Table 2.
Methods for Biofilm Analysis used with P. aeruginosa and S. aureus biofilms in vitro.
| Category of Evaluation | Principle/Target | Method Overview | Detection Method | Example Detection Settings | Example Model Systems | References |
|---|---|---|---|---|---|---|
| Viability | ||||||
| Colony counting | Viable cells are able to form colonies when plated on appropriate agar substrates | Dispersions of cells are spread or drop-plated. Colonies formed counted after appropriate growth period | Visual | Agar plate | [136,180,208] | |
| SYTO 9 | All Cells | Nucleic acids stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: 485 nm Em: 528 nm |
Microtiter plate | [150] |
| SYTO 9/PI | All cells (SYTO9) Dead/membrane permeable cells (PI) |
Nucleic acids stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: 485 nm Em: 528 or 645 nm |
Microtiter plate, flow cell, collagen model | [150,157,158,159] |
| Acridine orange | All cells (nucleic acids) | Nucleic acids stained and dispersed by vortexing in appropriate media | FS | Ex: 485 nm Em: 528 nm |
Microtiter plate | [150] |
| Ethidium bromide | DNA | DNA stained and visualized. Appears orange when excited | FS, FM | Ex: 210 or 285 nm Em: 605 nm |
Constant depth film fermenter (CDFF) and glass microscopy slide | [122] |
| Ziehl carbol fuchsin | Bacterial cells | Stains bacterial cells red/purple | LM | CDFF and glass microscopy slide | [122] | |
| DAPI | DNA | DNA stained and visualized. | FM, LM | Ex: 350 nm Em: 470 nm |
Microtiter plate and glass slide | [136] |
| Metabolic Activity | ||||||
| Tetrazolium Salts (INT, TTC, CTC, XTT, and MTT) | Reduction of Tetrazolium to formazan | Dissolved dye from stained biofilms recovered and quantified | AS | INT: 470 nm TTC: 405, 450, 490, 540 nm XTT: 450-492 nm (486nm) |
Microtiter plate, modified agar plate | [150,161,162,172] |
| Resazurin (Alamar Blue, PrestoBlue, CellTiter-Blue) | Reduction of Resazurin to resorufin | Reagent incubated with media and biofilms | FS, AS | Ex: 560 nm Em: 590 nm Abs: 570 and 600 nm |
Microtiter plate | [173,174,208] |
| Bioluminescent Assay (BacTiter) | Catalysis of ATP and luciferin by luciferase | D-luciferin is used in these assays as it undergoes conversion by luciferase to oxyluciferin a light generating compound when in the presence of ATP | L*S | Microtiter plate | [150,179,180,181,209] | |
| Fluorescein diacetate (FDA) | Cleavage of acetate by intracellular esterases | FDA converted to yellow fluorescent fluorescein | FS | Ex: 494 nm Em: 518 nm |
Microtiter plate | [182] |
| Biomass | ||||||
| Crystal violet | Stains negatively-charged molecules and polymers. All biomass (live, dead, and matrix) | Stained biofilms dissolved in appropriate solvent | AS | 550 - 600 nm | Microtiter plate | [102,185] |
| Congo red | Polysaccharides and cell membranes | Stained biofilms dissolved in appropriate solvent | AS | 500nm | Microtiter plate CDFF and glass microscopy slide |
[122,150] |
| Safranin | Nuclei and mucin red | Stained biofilms dissolved in appropriate solvent | AS | ~535 nm | Microtiter plate | [150,187] |
| Calcofluor white | beta-polysaccharides in matrix | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, AS | Ex: 360, 365, or 400 nm Em: 460, 435, 410 nm |
Microtiter plate CDFF and glass microscopy slide |
[122,150,188,189] |
| SYPRO Ruby | proteins | Biofilms stained and dispersed by vortexing in appropriate media | FS | Ex: 450 or 460 nm Em: 610 or 645 nm |
Microtiter plate | [150,188] |
| FITC | proteins and amino-sugars | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: ~488 nm Em: 500-550 nm |
Microtiter plate | [150,188,189] |
| Concanavalin A (Con A) conjugates | alpha-Mannopyranosyl and alpha-glucopyranosyl sugars | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: 543 nm Em: 550-600 nm |
[189] | |
| FITC-Con A | polysaccharides | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: 485 nm Em: 528 nm | Microtiter plate | [150] |
| Con A - Tetramethylrhodamine | Alpha polysaccharides | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FS, FM | Ex: 555 nm Em: 580 nm |
Biofilm reactor | [188] |
| Periodic acid-Schiff (PAS) | Stains polysaccharides | LM | Microtiter plate and glass slide | [136] | ||
| van Gieson | Stains collagen Fibers and Bacterial DNA | LM | Microtiter plate and glass slide | [136] | ||
| Vybrant DiD | Lipids and Membranes | Biofilms stained and visualized (M) or dispersed by vortexing in appropriate media (S) | FM | Ex: 644 nm Em: 665 nm |
Biofilm reactor | [188] |
| Turbidity | ||||||
| Turbidity threshold method | Quantification of dispersed cells | Measure absorbance of bacterial suspension and bacteria-free media and compare to a known dilution series | AS | 600 nm | Microtiter plate | [150,209] |
| MacFarland standards | Quantification of dispersed cells | Measure absorbance of bacterial suspension and McFarland Standards (mixtures of H2SO4+BaCl2 or latex particles) | AS | 625 nm | Microtiter plate | |
| Structure | ||||||
| Scanning electron microscopy (SEM) | Visualization of morphology and distribution of microorganisms and extracellular matrix (ECM) | Biofilms typically fixed and negatively stained (SEM) | SEM/Cryo-SEM/ESEM | Varies by instrument | Flat-bed perfusion system, collagen model | [158,181,209,210] |
| Confocal scanning laser microscopy (CLSM) | Isolation of 3D microbial community | Use applicable stains and dyes listed above to visualize various aspects of the biofilm | FM | Varies by stain/dye | Glass microscopy slide, flow cell | [209,211] |
| Fluorescent in-situ hybridization (FISH/PNA-FISH) | Visualize patterns of microbial colonization | Fluorescently labeled oligonucleotide probes hybridize to ribosomal RNA in cells that have been fixed and permeabilized | FM | Varies by stain/dye | Glass microscopy slide | [136,177,209] |
| Raman microscopy | Mapping of microorganisms and ECM Raman spectra | RM | Varies by instrument/target | Raman-neutral slide | [209] | |
| Mechanics | ||||||
| Atomic force microscopy (AFM) | Mapping of local and global adhesive and cohesive forces | Measure force-displacement curves | Varies by instrument/target | [97,212] | ||
| Micro-rheology | Measure behavior of isolated bacteria under different physical conditions | FM, LM | Varies by stain/dye | Flow cell | [202] | |
| Bulk rheology | Biofilms have viscoelastic properties | Measure viscoelastic properties of ECM matrix with microorganisms | Rheometer | Varies by instrument | Agar plate, colony system | [97] |
| Other | ||||||
| Agar disk/well-diffusion | Zone-of-inhibition of therapies measured | Agar plates inoculated with bacteria are exposed to a therapy within a defined area. Following growth period, area of new growth measured | Visual | Agar plate | [178] | |
Analysis: PI: propidium iodide; DAPI: 4′,6-diamidino-2-phenylindole; INT: 2-(p-iodo-phenyl)-3-p-(nitrophenyl)-5 phenyltetrazolium chloride; TTC: 2,3,5-triphenyl-tetrazolium chloride; CTC: 5-cyano-2,3-ditolyl tetrazolium chloride; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FITC: fluorescein isothiocyanate; PNA: peptide nucleic acid. Detection Methods: M: Microscopy; S: Spectroscopy; F: Fluorescence; A: Absorbance, L: Light; L*: Luminescence; R: Raman; Ex: Excitation wavelength; Em: Emission wavelength.