JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Published: April 22nd, 2022

DOI:

10.3791/62390

1Center for Biofilm Engineering, Montana State University

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

The definition of biofilm has evolved over the last few decades and encompasses microbial association with a variety of biological and/or non-biological surfaces, inclusion of noncellular components1 that display differing growth and genetic expression2 within a matrix. Biofilm provides protection from environmental stresses such as drying and may render the action of chemical disinfectants less effective resulting in the survival of microbes. The survivors within a biofilm can potentially provide a source of pathogenic microorganisms that are a public health concern3.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Vortexing and sonication

  1. Grow a mature P. aeruginosa ATCC 15442 biofilm grown according to ASTM Standard E25622.
  2. At the end of the 48 h growth period, prepare to treat the biofilm and sample coupons according to ASTM Standard E28718
  3. Aseptically insert autoclaved splash guards into sterile 50 mL conical tubes using flame-sterilized forceps. Repeat for all tubes that will receive treatment. Tubes for control coupons.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Validation/Confirmation of a Harvesting Method
Several studies that were conducted in our laboratory examined the ability of vortexing and sonication to effectively harvest biofilm grown in the biofilm reactor (ASTM E2562)2 using the Single Tube Method (ASTM E2871)8.

A P. aeruginosa ATCC 15442 biofilm was grown according to ASTM E25622 on borosilicate glass coupons. After 48 hours, four coup.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Minimum Information for Harvesting and Disaggregation Methods
To create reproducible biofilm data across the scientific community, it is imperative that authors include as much detail as possible regarding each of the growth, treatment, sampling and analysis steps of a biofilm method. The standardization of biofilm methods has aided in this endeavor as it allows the researcher to reference a specific method and any relevant modifications. However, many papers include only a sentence or two to descr.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We wish to acknowledge Danielle Orr Goveia, Blaine Fritz, Jennifer Summers and Fei San Lee for their contributions to this paper.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
50 mL conical vials Thermo Scientific 339652
100 mL glass beakers Fisher Scientific FB102100
5 mL serological pipettes Fisher Scientific 13-678-12D For adding treatment to vials containing coupons.
50 mL serological pipettes Fisher Scientific 13-678-14C For adding neutralizer to vials at the end of treatment contact time.
Applicator sticks Puritan 807
Hemostats Fisher Scientific 16-100-115
Metal spatula Fisher Scientific 14-373
PTFE policemen Saint-Gobain 06369-04
S 10 N - 10 G - ST Dispersing tool IKA 4446700 For homogenization of biofilm samples.
Scissors Fisher Scientific 08-951-20
Silicone Foley catheter, size 16 French Medline Industries DYND11502
Silicone tubing, size 16 Cole-Parmer EW96400-16
Splash Guards BioSurface Technologies, Inc. CBR 2232
T 10 basic ULTRA-TURRAX Disperser IKA 3737001 For homogenization of biofilm samples.
Tubing connectors Cole-Parmer EW02023-86
Ultrasonic Cleaner Elma TI-H15
Vortex-Genie 2 Scientific Industries SI-0236
Vortex-Genie 2 Vertical 50 mL Tube Holder Scientific Industries SI-V506

  1. Donlan, R. M. Biofilms: microbial life on surfaces. Emerging Infectious Diseases. 8 (9), 881-890 (2002).
  2. ASTM International. ASTM Standard E2562, 2017, Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor. ASTM International. , (2017).
  3. Azeredo, J., et al. Critical review on biofilm methods. Critical Reviews In Microbiology. 43 (3), 313-351 (2017).
  4. Gomes, I. B., et al. Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications. Critical Reviews in Biotechnology. 38 (5), 657-670 (2018).
  5. Goeres, D. M., et al., Simoes, M., et al. Design and fabrication of biofilm reactors. Recent Trends in Biofilm Sciences and Technology. , 71-88 (2020).
  6. Goeres, D. M., et al. A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor. Nature Protocols. 4 (5), 783-788 (2009).
  7. ASTM International. ASTM Standard E2871, Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single Tube Method. ASTM International. , (2019).
  8. Goeres, D. M., et al. Validation of a Biofilm Efficacy Test: The Single Tube Method. Journal of Microbiological Methods. , (2019).
  9. The development and validation of a standard in vitro method to evaluate the efficacy of surface modified urinary catheters. Theses and Dissertations at Montana State University Available from: https://scholarworks.montana.edu/xmlui/handle/1/15149 (2019)
  10. Hamilton, M. A., Buckingham-Meyer, K., Goeres, D. M. Checking the Validity of the harvesting and Disaggregating Steps in Laboratory Tests of Surface Disinfectants. Journal of AOAC International. 92 (6), 1755-1762 (2009).
  11. Conlon, B. P., Rowe, S. E., Lewis, K., Donelli, G. Persister Cells in Biofilm Associated Infections. Biofilm-based Healthcare-associated Infections. , (2015).
  12. ASTM International. ASTM Standard E2647, Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow. ASTM International. , (2020).
  13. Rosenberg, M., Azevedo, N., Ivask, A. Propidium iodide staining underestimates viability of adherent bacterial cells. Scientific Reports. , (2019).
  14. Stiefel, P., Schmidt-Emrich, S., Manuira-Weber, K., Ren, Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiology. , (2015).
  15. Kobayashi, N., Bauer, T. W., Tuohy, M. J., Fujishiro, T., Procop, G. W. Brief Ultrasonication Improves Detection of Biofilm-formative Bacteria Around a Metal Implant. Clinical Orthopaedics and Related Research. 457, 210-213 (2007).
  16. Lourenco, A., et al. Minimum information about a biofilm experiment (MIABiE), standards for reporting experiments and data on sessile microbial communities living at interfaces. Pathogens and Disease. , 1-7 (2014).
  17. Nascentes, C. C., Korn, M., Sousa, C. S., Arruda, M. A. Z. Use of Ultrasonic Baths for Analytical Applications: A New Approach for Optimisation Conditions. Journal of Brazilian Chemical Society. 12 (1), 57-63 (2001).
  18. Elma GmbH & Co. KG TI-H Ultrasonic Cleaning Units: Operating Instructions. Elma GmbH & Co. , (2009).
  19. Stamper, D. M., Holm, E. R., Brizzolara, R. A. Exposure times and energy densities for ultrasonic disinfection of Escherichia coli, Pseudomonas aeruginosa, Enterococcus avium and sewage. Journal of Environmental Engineering and Science. 7 (2), 139-146 (2008).
  20. Suslick, K. S. Sonochemistry. Science. 247 (4949), (1990).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved