Analytical Methods- Culture Test

Legionella are aerobic, fastidious bacteria; they have very strict requirements for growth.  Two of these requirements are the presence of iron and L. cysteine. They are weakly gram-negative and grow slowly compared to other bacteria.  Legionella are often overgrown by faster growing bacteria. Their growth can be inhibited by some other bacteria, most commonly P. aeruginosa, which is commonly found in biofilms. Legionella do not to grow on standard microbiological media used for aerobic plate counts or on selective agar used for detecting other, non-Legionella pathogens.

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 International Standards Organization (ISO). This method incorporates routine concentration, acid treatment and heat treatment of all potable and non-potable water samples to enhance the recovery of very low levels of Legionella in any sample. Buffered charcoal yeast extract agar (BCYE) with antibiotics is the base formulation to which additional supplements are added to control non-Legionella bacteria and recover more Legionella species.

The samples must be concentrated in order to recover low concentrations of Legionella. This is usually done by filtering the entire sample through a sterile membrane filter. The filter is then vortexed in sterile, distilled water. Aliquots are then taken for platingonto 4 different formulations of BCYE agar. Non-potable water often has a large concentration of bacteria that 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 release the Legionella so that they may be recovered, grown and quantified.

The BCYE plates are incubated at

35-37F (which is body temperature) rather than ambient temperature. Since Legionella bacteria grow slowly, incubation periods are specified as 7 days to confirm a positive result to no less than 10 days to confirm a negative result. For this reason, culture methods cannot be rushed.

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 colonies often exhibit a blue-white, green, or red auto fluorescence.

These suspect Legionella colonies are streaked onto BCYE plates that do not contain iron and cysteine. If suspect colonies do not grow on these 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.

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 water 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 per CFU/milliliter (CFU/ml) in the US.  Other countries commonly express results as CFU/liter (CFU/L).  While it is standard microbiological practice to express results as CFU/ml, this can be confusing to non-microbiologists.