Chapter 11 Lab Exercises
Section 17 Isolation of Biofilm Populations from Soil Crumbs by Flotation
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Isolation of Biofilm Populations from Soil Crumbs by
Flotation
Supplies Needed:
Quantity |
Description |
1 |
Pair tweezers |
4 |
Pasteur pipet |
1 |
Gram Staining Kit and or Spore staining kit per two
students |
1 |
Micropore filter funnel with sintered glass bottom
(without the membrane filter) OR a wide mouth Buchner
funnel with a sintered glass bottom |
1 |
Tygon or other flexible tubing to connect Buchner
and separatory funnels (approx 40 cm) |
2 |
ring stands and rings |
1 |
500 ml separatory funnel |
1 |
clean cover slip or microscope slide |
1 |
clamp to secure the base of the funnel |
As Necessary |
Multiple samples of well drained soil from various
locations, garden soil, compost, forest soil, desert
sand, greenhouse soil. |
Instructions:
- Assemble apparatus as shown in the diagram (from
Grossman and Lynn, 1967)
- In order to remove air bubbles from the system: Fill
the separatory funnel with about 300 ml of distilled water.
Remove the ground glass stopper from the separatory
funnel.
- Raise the separatory funnel and open the stopcock. The
water should fill the reservoir of the funnel and rise
through the sintered glass bottom of the micropore
filter.
-
Lower the separatory funnel until the water is just below
the surface of the sintered glass base of the micropore
filter.
- Weigh a soil crumb of between 0.01 and 0.5 grams
(Harris, 1971) and with forceps place it carefully on the
sintered glass base of the micropore filter.
- Slowly raise the water through the soil crumb by
elevating the separatory funnel. An observable film should
form on the surface of the water. The size of the film
roughly corresponds to the size of the soil crumb. Do not
disturb the funnel, as any agitation of the water layer is
likely to break up the film.
- Collect the sample biofilm on a cover slip, by
inserting the coverslip into the water next to the biofilm.
Move the coverslip so that it is under the film and then
slowly raise the coverslip. The biofilm will usually adhere
to the coverslip. Adherance can be facilitated by using a
Pasteur pipet to move the film into contact with the
coverslip.
- Allow the coverslip and biofilm to air dry.
- Heat fix the film and stain with your choice of
techniques. Gram stain and spore stains work well as do
methylene blue or crystal violet dye simple stains.
- Affix the cover slip to a slide using a tiny drop of
clear nail polish applied at the corners.
- Observe under oil immersion.
Observations:
- Is it possible to count the cells you observe within
the drop?
- Describe the diversity of the cell types you
observe.
- Compare your biofilm sample with those isolated from
different soil types and locations.
- If possible, estimate the number of bacteria obtained
per mass of the soil crumb you used.
Cells/gram of soil =
- Is your soil crumb still intact? If so attempt to
isolate sequential biofilms from the same soil crumb. What
happens to the number of cells in sequential biofilm
samples? Plot the cell number against the number of biofilm
samples isolated from a given soil crumb. How efficient is
this technique in recovering bacteria attached to the soil
matrix surface?
Illustrations:
Permissions
J. Lennox and M. Sellers, Penn State Altoona
Figure 1. Filter setup illustrated by Marcus
Sellers.
Permissions
D. Williams, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 2. Picture of filter setup in the lab
Acknowledgments:
This exercise was developed at Penn State Altoona College,
as a student generated independent study project and was
contributed by Marcus Bryan Sellers.
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