Cryosectioning of biofilms for microscopic examination |
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| Authors: | Feipeng P Yu Gayle M Callis Philip S Stewart Thomas Griebe Gordon A Mcfeters |
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| Affiliation: | 1. Center for Biofilm Engineering , Montana State University , Bozeman, MT59717, USA;2. Microbiology Department , Montana State University , Bozeman, MT59717, USA;3. Veterinary Molecular Biology , Montana State University , Bozeman, MT59717, USA;4. Chemical Engineering Department , Montana State University , Bozeman, MT59717, USA;5. Institut für Siedlungswasserbau , Universitat Stuttg?rt , Wassergüte und Abfalwirtschaft, Abtielung Chemie, Bandt?le 2, Stuttgart (Büsnau), 70569, Germany |
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| Abstract: | A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h. |
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| Keywords: | biofilm cryosectioning microscopy |
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