pyrophosphate, EDTA (Fletcher, 1980) and ethylene glycol-bis (B-aminoethyl ether)-N,
N-tetraacetic acid (EGTA) (Turakhia eta/., 1983) used in removal of adhered bacterial
cells and biofilms. The use of sonication after treatment with pyrophosphate may simply
have quickened the dispersal process by exposing more cations for further sequestering by
pyrophosphate. Exposed organic and inorganic anionic sites may then be neutralized by
excess sodium cations present in the suspending medium. In addition, sonication may
sever attached polymers. When the suspension is filtered, the water soluble complex ions
formed by pyrophosphate and polyvalent cations remain in the filtrate. Since the surface
charges have been neutralized, the bacteria and the organic and inorganic particulate
matter will be evenly distributed on the filter.
The results of these experiments indicate that most bacteria in seawater, surface sediment
and kelp blade samples can be dispersed by the following technique:
1. Preserve and fix the bacterial cells in seawater with formaldehyde (2-4 %, final
concentration). Samples such as sediment and kelp blades should be suspended in filter
sterilized seawater containing 2-4 % formaldehyde.
2. Add tetrasodium pyrophosphate (0.001 M, final concentration) to the seawater samples. Sediment and kelp samples are treated with 0.01 pyrophosphate. Incubate for 15-30
min.
3. Cool all samples to 0°C. Sonicate for up to 90 s at 100 W, using a ca. 4 mm diameter
probe tip immersed until 1 cm from bottom of tube.
ACKNOWLEDGEMENTS
We acknowledge the financial support of the natural sciences and research council of Canada.
BANKS W.J. and J. WALKER, 1977. Sonication of activated sludge flocs and the recovery of their bacteria on
solid media. J. Gen. Microbiol. 98 : 363-368.
BINGLE W.H, 1980. Methods for separating microorganisms from soil. M.Sc. Thesis, University of Saskatchewan, Saskatchewan. 90 pp.
CAHN A. and J.L. LYNN, 1983. Surfactants and detersive systems. In : Kirk-Othmer Encyclopedia of Chemical
Technology. (3rd ed.). Vol. 22. Wiley, New york. pp 332-432.
COAKLEY W.T., A.J. BAKER, and D. LLOYD, 1977. Disruption of microorganisms. In : A H. Rose and D. W.
Tempest (eds.). Advances in Microbial Physiology. Academic Press, London. pp. 279-340.
CONSIDINE D.M. and G.D. CONSIDINE (eds.), 1983. Van Nostrand’s Scientific Encyclopedia. 6th ed. Van
Nostrand Reinhold Cc. New York. 3067 pp.
CORPE W.A, 1974. Detachment of marine periphytic bacteria from surfaces of glass slides. Dev. Ind. Microbiol.
15 : 281-287.
COSTERTON J.W., G.G. GEESEY K.J. CHENG, 1978. How bacteria stick. Sci. Am. 238 : 86-95.
CUNDELL A.M., T.D. SLETTER and R. MITCHELL, 1977. Microbial populations associated with the surface of
brown alga Ascophyllum nodosum. Micro. Ecol. 4 : 81-91.
DALE N.G, 1974. Bacteria in interdial sediments : factors related to their distribution. Appl. Environ. Microbiol.
41 : 107-116.
DALEY R. J, 1979. Direct epifluorescence enumeration of native aquatic bacteria : uses, limitation and comparative accuracy. In : J. W. Costerton and R. R. Colwell (eds.) Native Aquatic Bacteria Enumeration, Activity, and
Ecology, ASTM STP 695. American Society for Testing and Materials, pp. 29-45.
257
N-tetraacetic acid (EGTA) (Turakhia eta/., 1983) used in removal of adhered bacterial
cells and biofilms. The use of sonication after treatment with pyrophosphate may simply
have quickened the dispersal process by exposing more cations for further sequestering by
pyrophosphate. Exposed organic and inorganic anionic sites may then be neutralized by
excess sodium cations present in the suspending medium. In addition, sonication may
sever attached polymers. When the suspension is filtered, the water soluble complex ions
formed by pyrophosphate and polyvalent cations remain in the filtrate. Since the surface
charges have been neutralized, the bacteria and the organic and inorganic particulate
matter will be evenly distributed on the filter.
The results of these experiments indicate that most bacteria in seawater, surface sediment
and kelp blade samples can be dispersed by the following technique:
1. Preserve and fix the bacterial cells in seawater with formaldehyde (2-4 %, final
concentration). Samples such as sediment and kelp blades should be suspended in filter
sterilized seawater containing 2-4 % formaldehyde.
2. Add tetrasodium pyrophosphate (0.001 M, final concentration) to the seawater samples. Sediment and kelp samples are treated with 0.01 pyrophosphate. Incubate for 15-30
min.
3. Cool all samples to 0°C. Sonicate for up to 90 s at 100 W, using a ca. 4 mm diameter
probe tip immersed until 1 cm from bottom of tube.
ACKNOWLEDGEMENTS
We acknowledge the financial support of the natural sciences and research council of Canada.
BANKS W.J. and J. WALKER, 1977. Sonication of activated sludge flocs and the recovery of their bacteria on
solid media. J. Gen. Microbiol. 98 : 363-368.
BINGLE W.H, 1980. Methods for separating microorganisms from soil. M.Sc. Thesis, University of Saskatchewan, Saskatchewan. 90 pp.
CAHN A. and J.L. LYNN, 1983. Surfactants and detersive systems. In : Kirk-Othmer Encyclopedia of Chemical
Technology. (3rd ed.). Vol. 22. Wiley, New york. pp 332-432.
COAKLEY W.T., A.J. BAKER, and D. LLOYD, 1977. Disruption of microorganisms. In : A H. Rose and D. W.
Tempest (eds.). Advances in Microbial Physiology. Academic Press, London. pp. 279-340.
CONSIDINE D.M. and G.D. CONSIDINE (eds.), 1983. Van Nostrand’s Scientific Encyclopedia. 6th ed. Van
Nostrand Reinhold Cc. New York. 3067 pp.
CORPE W.A, 1974. Detachment of marine periphytic bacteria from surfaces of glass slides. Dev. Ind. Microbiol.
15 : 281-287.
COSTERTON J.W., G.G. GEESEY K.J. CHENG, 1978. How bacteria stick. Sci. Am. 238 : 86-95.
CUNDELL A.M., T.D. SLETTER and R. MITCHELL, 1977. Microbial populations associated with the surface of
brown alga Ascophyllum nodosum. Micro. Ecol. 4 : 81-91.
DALE N.G, 1974. Bacteria in interdial sediments : factors related to their distribution. Appl. Environ. Microbiol.
41 : 107-116.
DALEY R. J, 1979. Direct epifluorescence enumeration of native aquatic bacteria : uses, limitation and comparative accuracy. In : J. W. Costerton and R. R. Colwell (eds.) Native Aquatic Bacteria Enumeration, Activity, and
Ecology, ASTM STP 695. American Society for Testing and Materials, pp. 29-45.
257