The microscope system automatically identifies and counts bacteria (shown here circled)

Scientists from the Preventative Health Flagship have developed an integrated microscope system to automatically count fluorescently tagged bacteria.

The automated system has been developed for use in human population studies which required reliable counts of particular bacterial species found in faeces that may be protective against colorectal cancer. The system is also expected to find a broad range of other applications.

Traditional counting of faecal bacteria involves collection of faeces from subjects, processing samples to remove food particles and other debris, and treatment of the crude bacterial preparations with a fluorescent dye that specifically stains the bacteria the scientists want to investigate. Then they load the slides, one at a time, on a microscope, focus in on the areas of interest and manually count the glowing bacteria.

But for large population studies, manual counting creates a bottleneck in the measurement process. Many subjects and many samples make the task a slow one. Further, manual counting is subjective. Different operators give different results.

To address the problem, CSIRO Biotech Imaging specialists Paul Jackway and Volker Hilsenstein and CSIRO biochemist Michael Conlon started with an ordinary fluorescent microscope. They modified the sample preparation methods and wrote new software to drive an automatically controlled stage and slide loader, enabling the microscope system to work on its own.

Image analysis algorithms were developed that determine which parts of a slide to count and then automatically focus, at high magnifications, on the areas of interest. Other algorithms determine which parts of the image represent bacteria and then count them. The result is a spreadsheet of counts that can be used by scientists to monitor trends in bacterial populations.

Microscope system showing microscope, slide loader and computer.

The development of the microscope was quite a challenge and took several years to optimise. For example, contaminating particulate matter sometimes fluoresces more brightly than the bacteria. Michael Conlon was able to modify the preparation of the samples to minimise these effects and allow the image analysis algorithms to work better. Also, CSIRO statisticians helped develop spatial sampling methods to obtain reliable counts of bacteria even if they are distributed in uneven patches on the slide.

Validation by comparing with human operators shows the automated microscope does its job well. But it could also have application in many other areas requiring high volumes of microscopy samples to be analysed reliably, round the clock, with little or no human intervention.

Potential uses could include:

• screening food samples for food poisoning bacteria

• counting parasites in livestock

• rapidly screening tuberculosis bacteria, since present testing methods are slow

Commercial applications are being explored.

"Robust Autofocusing for Automated Microscopy Imaging of Fluorescently Labelled Bacteria", Hilsenstein, V, in Proceedings of the Digital Image Computing: Techniques and Applications (DICTA 2005), pages 95-101, Cairns, Australia, 6-8 December 2005, IEEE Computer Society Press.

Carrie Bengston

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The Preventative Health Flagship is a CSIRO initiative and part of the National Research Flagships program that aims to deliver scientific solutions to advance Australia's most important national objectives. One of the largest scientific initiatives ever mounted in Australia, it aligns closely with the Federal Government's National Research Priorities. The initiative brings together our national research resources to deliver breakthroughs in fields ranging from healthcare to light metals and the environment.

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