or a plastic or metal screw cap for either. Bottle openings closed with plastic or glass stoppers shall be further protected from contamination by, e.g. aluminum foil.

The sample collector shall be trained in sampling procedures, such as wearing gloves and eye protection when collecting samples. It is important to sample water carefully, taking precaution to avoid contamination during the sampling procedure. Water samples are to be collected into sterile sample bottles containing chlorine neutralizer. Water samples shall be placed on ice and reach the Laboratory as soon as possible after collection, as testing must be completed within 24 hours.

Sample Preservation and Storage Water samples shall be placed on ice and reach the Laboratory as soon as possible after collection, as testing must be completed within 24 hours.

Method of Shipping and Duration and Receiving The Contractor shall place the sample into a cooler with ice for immediate delivery or shipment to the laboratory. The sample shall be received with a chain of custody (COC). The COC must include the name of the sample collector, date and time of sample collection, signature or initial of the sample collector, location of the sample and type of sample to be analyzed.

Analysis Method and Protocol:

Samples collected shall be incubated and presumptive colonies confirmed as legionella after morphological characteristic colonies on the selective culture media are identified by a subculture to demonstrate their growth requirement for L-cysteine and iron.

Please reference ISO 11731:2017, Water quality — Enumeration of Legionella for additional information.

Culture Media and Reagents Chemicals of analytical grade shall be used in the preparation of the culture media and reagents unless otherwise stated.

Prepare culture media with demineralized or distilled water.

Procedure The Contractor shall take care not to expose samples to adverse temperature condition (freezing or overheating) Membrane filtration technique may be used for detection of legionellae from water samples. For highly contaminated samples, dilute and use direct plating before and after pretreatment.

Record the volumes of samples diluted or processed. Concentration techniques shall be performed in samples when the number of legionellae is unknown.

Please reference ISO 11731:2017, Water quality — Enumeration of Legionella for additional information.

Membrane Filtration and Direct Placing of the Membrane Filter on Culture Media:

Water sample shall be filtered through a cellulose nitrate or mixed cellulose esters membrane filter. Some of the samples may be without treatment, or with acid and heat treatment. Acid treatment may be done directly or through a membrane filter in the funnel.

The filtered volume of the sample is recorded. The filtered volume depends on particulate content of the water.

The membrane filter shall be carefully removed with disinfected forceps and placed directly on the culture media to ensure that no air bubbles are trapped underneath.

Membrane Filtration Followed By a Washing Procedure:

Polycarbonate or polyethersulfone membrane filter shall be used to filter the water sample.

The volume of the water sample shall be recorded. Use disinfected forceps to remove the membrane filter from the stand. Care should be taken to avoid loss of residual deposit. Use screw cap sterile container with or without glass beads for the membrane filter. Add 5 ml to 10 ml of sterile diluent or sample to wash the micro-organism from the membrane filter and shake vigorously using a vortex mixer for at least 2 minutes. The container shall be placed in an ultrasonic water bath for a time interval that has been verified to determine the optimum time interval for maximum recovery.

Record the volume of the concentrate sample and divide the concentrate into three portions. One portion for untreated, one portion for treatment with heat and one portion for acid treatment.

Maintenance Plan for the Instrument The Contractor shall maintain equipment according to ISO 17031 protocol.

Calibration Procedure The Contractor shall calibrate equipment according to ISO 17031 protocol.

Quality Assurance and Quality Control The Contractor shall perform quality assurance and quality control according to ISO 17031 protocol.

Data Acquisition The data shall be acquired according to ISO 17031 protocol.

Fate of Sample (Destruction or Return) Every isolate of Legionella obtained (if any) shall be cryogenically preserved. Every isolate shall be genetically analyzed to determine the exact species and, in the case of L. pneumophila isolates, the Sequence Base Type (SBT) shall be determined using the standardized SBT protocol. If no legionella is detected, the sample can be discarded. Comment by Hughes, Casey (NIH/OD/ORF) [E]: Is this standard? Comment by Adjei, George (NIH/OD/ORF) [E]: Is standard

References https://www.cdc.gov/legionella/downloads/cdc-sampling-procedure.pdf ISO 11731:2017, Water quality — Enumeration of Legionella, https://www.iso.org/standard/61782.html ISO 19458, Water Quality – Sampling for microbiological analysis