. 2012 Oct 1;2(4):176–194. doi: 10.4161/biom.22905

Table 2. Techniques to study and quantify the microorganisms attached to a surface and the bacterial viability in biofilms.

Techniques	Advantages	Limitations
Colony Forming Units counting (CFU)	CFU plate counting is the most basic method for bacterial enumeration.⁶⁹^,¹⁷⁶^,¹⁷⁷ The washing is a very important part of a bacterial adhesion study using this technique, and its purpose is to remove the unattached and loosely adhered bacteria from the material surface. Methods for removing bacteria from substrata surfaces include homogenization, sonication, and the use of surfactants. According to the comparative study by McDaniel and Capone¹⁷⁶ sonication appears to be an efficient and safe way to remove bacteria from biomaterial surfaces. There are two basic ways to perform plate counting, the pour plate method and the surface spread method.¹⁷⁶^,¹⁷⁷	This technique is time consuming and involves tedious work, indirect and complicated procedures that give more uncertainty.¹⁷⁷ It detects only viable bacteria.¹⁷⁷
Light Microscopy	Technique for bacterial enumeration and observation. Normally bacteria are stained with dyes like crystal violet or fuchsin. Some special staining methods allow the observation of bacterial surface structures such as capsules, or appendages.¹⁷⁶^,¹⁷⁷ Light microscopy has been combined with a bacterial flow chamber to observe living attached bacterial cells in real time.¹⁷⁶^,¹⁷⁷ A transparent material forms part of the wall of a cell flow chamber so that the bacteria attached to the inner side of the material might be directly observed. The advances in image analysis make bacterial counting by light microscopy much faster and more efficient.¹⁷⁶^,¹⁷⁷	The substrata surfaces have to be translucent to be able to use light microscopy.¹⁷⁶^,¹⁷⁷
Epifluorescence microscopy	It allows to differentiate between live and dead bacterial cells on the surface, if certain fluorochromes are used.¹⁷⁸^,¹⁷⁹ Image analysis systems are used for determining the number of cells adhered.¹⁷⁸ It makes direct observation and enumeration possible for attached bacteria on an opaque surface. Relatively fast, easy method for biofilm characterization that is especially suitable for a large set of samples.¹⁷⁷^,¹⁷⁹ Wirtanen et al.¹⁸⁰ evaluated the efficacy of various disinfectants against biofilms of Pseudomonas aeruginosa and Pseudomonas fragi on stainless steel surfaces using epifluorescence microscopy coupled with acridine orange.	Two-dimensional imaging only.¹⁷⁹ The use of fluorochromes is necessary for viewing bacteria.¹⁷⁹ Limited to macroscopic investigation of bacteria-surface interactions.¹⁷⁹
Scanning Electron Microscopy (SEM)	SEM is a well-established basic technique to observe the morphology of bacteria adhered on a material surface, the material surface morphology, and the relationships between the two. It is also used to observe the morphology of bacterial biofilms on surfaces.¹⁷⁶^,¹⁷⁷^,¹⁸¹ Environmental SEM or Low Vacuum SEM do not require metal or carbon sputtering and is less prone to damaging the bacteria adhered on a surface or alter the surface characteristics of the specimen, therefore overcoming the referred drawbacks. Chemical composition of samples can be determined by using energy-dispersive X-ray (EDX) for elements with Z > 6.¹⁷⁹ SEM has previously been used to visualize biofilm development of S. epidermidis on contact lenses,¹⁸² extensive biofilms on endoscope tubing samples that had been sent for endoscope servicing¹⁸³ and the development of biofilms on catheters.¹⁸⁴	SEM has been used for the enumeration of adhered bacteria, but, because of the small field and time-consuming work, it is less adequate for this purpose.¹⁷⁷ It requires samples preparation for observation and the procedure for preparation can be tedious and labor intensive.¹⁷⁹ It requires the specimen to be conductive (essentially “metal sputtered”). Cannot differentiate between live and dead bacterial cells.¹⁷⁹ During sample preparation the drying step is considered to cause noticeable cell shrinkage and it exacerbates other undesirable outcomes, like damage and distortion of the biofilm.¹⁷⁹ It also requires specialist equipment, training and extensive samples preparation.¹⁷⁸
Confocal Scanning Laser Microscopy (CSLM)	CSLM is a three-dimensional technique using fluorescent molecular probes and laser beams to study in situ bacterial associations with surfaces.¹⁸⁵ It is used to visualize and count bacterial cells directly on transparent or opaque surfaces. It allows the examination of living fully hydrated biofilms in real time, and the simultaneous use of specific molecular probes allows to determine the identity (oligonucleotide probes) and the physiological state (live vs. dead) of the adherent bacterial cells.¹⁸⁶^,¹⁸⁷ This CSLM-based technique may be used to accurately assess the antibacterial properties of biofilm-resistant biomaterials.¹⁸⁶ This technique offers several advantages, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens.¹⁸⁷^-¹⁸⁹ Burnett et al.¹⁹⁰ observed the attachment of E. coli O157:H7 to apple tissue by confocal scanning laser microscopy. Lindsay et al.¹⁹¹ visualized co-cultured biofilms of Pseudomonas fluorescens M2 and Bacillus cereus DL5 on stainless steel surfaces.	The bacteria need to be colored or labeled with oligonucleotide probes for visualization.¹⁸⁶^,¹⁸⁷ Requirement of a CLSM to obtain the requested image quality is expensive.¹⁹²
Atomic Force Microscopy (AFM)	AFM has proved to be useful in imaging the morphology of individual microbial cells and bacterial biofilm on solid surfaces, both in dried and hydrated states.¹⁹³ It is used for mapping interaction forces at microbial surfaces.¹⁹⁴^-¹⁹⁹ AFM is a non-invasive microscopic technique capable of imaging surfaces at nanometer resolutions,¹⁸⁵ and three-dimensional images at high resolution.¹⁷⁹ Furthermore, as no stains or coatings are needed in this method, biofilms may be observed in situ.¹⁸⁵ Preparation of sample surface is not required.¹⁷⁹ AFM can be used preferencially to other methods, such as scanning electron microscopy, as the technique has several major advantages. Since the sample do not need to be electrically conductive, no metallic coating of the specimen is required. Unlike the case with the SEM, no dehydration of the sample is required, and biofilms may be viewed in their hydrated state. The resolution of AFM is higher than that of the environmental SEM, where images can also be obtained with hydrated samples, and extracellular polymeric substances may not be imaged with clarity.¹⁷⁹ Within the medical context, AFM has been used to observe the effect of modified catheter surfaces on bacterial biofilm development.²⁰⁰^,²⁰¹	The observation area is limited as compared with SEM.¹⁶⁴ It cannot differentiate between live and dead bacterial cells.¹⁶⁴ Imaging bacterial cells can be a time consuming task.¹⁷⁹^,²⁰²
Fourier Transform Infrared Spectroscopy (FTIR)	Spectroscopic techniques provide a wealth of qualitative and quantitative information about a given sample. FTIR spectroscopy measures the vibrations of chemical bonds within all the biochemical constituents of cells (i.e., proteins, lipids, polysaccharides, and nucleic acids) and thus provides quantitative information about the total biochemical composition of the intact whole microbial cell.²⁰³^-²⁰⁷ Differences in the structure and quantity of cell wall polysaccharide, lipids and protein are reflected in the FTIR spectra enabling differentiation between bacterial strains.²⁰⁸ The FTIR method is rapid, non-invasive, accurate, automated, inexpensive and quantitative, allowing users to collect full spectra in a few seconds per sample.²⁰⁸^,²⁰⁹ FTIR spectroscopy has shown to be an effective tool for analyzing bacterial strains.¹⁹⁴ This technique has also been shown to have sufficient resolving power for differentiation between CNS and S. aureus.²¹⁰^,²¹¹ Amiali et al.²⁰⁷ observed that FTIR spectroscopy had considerable potential as a rapid (1 h) and simple method for MRSA strain typing and monitoring in clinical settings.	Its efficacy in differentiating metabolic changes of differentially induced bacteria or genetically identical bacterial strains on different growth substrates remains untested.²⁰⁵
Radiolabelling	This technique is useful in the study of bacterial adhesion to irregular material surfaces. It is very sensitive and very accurate, allowing for rapid processing of a large number of samples.¹⁷⁷^,²¹² For example, it was shown that the radiolabelling of bacteria was very useful for the studies of bacterial adhesion to irregular material surfaces, such as the surfaces of particles or spheres.²¹³	It requires special laboratory space and techniques for handling radioactive materials and it carries potential risk to performers.¹⁷⁷
Contact Angle measurements	In the contact angle technique, a water droplet is applied to the surface of a dried lawn of bacteria. The angle formed where the water contacts the organisms is proportional to the surface hydrophobicity of the bacteria.²¹⁴ Analysis is very quick to perform.¹⁷⁹ Fonseca et al.⁶⁵ evaluated the hydrophobicity of S.epidermidis RP62A (ATCC 35984) using contact angle measurements.	Contamination of test surface may cause error in the obtained values.¹⁷⁹
Molecular Biological techniques	It can identify the total community of bacteria attached to a surface. It offers a very sensitive method for detection of specific genes or species. A species of bacteria can be viewed in a heterogeneous community by fluorescently labeling by oligonucleotide probes.¹⁷⁹ Castonguay et al.²¹⁵ employed quantitative polymerase chain reaction (PCR) in their studies for confirmation of the presence of the two bacteria in a mature biofilm.	When using oligonucleotide probes, there is a requirement that they must bind specifically to the bacterial DNA sequence.¹⁷⁹
Colorimetric biomass assay (crystal violet)	This assay is used for quantification of biofilm biomass and crystal violet (CV) is frequently used. CV is a basic dye that stains both living and dead cells, by linking to negatively charge surface molecules and polysaccharides in the extracellular matrix.¹⁷⁰^,²¹⁶ CV assay is cheap, straightforward and is commonly used for the quantification of biofilms formed by a broad range of microorganisms.¹⁶⁹	It cannot differentiate between live and dead bacterial cells.¹⁶⁹ Moreover, because both living and dead cells, as well as matrix, are stained with CV, this method provides no information about viability.²¹⁷
Syto 9 assay	The fluorogenic dye Syto9 is a nucleic acid stain, which diffuses passively through cellular membranes and binds to DNA of both viable and dead cells.²¹⁸ As DNA is also a substantial part of the extracellular matrix,²¹⁹ this staining will provide information on total biofilm biomass. Syto9 has previously been used in CLSM studies of biofilm composition and morphology.²²⁰ This stain has also been used for the routine quantification of bacterial and yeast biofilm biomass.²²¹^,²²²	This assay includes high costs of Syto9.¹⁶⁹ It cannot differentiate between live and dead bacterial cells.¹⁶⁹
Resazurin assay	Resazurin is a common metabolic activity indicator that has been shown to be effective in assessing bacterial viability²²³ and in biofilm quantification.¹⁶⁹ Resazurin, the main component of Alamar Blue, is a blue redox indicator that can be reduced by viable bacteria in the biofilm to pink resorufin,²²⁴ thus continued growth maintains a reduced environment (pink) and the extent of conversion from blue to pink is a reflection of cell viability.²²⁵ Peeters et al. showed that resazurin viability assay is a good alternative for quantification of microbial biofilms grown in microtiter-plates.¹⁶⁹	It is necessary to construct a calibration curve.²²⁶
Fluorescein diacetate (FDA) assay	Viable microbial cells are capable of converting non-colored, non-fluorescent fluorescein diacetate (FDA) into yellow, highly fluorescent fluorescein by non-specific intra- and extracellular esterases. FDA has been used for the quantification of biofilm biomass and viability.²²¹^,²²⁷	It is necessary to construct a calibration curve.¹⁶⁴