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Biofilm consists of other bacteria, blue green algae, amoeba, and protozoans. Biofilm protects Legionella from direct exposure to ultra-violet (UV) light, desiccation, and the chemicals used to control its growth. In addition, the Legionella bacteria are ingested by the protozoans and amoeba and will continue to multiply inside these organisms. Once these organisms die large numbers of Legionella bacteria will be released into the surrounding environment.
Because biofilm protects the organism and enhances Legionella multiplication, incorporating swabs in your sampling protocol is very important. Swab sampling of biofilm found in cooling tower sumps, potable water faucets, showerheads, and whirlpool spa filters is necessary to obtain a true picture of the presence of Legionella. Very often, biofilm swab samples demonstrate the presence of Legionella undetected by water sampling alone.
While collecting air samples for Legionella mimics the route of exposure, it is generally not recommended for routine monitoring purposes. Legionella are unlikely to survive the exposure to UV light and dessication for long periods of time. In addition, the bacteria may be killed from the impaction of the bacterial cells on the collection media.
When taking samples around cooling towers a high efficiency particulate respirator, safety glasses, and gloves should be worn. Take care not generate any aerosols when collecting potable water samples. Only sterile, appropriately preserved bottles and swabs obtained from your lab should be used. Potable water bottles should be preserved with sodium thiosulfate to neutralize chlorine in the water sample. These preserved bottle should also be used when collecting water from cooling towers that have been treated with chlorine diozide.
After collecting a water sample, be sure to leave an air space in the bottle. Since Legionella require oxygen for their survival, an air space in the bottle will ensure that aerobic conditions are maintained during shipment to the lab.
Samples should be packed and shipped to minimize the multiplication of non-legionella bacteria. Since Legionella remain viable at low temperatures, using an insulated cooler with freezer packs is recommended. Samples should be shipped overnight to the lab.
Analytical Methods- Culture is the "Gold Standard" Worldwide
Legionella are aerobic, fastidious bacteria; they have very strict requirements for growth. Two of these requirements are iron and L. cysteine. They are weakly gram negative and grow slowly compared to other bacteria. Legionella are often overgrown by faster growing bacteria, inhibited by some bacteria, or will not grow on standard microbiological media. For this reason labs should use methods that are selective for isolating and identifying the organism.
Currently the definitive method worldwide for identifying Legionella in clinical and environmental samples is the culture method. This method uses an improved procedure developed by the CDC when it first isolated the organism after the American Legion outbreak in Philadelphia in 1976. The method uses buffered charcoal yeast extract agar (BCYE) as the base formulation.
For potable water, the samples must be concentrated in order to enhance the quantitation limit. This is usually done by filtering the entire 1000 ml through a sterile membrane filter. The filter is then vortexed in 5 mL of sterile, distilled water.
Non-potable water often has a large concentration of bacteria that surpasses or inhibits the growth of Legionella. Since Legionella are more resistant to acidic pH levels, these samples are pretreated with a buffered acid solution to eliminate the non-legionella bacteria.
Equal sample aliquots are then plated onto the BCYE agar containing iron and L. cycteine (BCYE+), BCYE agar with Polymixin B, Cycloheximide, Vancomycin (PCV), PCV with Glycine (GPCV), and PCV without iron and L. cysteine (PCV - ). These plates are incubated at 35-37 0 F. Because the Legionella bacteria from environmental
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samples may grow slowly, the plates are incubated for 10-14 days.
After 72 to 96 hours, the colonies are examined using a dissecting microscope with UV light. Legionella colonies appear as convex, circular white colonies having a center that resembles ground glass. The edges of the colonies often exhibit a blue, green, purple or red autofluorescence.
These suspect Legionella colonies are streaked onto BCYE plates that do not contain iron and cysteine. If these colonies do not grow on the BCYE - plates, they are presumptively identified as Legionella.
The presumptive colonies are then analyzed using Direct Fluorescence Antibody (DFA) technique to confirm the identification of species and identify the sertotypes. Since Legionella in environmental samples grow slowly, a confirmed non-detected sample result should be provided only after the 10th incubation day.
Due to cross reactivity and the potential for false positive and false negative results, DFA should be used only on pure colonies obtained after incubation. DFA should not be used directly on environmental samples as some laboratories claim.
While 90% of the outbreaks in the US are caused by L. pneumophila serotype 1, there are other serotypes of L. pneumophila and even other Legionella species that can cause the disease. Not all labs employ the same method for isolating the organism. Ascertain whether your lab uses the method to give you the level of identification and quantitation you need. The results for the culturable method are expressed as Colony Forming Units (CFU) /ml or per volume sampled. Even though some labs prefer to express the results as CFU/volume sampled, it is standard microbiological convention to express results as CFU/ml. CFU/ml is also the concentration used in the OSHA and other international guidelines.
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