Chapter 11 Lab Exercises
Section 6 Collecting Soil Biofilms by the Buried Slide Technique
Page 3 Collecting Soil Biofilms by the Buried Slide Technique - Instructions for Students
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Collecting Soil Biofilms by the Buried Slide Technique
Introduction
This exercise describes a method for collecting soil
biofilms on glass slides through direct contact between soil
and microscope slide, in the field or in the laboratory.
This technique is described in “Methods for Studying the
Ecology of Soil Micro-organisms” (Parkinson et al.1971).
In this exercise a microscope slide is placed in contact with
soil. Organisms representative of the soil population will form
a biofilm on the slide. Parkinson (1971) cautions not to assume
that the microbial population observed on the glass slide is
similar to that of the undisturbed soil. The glass slide alters
the dynamics of the soil ecosystem by providing a new and novel
substrate. The organisms that colonize the slide can be assumed
to be soil microorganisms, but the proportion, distribution and
relationships of the organisms may be drastically
altered.
Supplies Needed:
Materials
Quantity |
Description |
As Necessary |
1 x 3 inch glass microscope slides, cleaned and
sterile |
As Necessary |
Staining kits |
Equipment
Quantity |
Description |
1 |
sterile forceps |
1 |
long bladed spatula or knife |
1 |
microscope |
1 |
clay or plastic flowerpot or a plastic cup |
Instructions
- Clean the microscope slides with acid alcohol and heat
sterilize them in a Bunsen burner flame or wrap them in
aluminum foil and sterilize them by autoclaving.
Note: To enumerate the cells collected
from soil, use preprinted slides such as those available
from Erie Scientific Co. These slides have circular areas
of known surface area (πr2 ) (Figure 1).
Permissions
Image used courtesy of Erie Scientific Company http://www.eriesci.com/index.aspx
Figure 1. Preprinted slides with clear spots
of known diameter may be used to harvest
biofilms.
- Choose an inconspicuous site for placing
the microscope slides so that they will not be
intentionally or inadvertently disturbed.
Alternatively, bring a soil into the laboratory
by using a garden bulb planter (Figure 2).
Select a soil sampling site, then twist the
bulb planter into the soil to remove a soil
core. Place this core into a clay pot, plastic
pot or a plastic cup of appropriate size. If a
plastic cup is used, be sure to punch some
holes in the bottom of the cup to permit water
drainage (unless, of course, there is interest
in sampling biofilms from waterlogged and
anaerobic soil).
Permissions
J. Lennox, Penn State Altoona
Figure 2. Bulb planter technique
for obtaining a soil sample
- With a sterile knife or spatula, make a
slit in the soil core sample (dip knife or
spatula blade in alcohol and flame). Widen the
slit by moving the knife from side to
side.
- Carefully insert one or more sterile slides
into the slit using sterile forceps.
- Firm the soil up around the slide to ensure
that the soil is in close contact with the
slide. Then lightly water the soil.
- Cover the pot or cup with a Petri dish lid
or plastic wrap and label it. If this exercise
is being carried out in the field, mark the
site inconspicuously so as not to attract
unwanted attention.
- The slides should remain in place for 1 - 3
weeks, after which the slides may be removed
from the soil. This is best done by removing
the soil core from its container and carefully
breaking the soil away from the slide.
- Wipe one side of each slide clean with a
paper towel.
- The slide may now be stained for
observation under the microscope. The
“Flow Through Gram Stain” described
elsewhere in this collection, or any of a
number of stains can be used for visualizing
the soil biofilms. Ruthenium red and Alcian
blue are recommended for staining extracellular
polysaccharides.
Classical simple and differential stains such as Crystal
violet, Gram stain and spore stain may be used on these slides.
Note: Since biofilms are 98% water, staining
without heat fixation is preferred. Heat fixation usually
employed to attach cells to the slide, dehydrates the biofilm
so that its structure is greatly altered.
Observations:
- View the slide under high dry or oil immersion
microscopy.
- Can you distinguish the biofilm cells and matrix from
the inorganic soil components which may be adhering to the
slide?
- Is it possible to see the matrix material in which the
cells are embedded?
- Is there any discernable organization to the biofilm on
the slide? Are the cells uniformly distributed or are they
clustered in microcolonies?
- Is there any evidence of channels within the
biofilm?
- Is there any association between the organic and
inorganic components of the biofilm?
Questions:
- In viewing the stained or unstained slide under the
microscope, how do you know a biofilm is present?
- Why is the biofilm on the slide not necessarily
representative of the biofilm in the soil?
References
Microorganisms in surface films from soil crumbs
Harris PJ
Soil Biol Biochem 1972; 4:105-106
Methods for Studying the Ecology of Soil Micro-organisms
in Parkinson D, Gray TRG, Williams ST (eds.)
Blackwell Scientific Publications, Oxford, 1971, pp 30-31
This material is based upon work supported by the National
Science Foundation under Grant No. 0618744, and in part by the
Waksman Foundation for Microbiology. Developed in collaboration
with
Dr. John Lennox, Penn
State Altoona. Any opinions, findings, and conclusions or
recommendations expressed in this material are those of the
author(s) and do not necessarily reflect the views of the
National Science Foundation.
©2002-2008 Center for Biofilm Engineering,
http://www.biofilm.montana.edu