Chapter 11 Lab Exercises
Section 13 Measurement of Biofilm Thickness
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Measurement of Biofilm Thickness
Supplies Needed:
Quantity |
Description |
1 |
glass cover slip |
1 |
felt tip marker |
1 |
microscope - modified to make depth
measurements |
1 |
metric feeler gauge - used to calibrate the
microscope (optional)
|
Introduction
The thickness of biofilms is an important
parameter for scientists and engineers to know because it is
related to the rate of growth of the biofilm and the extent to
which biofilms interfere with human engineered devices. The
rate at which nutrients and antimicrobics penetrate biofilms is
related to their thickness, as is the rate to which they
respond to antimicrobics like disinfectants and antibiotics.
The degree to which biofilms interfere with the fouling of
pipes and fluid delivery systems and interfere with the
function of devices like heat exchangers is also due, in part,
to the depth of the biofilm (see Figure 1).
Permissions
Permission pending, Jean Barbeau, author. ASM MicrobeLibrary
Figure 1. This biofilm cross section was obtained
with electron microscopy from the interior wall of a
dental office evacuation system. The biofilm shown is
approximately 36 µm thick.
Depending upon age, nutrient concentrations and the rate of
sloughing of biofilm fragments, a biofilm may vary in thickness
from a few micrometers to several hundred micrometers. This
exercise describes a technique and apparatus by which one can
measure the thickness of living biofilms in a non-destructive
manner over time.
Instructions
In this exercise you will use a modified
microscope to measure the depth of a biofilm. Notice that the
microscope has a piece of polar coordinate graph paper fixed to
its base surrounding the fine and course adjustment knobs.
Attached to the fine adjustment knob is a thin piece of wire
that serves as a pointer.
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 2. A microscope modified to measure
depth.
As the fine adjustment knob is turned, the pointer scribes an
arc which can be recorded as degrees of arc on the graph
paper.
Calibrating the microscope to measure depth
Your
instructor will provide you with a metric feeler gauge normally
used by automobile mechanics and engineers to measure the gap
between two points (spark plug gap for example). The leaves of
this gauge are precisely machined and are very delicate, so
"handle with care."
- With transparent tape, fix the 0.04 mm feeler gauge
tightly to the surface of a microscope
slide. Along one edge of the gauge, on the slide, make a
mark with a glass marking pen. This will make it easier to
focus the microscope on the surface of the slide.
- Mount the slide with feeler gauge attached into the
mechanical stage of your microscope and focus on the top
surface of the metal gauge at the edge closest to the mark
you made earlier.
- When the microscope is in focus on the gauge, record
the angle in degrees shown by the pointer on the polar
coordinate graph paper.
Top of gauge = ___________ degrees.
- Now, carefully focus down so that the microscope is in
focus on the top surface of the microscope slide. Look for
the mark you made earlier. Record this angle.
Top of slide = _____________ degrees.
Determine the difference in degrees arc between these two
readings.
We now have the depth of the feeler gauge (0.04mm or 40
µm) in terms of degrees of arc from the graph paper.
If for example, 82 degrees of arc moves the lens from the
surface of the gauge to the surface of the slide (82
degrees of arc = 0.04 mm or 40 µm; therefore 1 degree
of arc = 0.49 µm or approximately 0.5 µm).
With this simple device, you can now measure the thickness
of living biofilms non-destructively over time.
Biofilm thickness measurement
- Place the biofilm (either on a glass microscope slide
under a coverslip or in a flow cell, on the stage of the
microscope. Focus on the very top of the biofilm and record
the reading of the pointer on the graph paper.
Top of biofilm = ___________ degrees.
- Carefully focus on the substrate level (base of the
biofilm) and record that reading as well.
Base of biofilm = ___________ degrees.
- The thickness of the biofilm is the difference is
degrees of arc times the length value you have determined
for one degree of arc through your calibration.
Thickness of biofilm = ___________________.
Refractive index correction
- From your earlier experiences in microscopy you have
studied the concept of refractive index. You will recall
that different transparent materials have different Indices
of Refraction (n). In practice this means that as light
passes through these materials it bends to different
degrees. Air has about the same index of refraction as the
vacuum of space and is set as the standard with an n of
1.00. Water on the other hand has an n of 1.33. So light
passing from air into water is bent. You have probably
observed this phenomenon while observing a stick placed
into water. At the air/water interface the stick appears to
bend.
- This same concept implies that the depth of the biofilm
you have measured (optical thickness) is not quite accurate
and to get the "real" length or physical length, a
correction factor is required (See Figure 3 and
Reference).
-
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 3. Light paths from the
substratum-biofilm interface, through the
biofilm sample and air to the objective lens.
(Modified from Bakke and Olsson,
see
reference)
- The physical length Lf ≈ kfyf where yf = the
optical thickness measured and kf = a proportionality
constant function of the refractive indices of the biofilm
and air.
- Since the proportionally constant kf ≈ nf / nf
(Refractive index of the biofilm [nf = 1.33 or
approximately that of water] divided by the refractive
index of air [nf = 1.0]) the proportionality constant =
approximately 1.33.
- The actual length (Lf ≈ kfyf) is therefore the
measured optical distance (yf) multiplied by the constant
1.33 (kf).
References
Biofilm Thickness Measurements by Light Microscopy
R. Bakke and P.Q. Olsson
J Microbiological Methods 1986; 5:93-98.
Educational Program Curricula and Teaching
Resources
Supported in part by the Waksman Foundation for
Microbiology
Developed in collaboration with Dr. John Lennox, Penn State
University-Altoona
©1999-2008 Center for Biofilm Engineering,
http://www.biofilm.montana.edu