December 19, 2018

Controlling Legionella: updated water testing methods

By Patricia Lee, Senior Product Leader

The need to control Legionella and conduct investigations into outbreaks of Legionellosis/Legionnaires, is an ever-increasing public health concern. Delve into water testing and the updated membrane filtration methods of ISO 11731.

Legionella control is a global public health concern. Regulatory bodies such as the International Standard Organization (ISO) have recently updated the recommended testing methods to help detect Legionella in order to prevent or investigate outbreaks.

In this blog, I’ll talk about what makes the Legionella bacteria a public health concern: the sources of infection, how it spreads, and its effect on people. I’ll also outline the updated water testing protocols, describing two different filtration methods for identifying the presence or absence of Legionella bacteria.

Some facts about Legionella

Legionella is a pathogenic group of gram-negative, waterborne bacteria. One species, L. pneumophila, can cause a severe form of pneumonia called Legionnaires’ disease, which the CDC estimates affects 5,000 people annually in the United States.

High fever, cough, and muscle aches are some of the symptoms that come with this serious respiratory infection. It’s especially dangerous for smokers, the elderly, and those suffering from asthma, chronic lung disease, or those who have a suppressed immune system.

The primary source of Legionella bacteria is a building’s drinking water supply, with large buildings such as hospitals and hotels common locations for outbreaks. The bacteria enter a water supply, multiply, and become distributed throughout the system.

Exposure to the Legionella bacteria happens when individuals inhale or aspirate water droplets from fountains, mist machines, humidifiers, cooling towers, showers, and sinks.

Water sample testing for Legionella

Authorities investigate all cases of Legionnaires’ disease thoroughly to reduce the risk of further spread. It’s essential that they can locate the source of the infection quickly to make sure that the affected water system is treated as speedily as possible.

Ongoing monitoring for Legionella bacteria in water systems is also essential for public health reasons and Legionella control. Monitoring helps identify environmental sources that pose a risk for legionellosis before there is any transmission to the public.

Legionella monitoring is also useful for validating control measures already in place and verifying that these control measures work effectively.

Updating the Legionella monitoring standards

Regulatory bodies updated the recommended water sample testing procedures for Legionella in 2017. ISO 11731:2017 outlines two culture methods for the isolation of Legionella and estimation of their numbers. These methods are applicable for most samples, where you expect low to moderate levels of Legionella. Both methods can be used for all types of water sample, including potable, industrial, waste, and natural waters.

For water samples containing high concentrations of legionellae, you might be able to go direct to plating and culturing, or just perform a simple dilution first. In most cases, however, the legionellae in the water sample will need to be concentrated.

The first step of both Legionella monitoring methods outlined below uses membrane filtration by vacuum or positive pressure to concentrate the bacteria in the water sample. To minimize the growth of other bacteria also concentrated by the membrane filtration step, portions of the water samples are subjected to heat and/or acid treatment.

Let’s look at an overview of these two methods, and some advantages and disadvantages of each.

Method 1. Culturing directly on the membrane

After concentrating the sample, the Legionella bacteria are cultured by placing the membrane filter directly onto culture media. The growing colonies can then be counted directly on the membrane. The ISO 11731:2017 guidelines state that black membrane filters contrast better with the white Legionella colonies than light-colored membrane filters, making these a good option for this method.

This method involves:

  1. Filtering the water sample through a nitrocellulose or mixed cellulose esters membrane filter to concentrate the legionellae.
  2. Carefully removing the membrane filter from the stand using disinfected forceps.
  3. Placing the membrane filter directly on the culture media (e.g. Buffered charcoal yeast extract (BCYE) agar or Glycine vancomycin polymyxin B cyclohexamide (GVPC) agar).

This is an easy method that has a low limit of detection, but there are a few tips that can help your water test go swimmingly:

  • Filtering a suitable volume of water, based on the particulate content and desired detection level.
  • Making sure you place the membrane the right way up on the culture media, leaving no trapped air bubbles.
  • Using black nitrocellulose membranes to improve the contrast with the bacterial colonies.
  • Using filter papers with high contrast grids to simplify colony counting.

Method 2. Washing and eluting before plating onto culture media

Once you have a concentrated sample, washing and eluting the legionellae from the membrane before plating distributes the cells evenly on the agar, simplifying colony counting.

This method involves:

  1. Filtering the water sample through a polycarbonate track etched membrane (PCTE membrane) or polyethersulfone membrane filter to concentrate legionellae.
  2. Removing the membrane filter from the stand with disinfected forceps.
  3. Placing the membrane filter in a screw cap sterile container with or without sterile glass beads
  4. Washing the microorganisms from the membrane filter using sterile diluent and shaking vigorously using a vortex mixer, or placing the container in an ultrasonic water bath.
  5. Plating the sample onto growth medium for culturing (e.g. Buffered charcoal yeast extract (BCYE) agar or Glycine vancomycin polymyxin B cyclohexamide (GVPC) agar).

Compared to the first method, this process is more time-consuming and has a lower recovery rate. To help aid in sample elution and maximize the number of cells plated, membrane filters can be cut into pieces using sterile scissors before washing.

Choosing a water sample testing method

The choice of method is ultimately down to the individual laboratory, as it depends on the origin and characteristics of the samples. For example, whether the water has low, high, or extremely high background. The laboratory also needs to take the desired lower limit of detection into consideration. Table 1 summarizes the advantages and disadvantages of each approach.




Concentrating the sample and culturing directly on the membrane

Easy to perform
Low limit of detection

Difficult to count individual colonies (due to outgrowth of neighboring Legionellae and other microorganisms)
Membrane might influence growth

Concentrating the sample and washing and eluting from the membrane before plating

Easy to count individual colonies
Low limit of detection

Lower recovery than culturing directly on membrane

For assistance with any aspect of your water testing workflow or membrane filtration techniques for microbiological analysis, contact Cytiva Support.