The fact is that recovering these bacteria from the environment is not an easy task. The sampling part of it is the easiest and was already completed. However, once the samples reach the Centers for Disease Control and Prevention (CDC) there is a complex process that takes place.
The following is a summary based on the CDC’s laboratory procedures for the recovery of Legionella from the environment. Based on the extensive protocol, this is what could be happening in the laboratory during the 10 days.
Samples are segregated upon arrival depending on their origin (exact sampling locations). The samples from potable waters generally have low bacterial densities and are either plated directly into the special plating media or concentrated to improve the chances of detecting the bacteria. The samples are concentrated by filtration, the water samples are filtered and the particles left behind in the filter are suspended into solution and then plated. Non-potable waters (i.e. cooling towers) generally do not require concentration but do require special treatment to eliminate natural contaminants.
Media plates are specially prepared with the right chemicals/additives that will be best suited to allow the Legionella bacteria to grow into colonies that can later be studied for specific identification. Basically, the media is enriched with the nutrients best suited to favor the growth of Legionella if present in the samples.
Once the samples are all inoculated in the appropriate media plates, they are incubated. The incubated plates are checked every 24 hours up to 96 hours. The timeframe is dependent on the time it takes the bacteria to grow into colonies for follow-up testing. Only the positive samples go to the next steps.
The follow-up from the first media plates is sub-plating specific colonies. The microbiologists select the colonies with the known physical characteristics of Legionella colony formations for sub-plating into new media plates. This new media plates are incubated at 35 degrees C with and without carbon dioxide exposure. In addition, pre-treatment with acidic buffer or heat can be used in the protocol depending on the results obtained from initial plating. The purpose of sub-plating is to obtain pure cultures, free of contaminants. This secondary incubation can take up to 7 days with daily observations. Some protocols for the harder to grow Legionella strains can last up to 6 weeks of incubation. Positive identification is made from plate visual examination of colonies that have specific characteristics. Subsequently, further special testing is performed to arrive at the bacterium subtype.
All that is what takes so long. Microbiology’s time to results is heavily dependent on bacterial growth rates. That growth can not be speed up. However, the science of molecular biology is moving very quickly in the development of polymerase chain reaction (PCR) techniques to speed up the process of identification of many bacteria. The time is not too distant when the number of days for results will be a fraction of what it is today. For now we have to wait.
For more specific information http://www.cdc.gov/legionella/files/LegionellaProcedures-508.pdf
Comments
Maria, Your article is helpful, and its great to see more detailed discussions on the laboratory end of outbreak control.
Faster testing methods are already available and in use. As you note PCR technology is moving at quite a pace - and is already recognised for regulatory compliance use in some European states. In addition we have developed fast, on site, methods (see www.hydrosense.biz) which can provide an early warning of Legionella pneumophila sg1 (causative agent of 90% of community acquired Legionnaires' disease) collonisation. We are not trying to replace traditional lab testing but to offer the ability to make informed risk assesments based on up-to-date information.
Experience suggests that the US are a bit slower to adopt faster test innovations for Legionella than in other parts of the world.
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