Table 3.

Summary of major direct and indirect methods for characterization of biofilms.

	Time to Complete	Specialized Equipment Required	Biofilm Preparation	Notes	References
Plate Count, Viable cell enumeration	1–3 days	Incubator:- Consumables: disposable petri plates, culture flasks, suitable agar and medium	Cells are removed from the substrate, homogenized, resuspended in liquid medium, diluted, and aliquots are plated, incubated and counted.	Most readily adaptable to liquid/ planktonic cultures. This method only quantifies live cells, and an assumption is made that each colony derives from one original cell. The differing metabolic states of living cells in the biofilm may complicate determination of accurate number of cells in the biofilm. Must be confirmed by cell mass or surface area.	[21,23,25]
Light Microscopy	Minutes	Compound, brightfield microscope	Biofilm can be grown directly on a transparent substrate, such as a slide or coverslip, stained and observed directly.	Counting or observing mature biofilms is limited, as accumulation of extensive biofilm mass prevents observation of individual cells. Can be used in conjunction with dry mass measurements to acquire biofilm thickness and quantifying specific visual characteristics of the biofilm.	[19,52]
Manual Cell Counting Using a Microscope	Minutes to hours	Hemacytometer: Brightfield or Fluorescent Microscope, Cell Stains	Biofilm must be removed from the substrate and homogenized.	Relatively inexpensive, reusable, and easy to learn, but tedious. It does not distinguish live and dead cells, and motile cells are extremely difficult to count accurately unless fixed (killed).	[16,27,28]
Automated Cell Counting, Coulter Counter	One to a Few Hours	Coulter counter	Biofilm must be removed from the substrate and homogenized	Coulter counter quantifies cells and particles and can distinguish entities by size. Very small cells may be difficult to count accurately.	[31,32]
Automated Cell Counting; Flow Cytometry	A few hours – more labeling and separation may take longer.	Flow cytometer: Antibodies or fluorescent stains	Biofilm must be removed from the substrate and homogenized. Flow cytometry requires cell populations to be labeled with a separate fluorophore for each cell type to be isolated. May not be efficient if you are only trying to count cells.	Flow cytometry can distinguish different cell types. This can be expensive, and requires technical expertise.	[33,34]
Fluorescent Microscopy and Staining	20 – 30 minutes.	Brightfield/ Fluorescent Microscope: Fluorescent stains, antibodies or endogenous fluorescent proteins	Biofilm can be grown directly on a slide or coverslip, stained, and observed in situ. Biofilm is stained with view of the desired outcome.	Some stains are potential mutagens. Stain should be chosen carefully--not all stains penetrate the cell membrane, and not all are compatible with maintaining a living biofilm.	[14,27,37,140,140]
Confocal Fluorescent Microscopy	One to a few hours.	Confocal Fluorescent Microscope: Fluorescent stains, antibodies or endogenous fluorescent proteins	Can image in place biofilm (on a coverslip, e.g.). Cells must be labeled. Fluorophores can be selected according to a variety of purposes, such as distinguishing live and dead cells, staining nuclei/DNA, etc.	Biovolume can be calculated with appropriate software and computing capability. Usually requires a dedicated technician to run and maintain the instrument. Can image any cell or particle that has a fluorescent label that can be detected by the microscope. It is better used for structures and 3D architecture than counting cells. Can image within the thickness of the biofilm and assemble z-stacks.	[20,35,38,41,44,126,170]
Determination of Dry Mass	Three – four hours.	Analytical Balance: Lab oven capable of reaching 100 °C	Film on substrate is dried, massed, then cleaned. Substrate is massed again.	Film area should be measured; thickness can be measured to give dry mass per unit of wet volume.	[42,43,48,63,88]
Total Organic Carbon	15 minutes per sample.	TOC instrument	Homogenization and resuspension.	A standard protocol can discriminate between carbon in EPS and cellular carbon.	[17,24,54]
Crystal Violet Assay	Two – four hours over two days.	Plate reader or UV/VIS spectrophotometer: Gram Stain	Indirect measure of biofilm growth. Cells are stained with crystal violet, washed, and the absorption of CV measured. Higher absorption relates to more biofilm mass.	Individual wells are somewhat variable, so controls, standards, and replicates are important. A 96-well plate adaptor is required. Also requires standard 96-well plates with flat bottoms.	[25,58,59,66,88]
Tetrazolium Salt Assay	Two – four hours.	Tetrazolium Salt:- Evaluation method: Microscopy, Flow Cytometry, Spectroscopy, etc.	Direct or Indirect measure of biofilm growth. Cells are incubated with tetrazolium salt that is metabolically converted to formazan derivative. Evaluated via spectroscopy, microscopy or flow cytometry depending on the solubility of the formazan.	Insoluble formazan salts will be trapped in the cell membrane allowing for direct individual cell analysis. Soluble formazan may be collected from the media and quantified for an indirect quantification. Only living cells will convert the salt to formazan providing a measure of viability.	[23,27,63,65–68]
ATP Bioluminescence	A few hours.	Assay Kit: Luminometer	Incubation of biofilm on soy broth for up to 5 days.	The reagent is stable for one day at 15 to 25 °C or for one week when stored at 0 to 4 °C.	[73,75,77,78]
Total Protein	A few hours	Assay Kit: UV-Vis spectrophotometer	Biofilms are scraped from their substrates and homogenized in a liquid suspension, often using a commercial homogenizer.	Protein determination methods are subject to interference from other substances potentially present, such as certain ions, detergents, reducing agents, or other species.	[88,90,91,94]
QCM & QCMD	Minutes	Quartz crystal microbalance Bioreactor	For accumulation measurement, a calibration between frequency and cell number must be done.	Material property information from QCMD requires an appropriate theoretical model.	[96,99–101,104,106]
SEM	Hours to Days	Scanning electron microscope: Sputtering Coater	Biofilm can be grown on a coverslip (or other substrate) and directly imaged on the microscope. Samples must be fixed, dried, and coated with metal (Pt-Pd).	Toxic chemicals may be involved in some fixation techniques. Usually requires a maintenance contract and special housing conditions.	[21,39,88, 124–126,131,135,137]