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Comparison of microscope techniques for the examination of biofilms

Comparison of microscope techniques for the examination of biofilms
Comparison of microscope techniques for the examination of biofilms

Biofilms developed on solid supports suspended within a continuous culture model water system. The system inoculum was composed of a diverse consortium of microorganisms obtained from a naturally occurring aquatic source. Biofilms were removed and either directly visualised or fixed and stained prior to examination. Several techniques for microscopy examination of biofilms in situ on the substrata supporting their growth have been used in this study. These have included transmission electron microscopy (TEM), scanning electron microscopy (SEM), environmental scanning electronmicroscopy (ESEM), episcopic differential interference contrast microscopy (DIC) with and without fluorescence, Hoffman modulation contrast microscopy (HMC), atomic force microscopy (AFM) and scanning confocal laser microscopy (SCLM). A comparison of the advantages of the individual techniques to visualise biofilms is discussed. Cross-sections prepared for TEM analysis gave useful information about the spatial relationships of microorganisms within the biofilm matrix, whilst SEM enabled the surface topology of the biofilms to be examined at a high magnification. The preparation required for TEM and SEM, may however, result in the inclusion of artefacts. ESEM and AFM allow direct visualisation of intact hydrated specimens at high magnification. AFM images may be rotated and manipulated to provide accurate measurement of individual microorganisms with relative ease. SCLM was used to investigate not only the presence and the viability of the biofilm consortium but also biofilm/substrata interactions. Light microscopy techniques, although unable to reproduce the high magnification of the methods described above, are still of importance in the examination of intact biofilms. HMC allows the in situ examination of biofilms, a clear image is produced without artefacts. DIC may be used to examine biofilms on opaque surfaces and if used in conjunction with fluorescent vital stains can be used to assess the viability of the microbial population.

atomic force microscopy, biofilms, electron microscopy, fluorescence microscopy, light microscopy, scanning confocal laser microscopy
0167-7012
57-70
Surman, S. B.
3141c462-c96c-4adb-b176-39ca443f6804
Walker, J. T.
2bb5ed4e-d929-47e4-97ba-70641716acd7
Goddard, D. T.
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Morton, L. H.G.
922ad180-4bfc-4894-b83c-8e468d94a9d9
Keevil, C. W.
cb7de0a7-ce33-4cfa-af52-07f99e5650eb
Weaver, W.
dc69a0ed-0acb-48d1-bad3-7cad3b3581d0
Skinner, A.
18164806-3926-43b4-8dda-8a2635bad76c
Hanson, K.
fe27ab72-eddf-4d80-a576-398d007528de
Caldwell, D.
0a8be36b-14f7-4371-8839-67ec67b41505
Kurtz, J.
0758951b-fd09-4416-873b-964a071d04d4
Surman, S. B.
3141c462-c96c-4adb-b176-39ca443f6804
Walker, J. T.
2bb5ed4e-d929-47e4-97ba-70641716acd7
Goddard, D. T.
45e161e7-7e9e-456a-b75a-3feaf7dcda4a
Morton, L. H.G.
922ad180-4bfc-4894-b83c-8e468d94a9d9
Keevil, C. W.
cb7de0a7-ce33-4cfa-af52-07f99e5650eb
Weaver, W.
dc69a0ed-0acb-48d1-bad3-7cad3b3581d0
Skinner, A.
18164806-3926-43b4-8dda-8a2635bad76c
Hanson, K.
fe27ab72-eddf-4d80-a576-398d007528de
Caldwell, D.
0a8be36b-14f7-4371-8839-67ec67b41505
Kurtz, J.
0758951b-fd09-4416-873b-964a071d04d4

Surman, S. B., Walker, J. T., Goddard, D. T., Morton, L. H.G., Keevil, C. W., Weaver, W., Skinner, A., Hanson, K., Caldwell, D. and Kurtz, J. (1996) Comparison of microscope techniques for the examination of biofilms. Journal of Microbiological Methods, 25 (1), 57-70. (doi:10.1016/0167-7012(95)00085-2).

Record type: Article

Abstract

Biofilms developed on solid supports suspended within a continuous culture model water system. The system inoculum was composed of a diverse consortium of microorganisms obtained from a naturally occurring aquatic source. Biofilms were removed and either directly visualised or fixed and stained prior to examination. Several techniques for microscopy examination of biofilms in situ on the substrata supporting their growth have been used in this study. These have included transmission electron microscopy (TEM), scanning electron microscopy (SEM), environmental scanning electronmicroscopy (ESEM), episcopic differential interference contrast microscopy (DIC) with and without fluorescence, Hoffman modulation contrast microscopy (HMC), atomic force microscopy (AFM) and scanning confocal laser microscopy (SCLM). A comparison of the advantages of the individual techniques to visualise biofilms is discussed. Cross-sections prepared for TEM analysis gave useful information about the spatial relationships of microorganisms within the biofilm matrix, whilst SEM enabled the surface topology of the biofilms to be examined at a high magnification. The preparation required for TEM and SEM, may however, result in the inclusion of artefacts. ESEM and AFM allow direct visualisation of intact hydrated specimens at high magnification. AFM images may be rotated and manipulated to provide accurate measurement of individual microorganisms with relative ease. SCLM was used to investigate not only the presence and the viability of the biofilm consortium but also biofilm/substrata interactions. Light microscopy techniques, although unable to reproduce the high magnification of the methods described above, are still of importance in the examination of intact biofilms. HMC allows the in situ examination of biofilms, a clear image is produced without artefacts. DIC may be used to examine biofilms on opaque surfaces and if used in conjunction with fluorescent vital stains can be used to assess the viability of the microbial population.

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More information

Accepted/In Press date: 27 July 1995
Published date: March 1996
Keywords: atomic force microscopy, biofilms, electron microscopy, fluorescence microscopy, light microscopy, scanning confocal laser microscopy

Identifiers

Local EPrints ID: 431346
URI: https://eprints.soton.ac.uk/id/eprint/431346
ISSN: 0167-7012
PURE UUID: e71b5cfb-e2eb-4a6d-a491-6a3efc8ff3ef
ORCID for C. W. Keevil: ORCID iD orcid.org/0000-0003-1917-7706

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Date deposited: 29 May 2019 16:30
Last modified: 12 Nov 2019 01:51

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