Isolation of Biofilms from Soil Crumbs

Isolation of Biofilms from Soil Crumbs

A Contribution to the On-line Biofilms Laboratory Manual

Marcus Bryan Sellers

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.

Equipment:

Micropore Filter Funnel with sintered glass bottom (without the membrane filter) or a wide mouth Buchner funnel with a sintered glass bottom

Tygon or other flexible tubing

Ring stands and rings

500 ml Separatory funnel

Clean Cover slips or microscope slides

1 clamp to secure the base of the funnel

Procedure:

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.
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.s
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. 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 nut disturb the funnel, as any agitation of the water layer is likely to break up the film
Collect the film on a cover slip, let it air dry, heat fix and stain. Affix the cover slip to a slide using a tiny drop of clear nail polish applied at the corners.
Observe under oil immersion.

Bibliography

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.