Measuring Biofilm Depth

Depending upon age, nutrient concentration and the frequency of sloughing of film segments, a biofilm may vary in thickness from a single cell to several hundred micrometers. Although measurements of length and width are commonly made using ocular or filar micrometers calibrated to external standards (e.g. A stage micrometer), measurements of depth represent a more difficult task.

Figure 1. This biofilm cross section was obtained with electron microscopy of the interior wall of a dental office evacuation system. The biofilm shown is approximately 36 mm thick. This picture is part of the ASM biofilms collection and was prepared by Jean Barbeau of the Faculty of Dentistry, University of Montreal, PO Box 6128, Station C, Quebec, H3C 3J7 Canada.

Some microscopes are equipped with a measuring device attached to the fine adjustment knob. Our old AO Spencer microstar Series 10 scopes for example had a series of graduations in which one complete turn of the fine adjustment knob advanced the lens 200 mm. As there were 200 divisions on the knob each division represented an advance of 1 mm. One merely focuses on the top of an object and then again at its base and records the number of fine adjustment knob divisions which have passed the registration line. Unfortunately, all microscopes do not come so equipped.

An alternative, if somewhat more awkward, method is shown in Figure 1. Here a piece of polar coordinate graph paper has be copied onto a piece of heavy card stock, cut out and mounted over the fine and course adjustment knob of a microscope.

A pointer bent from a piece of thin wire is fitted to the fine adjustment knob of the microscope and held in place with tape. The degree to which the fine adjustment is turned can now be measured in degrees of arc.

This simple device can be calibrated by measuring the number of degrees of arc required to focus on the top and bottom surface of an object of known thickness. Thanks to the engineering department down the hall I had access to a set of metric feeler gauges which I could tape to a microscope slide on the stage of the microscope. It was an easy task to focus on the surface of, for example the 0.04 mm gauge and then on the surface of the slide to which it was tightly secured with tape. Placing a mark on the slide with a felt tip pen made focusing on the blank slide much easier.

In this case I found that 82 degrees of arc would move the lens from the surface of the gauge to the surface of the slide. That is 82 degrees of arc = 0.04 mm or 40 mm. One degree of arc therefore equals 0.49 mm or approximately 0.5 mm.

You don't have a metric feeler gauge, not to worry. A glass cover slip such as Thomas Red Label # 2 is produced to a thickness tolerance of 0.19 to 0.23 mm. The one I used had a measured thickness (thanks to those engineers again) of 0.21 mm. Placing a felt tip mark on top and bottom of the cover slip at right angles made focusing easier.

With this simple device, students can gather more information about the thickness of biofilms or any other object they can bring into focus under a microscope. If you have an old, little used scope in you lab you might set it up as a dedicated instrument for just this purpose.

With it, one can measure the thickness of living biofilms non-destructively over time.