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Analytical Methods- Culture Test


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 and can be inhibited by some bacteria. Legionella will not grow on standard microbiological media used for aerobic plates or for detecting other bacteria. For this reason labs should use methods that are selective for isolating and identifying the organism.


Currently the most recognized method in the US for identifying and enumerating 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. Aliquots are then taken of this distilled water for plating onto 6 different formulations of BCYE agar.


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.








Samples containing large amounts of protozoans such as municipal wastewater or wastewater from paper mills require heat pretreatment. Heat pretreatment is needed to kill the protozoans in order to expose the Legionella so that they may be grown and quantified.


The BCYE plates are incubated at 35-37 0 F. Because the Legionella bacteria from environmental samples grow slowly, turnaround times for releasing results are typically after 7 to 10 days of incubation.


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 auto fluorescence.


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) or Latex Agglutination to confirm the identification of species and identify the serotypes. Since Legionella in environmental samples grow slowly, a confirmed negative sample result should be provided only after the 10th incubation day.



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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. According to CDC, DFA should not be used directly on environmental samples as some laboratories claim.


While it is estimated that 85% 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 and identifying 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. While it is standard microbiological practice to express results as CFU/ml, this can sometimes be confusing to non-microbiologists.


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