Legionella Sampling in the Environment
A. Legionella Sampling in Air:
A1. How to Handle Microbiological Media (AGAR PLATES)
- Agar plates must be kept refrigerated or in a cooler box with ice packs until ready to use
- The plates must be allowed to warm up to room temperature before taking a sample (approx. 20 minutes)
- Do not remove the lid from the plate at anytime except during sampling
- The plates must be shipped back to MBL on ice with OVERNIGHT PRIORITY. Refreeze and reuse the original icepack (this type of icepack is stable for 24 hours)
- Adequate packing material must be used to protect the plates. The weight of the icepack can crush the plates during shipping
- Plates must not come into direct contact with the ice, as the tests will be invalid if the media freezers
- If there is any delay in sending the agar plates to MBL, they should be refrigerated until ready for overnight delivery
- A Chain of Custody (CoC) form must accompany the plates. Note on the CoC the date collected and the date sent to the laboratory
A2. Materials Needed for Legionella Sampling in the Air
- Latex gloves
- 70% Isopropyl alcohol to sterilize sampler after every other sample
- SKC QuickTake 30 pump with Andersen N-6 single stage Impactor
- BCYE (Buffered Charcoal Yeast Extract) agar
A3. Sampling Procedure
- Allow agar plates to reach room temperature before use
- Attach one end of tubing to the intake of the sampling pump (QuickTake 30) and the other end to the inlet of the sampler (Andersen N-6 single stage impactor)
- Calibrate the flow rate of the vacuum pump (will be done by MBL)
- Wear gloves, open the impactor and wipe the inside surfaces with 70% Isopropyl alcohol and allow to air dry
- Place Petri dish containing agar into the sampler
- Remove the cover of the Petri dish and place into a sterile sample bag to minimize contamination
- Close the sampler
- Set timer to appropriate time depending on environmental conditions (sampling time is between 2-5 minutes)
- Turn on the pump
- When the time is up, the pump will stop automatically
- Disassemble sampler and place cover back onto agar plate
- Secure lid onto Petri dish with masking tape or Parafilm (avoid using electrical, packing, transparent and duct tape)
- Using a fine felt pen, write the sample number on the bottom of the Petri dish
- Record all appropriate information on the Chain of Custody
B. Legionella Sampling from Surfaces:
B1. Swab and Material sampling
- Collect a surface sample with a sterile swab where there is scale build up or on water outlets.
- Swabs of faucet aerators and shower heads should be taken before the water sample from these sites with the aerator or shower head removed if possible.
- Pre-wet the swab with water from the sampling site and immerse swab in 3-5 ml of water taken at the same time to prevent drying during transport.
- Collect samples of sludge, slime, or sediment with swabs or in sterile containers.
C. Legionella Sampling from Sampling for Legionella:
C1. Materials Needed for Legionella Water Sampling
- Use sterile screw-capped plastic bottles for sampling. Bottles should routinely contain a sufficient amount of sodium thiosulfate as chlorine neutralizing agent in the water system
- A 1 litter water sample is recommended for potable water sampling and 250 ml for non-potable water sampling
- Waterproof marker ( to label bottles)
- Respirator (wear appropriate respiratory protection during the examination of water systems if a significant potential exists for exposure to high concentrations of contaminated aerosols)
C2. How to Sample for Legionella in Water
- Wear gloves and, where necessary, respiratory protection
- Use sterile screw-capped plastic bottles containing sodium thiosulfate
- Collect water, leaving 2-3 cm air space in the bottle to facilitate mixing by shaking
- Tightly cap the bottles to minimize leakage during transportation to the lab
- Label sample container appropriately
- Record sample information on the chain of custody
- Place the samples in insulated boxes or on freezer packs to protect specimens from extreme temperature fluctuations in the summer months
- Ship samples by overnight express courier to MBL Inc
D. Types of Samples:
D1. Potable Water
Collect 2 water samples from shower heads, tap water faucets, respiratory therapy equipment, water fountains, etc. First a pre-flush or first draw sample by draining the first 1 L of water from the faucets or flush drains into a bottle. Allow the water to run for approximately 1 minute and collect the second draw of 1 L of water.
D2. Non-potable Water
Collect minimum 300 mL of water from cooling towers, chillers, condensate pans, surface water in reservoirs, sprinklers, hot water storage tanks, evaporative condensers, whirlpool spas, decorative fountains, ultrasonic mist machines, humidifiers, etc. Collect non-potable water samples from the bottom or side of the vessel or reservoir.
D3. Swab Sampling
Swab samples should be taken of shower heads, pipes and taps. Furthermore, sludge, slime or sediments within building water services, cooling towers, humidifiers or spas can also be collected, particularly where accumulation occurs. These samples allow sampling of biofilms, which frequently contain Legionella. Swabs of faucet aerators and showerheads should be taken along with water samples from these sites. When sampling cooling towers, whirlpool spas or fountains, look for areas of biofilms and take a swab sample of the biofilms. Swabs are not considered appropriate for sampling hot water tanks and routine testing of cooling towers.
E. Water Sampling Containers:
Chlorinated water should be treated with sodium thiosulfate (available – please let us know) to neutralize the chlorine. Water samples should be brought to the lab ideally within a few hours of sampling and should be stored at room temperature (20 + 5°C) in the dark. (Please contact us to discuss your needs and how we can help).
F. Potential Sources or Sampling Sites for Legionella:
F1. Potable water system
- incoming water main
- water softener
- holding tanks, cisterns
- water heater tanks (at the inflows and outflows)
F2. Potable water outlets, especially those in or near patient rooms
- faucets or taps
F3. Cooling tower, evaporative condenser
- makeup water (e.g., added to replace water lost because of evaporation, drift, leakage)
- basin ( i.e., area under the tower for collection of cooled water)
- sump ( i.e., section of basin from which cooled water returns to heat source)
- heat sources ( e.g., chillers)
F4. Humidifiers (e.g., nebulizers)
- bubblers for oxygen
- water used for respiratory therapy equipment
F5. Other sources
- decorative fountains
- irrigation equipment
- fire sprinkler system (if recently used)
- whirlpools, spas
- Arnow, P.M., Weil, D., and M.F. Para. 1985. Prevalence and significance of Legionella pneumophila contamination of residential hot- tap water systems. J Infect Dis. 152: 145-51.
- Barbaree, J.M., Gorman, G.W., Martin, W.T., Fields, B.S. and W.E. Morrill 1987. Protocol for sampling environmental sites for Legionellae. Appl Environ Microbiol. 53: 1454-8.
- [CDC] Procedures for the recovery of Legionella from the environment. Atlanta, GA: US Department of Health and Human Services, Public Health Service, CDC.
- Joly, J.R. 1993. Monitoring for the presence of Legionella: where, when, and how? In: Legionella: current status and emerging perspectives. Barbaree JM, Breiman RF, Dufour AP, eds. Washington DC: American Society for Microbiology Press, 211-6.