Flow cytometer workshop at NIOZ

15-02-2013  This week, an international workshop on the comparison of various methods of counting small organisms in ballast water was organized at the Royal Netherlands Institute for Sea Research (NIOZ) on Texel. Ship-owners and the shipping inspectorate are looking for a method that is quick, reliable and cheap.

For four days, over 25 participants from eight different countries went into the results of the different measuring methods. Many participants had brought their own flow cytometer so that they could compare each other's measuring methods. A flow cytometer is a device that measures the number of microscopic organisms in water samples.

The International Maritime Organization (IMO) will presently require ship-owners worldwide to clean their ballast water before it is discharged. Ballast water in ships serves to add stability. When a ship is loaded with cargo, the ballast water is no longer needed and it is discharged. In the past, quite a few organisms have been transported in this way from one region to another, sometimes with dramatic consequences. The warty comb jelly, for instance, could spread unchecked in the Black Sea. There it fed on fish larvae, which strongly affected fishery.

To prevent this in the future, ships will soon be required to clean their ballast water and make sure that there are practically no living organisms left. That is why methods are being developed for checking that the ballast water is indeed sufficiently clean. This is a complex matter because the organisms in question are tiny, measuring between 0.002 and 0.050 mm.

During the workshop, 9 flow cytometers and a few microscopes, including a holographic microscope, were set up. First, water with tiny synthetic balls was tested, next several water samples with cultures grown at NIOZ were tested, and finally water from the Wadden Sea.

The tiny synthetic balls were easiest to measure: these balls were all of equal shape and size. All flow cytometers could easily detect them. The smallest balls measuring 0.002 mm were more difficult to count under the microscope, as they were hard to see. Some of the flow cytometers were blocked by the 0.050-mm balls. The microscopes were more suitable for counting these larger balls.

The larger the algae and the more varied their shape, the more difficult is was to come up with a reliable count with the flow cytometers. The most difficult was the water from the Wadden Sea, which contains many different species in different sizes. The larger algae were easier to count with the microscopes.

An important conclusion of this workshop is that there are no good and bad flow cytometers. The results of the various devices are similar. For a good analysis of ballast water, it is best to use both a flow cytometer and a microscope. A flow cytometer is fast but not particularly suitable for counting the larger algae; a microscope is much more suitable for this, but this method is much slower. So one single method that is fast, cheap as well as reliable has not been found yet.

The next challenge is assessing if the organisms are dead or alive. This can be made visible by adding a stain. Research is currently carried out into the possibility of using different stains to stain the dead and living organisms differently.