Isolation of Biofilm Populations from Soil Crumbs by Flotation

Instructor Version (go to Student Version)

Subject Area(s)	microbiology
Intended Audience	high school biology, independent study/science fair, introductory undergraduate microbiology
Type	laboratory exercise
Revision Date	September 8, 2005

CONTENT

Biofilms develop on surfaces that are in contact with liquids. What better place to find biofilms than soil? This exercise is an adaptation of a technique first described by Grossman and Lynn (1967) as modified by Harris (1972). In this procedure, biofilms form discrete films on the surface of the suspending water layer. These films can be transferred to microscope slides or coverslips, Gram stained and examined under oil immersion. Harris (1972) reported being able to count the number of bacteria removed with the biological film thus obtained. This number does not correspond to the total biofilm population, however, since repeated isolations from the same soil crumb will yield additional bacteria.

Although not part of the original protocol, the biofilms can also be harvested with a sterile pipet for quantification and isolation of component organisms.

PREREQUISITES

Students should be able to define biofilm, be able to describe the differences between biofilm (surface attached) and planktonic (free-floating) cells, and be able to describe why bacteria tend to grow on surfaces.

INSTRUCTIONAL OBJECTIVE

Given materials readily available in many school laboratories and detailed instructions, the student will be able to stain (Gram, Capsule, Alcian Blue) and examine biofilms under oil immersion. They will also be able to harvest the biofilm with a pipette for quantification by dilution and plating techniques.

INSTRUCTIONAL PROCEDURES

1. Students will be provided with all materials required to assemble the apparatus described in the detailed student instructions.
2. Students will be given an opportunity to review the materials and instructions and ask questions concerning the procedure.

MATERIALS AND EQUIPMENT

Quantity	Description
1	Pair tweezers
1	Pasteur pipet
1	Gram Staining Kit and or Spore staining kit per two students
1	sterile pipettes, 1 ml size
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 funnel to its base
As Necessary	Multiple samples of well drained soil from various locations: garden soil, compost, forest soil, desert sand, greenhouse soil.

ASSESSMENT / EVALUATION

The exercise may be assessed by microscopic examination of the stained biofilm sample produced by each student. Does the stained biofilm sample appear to be entire or is it a fragment of the film lifted from the surface of the water?

Note: Getting an entire film is difficult and requires practice.

FOLLOW-UP ACTIVITIES

1. Suggestions for class discussion: Why would this film NOT be considered a biofilm?"
2. The floating biofilm sample produced by this technique is also amenable to harvesting by pipette for plate counting, and isolation of individual microbial components of the soil microbial population. Thus this exercise can provide working material for many of the other exercises in this set including:
3. Colorimetric Measurement of Biofilm Density
4. Drop Plate Method for Counting Biofilm Cells
5. Harvesting and Dispersing of Cells from Biofilms

REFERENCES

Grossman, R. B. and W. C. Lynn. (1967) Gel-Like Films that May Form at the Air-Water Interface in Soils. Proceedings of Soil Science Society of America, 31, 259-262.

Harris, P. J. (1972) Micro-organisms in Surface Films from Soil Crumbs, Soil Biology and Biochemistry, 4, 105-106.

Acknowledgments:

This exercise was was developed at Penn State Altoona College, as a student generated independent study project and was contributed by Marcus Bryan Sellers.

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Educational Program Curricula and Teaching Resources

Supported in part by the Waksman Foundation for Microbiology
Developed in collaboration with Dr. John Lennox, Education Editor, Penn State Altoona
© 1999-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu