Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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.......

Discussion

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.......

Materials

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