Journal of Indusb'ial (1995) 15, 263-276 1995 Society for Industrial Microbiology0169-4146/95/$12.00

Continuous nondestructive monitoring of microbial : a review of analytical techniques DE Nivens 1,2, RJ Palmer Jr 2 and DC White z3,4

I Microbial Insights Inc, Knoxville, TN; 2Center for Environmental Biotechnology; 3Department of Microbiology, University of Tennessee, Knoxville, TN; 4Environmental Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

A fundamental requirement for the understanding and control of biofilms is the continuous nondestructive monitor- Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 ing of processes. This paper reviews research analytical techniques that monitor biofilm processes in a continuous nondestructive manner and that could also be modified for industrial applications. To be considered 'continuous' and 'nondestructive' for the purpose of this review a technique must: (a) function in an aqueous sys- tem; (b)not require sample removal; (c) minimize signal from or contaminants in the bulk phase; and (d) provide real-time data. Various microscopic, spectrochemical, electrochemical, and piezoelectrical analysis methods fulfill these criteria. These techniques monitor the formation of biofilms, the physiology of the microorgan- isms withiin biofilms, and/or the interaction of the biofilms with their environment. It is hoped that this review will stimulate development and use of biofilm monitoring techniques in industrial and environmental settings.

Keywords: biofilm; on-line monitoring; nondestructive monitoring; microscopy; Fourier-transform spectrometry; bio- luminescence; microelectrode; quartz crystal microbalanee

Introduction within municipal delivery systems (1 x l01 to 2.2 x 106 colony forming units per cm 2) [79] to the centi- A biofilm has been defined as ' ... cells immobilized meters-thick sludge blankets in sewage treatment sys- at a substratum and frequently embedded in an organic tems. A biofilm in a biotechnological treatment process polymer matrix of microbial origin ... which is not may be a bacterial monoculture, while natural biofilms necessarily uniform in time or space.., composed of a such as the periphyton covering streambed surfaces tend significant fraction of inorganic or abiotic substances to be made up of many species. The composition of a held together by the biotic matrix' [24]. Microbial com- biofilm typically includes (besides the organisms munities at gas-solid, gas-liquid, and liquid-liquid inter- themselves) trace organics which concentrate at surfaces, faces also exist [94], but are not considered here. Bac- extracellular polysaccharides elaborated by the , teria and microeukaryotes (fungi, algae, and protozoans) and particulate matter trapped in the polysaccharide readily attach to surfaces to form biofilms, and in nature matrix. The complexity and variability of biofilms can most are attached rather than free in the significantly increase the difficulty of analysis. bulk-liquid phase. The importance of microbial biofilms Application of classical bacteriological techniques to the has been established in, for example, aquatic, , indus- study of industrial process problems such as biofouling and trial, and clinical environments [1,26,45,73]. Advantages corrosion has demonstrated that microorganisms can play for microorganisms within biofilms include increased a significant role in the corrosion of metals [89], the availability of nutrients concentrated on substrata, fix- deterioration of concrete [40], and the clogging of pipelines ation in a flowing system increasing the availability of and filters [116,132]. Heterogeneous distribution of dissolved nutrients, and the creation of 'microniches' microbes and/or their metabolic activity can promote such as anaerobic sites within an aerobic environment. microbiologically influenced corrosion. This multibillion Attachment can prevent transfer to a hostile environment dollar problem [106] is not well understood even after dec- such as, for oral microorganisms, from the hospitable ades of research. Microbial biofouling is ordinarily the pri- conditions of the mouth to the acidic anaerobic gut. The mary step in macrofouling (eg barnacle attachment) and disadvantages of within a biofilm can include compe- results in a substantial decrease in the hydrodynamic tition for nutrients and the availability to predators of a efficiency of ship hulls [11]. Biofilms can seriously concentrated, sessile . The structure, function, decrease the efficiency of heat transfer units [23]. Plank- and physiology of biofilms is beyond the scope of this tonic organisms in ultra-pure water are often a result of work, and ihas been extensively reviewed [23-25,94]. biofilm growth elsewhere in the system [107]. Biofilm for- The characteristics of individual biofilms are extremely variable. The density ranges from the mation on medical implants can be life-threatening because the body's natural defenses, even when augmented by extremely sparse communities of oligotrophic bacteria potent antibiotics, are usually ineffective against these infections [26]. Correspondence: DE Nivens, Microbial Insights, 201 Center Park Dr, In other biotechnological or processes, it Suite 1140, Knoxville, Tennessee 37922-2105, USA is an advantage to promote biofilm formation and maintain Received 23 January 1995; accepted 8 June 1995 the active biomass. Municipal wastewater treatment is Continuousnondestructive monitoring of biofilms DE Nivenset al 264 largely dependent upon biodegradation of organic waste by digital microscopy have been complemented by rapid microbial biofilms and aggregates [83]. In situ biodegrad- advances in the number and selectivity of dyes used to ation of organic wastes in subsurface aquifers requires the detect biochemical and physiological characteristics of development of sufficiently active subsurface microbial cells. Many of these dyes have a low toxicity and can be biofilms [61]. The stability and of many indus- washed out of the cells (eg fluorescein, certain rhodamines) trial bioprocessing and food procedures [139] thereby making repetitive (temporal is increased by the presence of appropriate microbial measurements) possible (R Palmer and J Almeida, Univ. biofilms. Biofilms are even implicated in the maintenance Tennessee, unpublished). of health in vertebrates--the attached to the Some of the earliest microscopic observations that could inner lining of the gut produce vitamins and aid in diges- be termed on-line were in situ microscopy of algae and tion [127]. bacteria in a pond [126], and temporal studies of soil bac-

Consequently, it is important that biofilm microbial ecol- teria in capillary channels [5,110]. Staley [126] utilized a Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 ogy be understood so that biofilm development can be pre- partially immersed microscope to observe the colonization vented or advantageously manipulated. Unfortunately, the of a slide by bacteria and algae. The capillary technique, most commonly applied methods are destructive, requiring which was used to follow the growth of bacteria with dis- either samples of the biofilm-substratum system, or removal tinctive morphologies such as Caulobacter and Pedomicro- of the biofilm from the substratum. This often results in bium, has never been widely used because it requires speci- disruption of the biofilm and usually requires the biofilm alized glassware and because conditions within the to pass through an air-water interface which can damage capillary (Eh, pH, tension) could not be controlled and/or contaminate the sample. The three-dimensional or monitored. architecture of a biofilm will be lost if analysis procedures The problems of microscopy under field conditions are require fixed and dried samples. Methods for removing difficult, and a more convenient way to examine attached biofilms from the substratum are inefficient and may cause microorganisms is through the use of flowcells. Laminar lysis of the cells. If organisms recovered through biofilm flow conditions can be created in parallel-plate flowcells, removal are regrown ex situ, they are then subject to severe and microscopy of the biofilm that develops on a glass sub- selection by the medium [1,117,135]. However, the greatest stratum can be performed [21]. The flowcells, which are problem with destructive analyses is that the inherent varia- simple, versatile, and inexpensive modifications on a design bility of successive samples may completely mask the originated by Berg and Block [9], can be adapted for development of the biofilm over time. microscopy of biofilms under a wide variety of conditions. The problems outlined above can best be overcome by Movement of Pseudomonas fluorescens within the hydro- use of continuous nondestructive monitoring techniques. dynamic boundary layer was examined photomicroscop- For the purpose of this review, a technique is classified ically [75]. Images were analyzed using the 'turnkey' IBAS as continuous and nondestructive if it allows direct system (Kontron, West Germany). The same techniques measurement of biofilm parameters in an aqueous system were used to examine the effect of laminar flow velocity in real time. Furthermore, the technique should be nonin- on attachment and growth rate of a wild-type P. fluorescens vasive and should minimize interferences from microor- and of a motility-mutant that lacked flagella [70]. Also, the ganisms in the bulk phase. In an industrial situation, attachment, colony formation, and motion of Vibrio para- these techniques could be used on-line (monitoring of a haemolyticus strains on a glass surface was followed under process via a sampling tube) or in-line (direct monitoring high- and low-viscosity conditions using similar techniques of the process). [77]; a wide range of phenotypic variability was seen among the various strains. Long-working-distance optics have been used in conjunction with thicker flowcells to examine the influence of particle (streptococcal cells, poly- Microscopic techniques styrene lattices) surface characteristics on attachment to Visualization of microorganisms has always been critical glass [122]. Automated data collection for this system was to understanding microbial interrelationships, particularly realized by integration of a charge-coupled-device camera those that occur on surfaces. In many industrial appli- with an image analysis system and continuous, real-time cations, it is the interaction of microorganisms with sur- measurements of streptococcal deposition were performed faces that creates process problems. Despite the huge var- [ 123]. The effects of different substrata (fluoroethylene-pro- iety of techniques available, microscopy remains under- pylene copolymer, polymethylmethacrylate, glass, mica) on utilized in biofilm studies. However, the potential for on- attachment of streptococci [124] were also analyzed. line microscopic investigation of industrial problems has Measurement of microbial co-adhesion in a flowcell has never been greater, especially in light of rapid advances in been described [12]. Actinomyces cells were allowed to computer hardware and software. For example, the field of adhere to the glass substratum, images were captured after view in a microscope can now be rapidly converted to a an adherence phase, then streptococcal cells were intro- digital image at a resolution approaching that obtained by duced and images were captured after these cells had sub- photomicrography. The digital images can be manipulated sequently adhered. Subtraction of the Actinomyces-only with image analysis software, thereby permitting cells in a image from the Actinomyces-plus-Streptomyces image gave field of view to be counted automatically in a fraction of an image that was then used to determine the number of the time needed for other enumeration procedures. The high Streptomyces cells inside a 0.16-/xm shell around each Acti- sample throughput and compact data storage offered by nomyces cell (co-adherence). Continuousnondesb'uctive monitoring of biofilms DE Nivenset al 265 These studies demonstrate the uses of microscopy, flow- brane potential) in three dimensions within living biofilms cells, and image analysis. It is important to recognize that in real time. Detail on these varied topics is outside the use of a flowcell establishes certain parameters, such as the scope of this article. The reader is referred to excellent hydrodynamic regime, that may not exist in the system to sources on theory and practice [13,108]. A multitude of be examined or that exist only at certain locations, such as fluorochromes exist for neuro- and cell biological purposes at a pipeline wall. Despite this constraint, flowcells have [59]. The potential utility of these probes in biofilm studies many practical advantages as on-line systems including is not known, although some stains have been used to simplicity of construction, low cost, and ease of integration investigate bacteria in a manner analogous to the eukaryotic into industrial systems. For example, microbial applications [95]. structure within a bioreactor can be examined simply by The pioneering work of Caldwell, Korber and Lawrence attaching Lhe flowcell to a loop in the reactor line [69]. established the value of CLM in microbiological research

Integration of flowcells into other systems could be as sim- [19]. Using a simple exclusion-staining method [20], Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 ple. This type of on-line investigation provides data on the biofilms grown in flowcells were optically sectioned, and actual community as it develops in situ under the conditions the spatial arrangement of organisms within biofilms was within the cell, not on the potentially artifactual simulations visualized in three dimensions [76,138]. Physical para- of microbial biofilms created by use of pure cultures or of meters, such as coefficients [78], can be measured defined cocultures. within the biofilm matrix. Palmer and Caldwell [106a] have Geesey and colleagues recently used plasmid-borne /3- used CLM to examine the three-dimensional structure of a galactosidase (lacZ) activity fused to an alginate promoter dental-plaque-analogue biofilm (Figure 1). In this 'on-line (algC) for analysis of alginate production during attach- in vitro mouth', natural saliva was used to establish unde- ment of Pseudomonas aeruginosa [29,30]. Beta-galactosid- fined, mixed-species biofilms similar to those occurring in ase activity and, therefore, algC promotion can be followed vivo. Three-dimensional interrelationships of fluorescein- at the lewfl of a single cell by providing nonfluorescent stained organisms within these films were analyzed, and methylumbelliferone-tagged /3-galactopyranoside, a differences in the intensity of staining between morphologi- fluorogenic substrate that is cleaved by the lacZ gene pro- cally identical cells were interpreted as differences in ceUu- duct to yield the fluorescent compound, methylumbelli- lar chemistry. Fluorescence Recovery After Photobleaching ferone. Methylumbelliferone appears not to diffuse out of (FRAP) has been used to measure accumulation of a fluor- cells, thereby permitting detection of tacZ expression with escent substrate in the extracellular polymer of a mixed- an epifluorescence microscope. Using this single-cell flu- species biofilm; the uptake of the substrate was inhibited orescence assay of gene expression, it was demonstrated in the presence of KCN [137]. that attaching cells appeared to undergo a lag in lacZ trans- lation, and that the majority of cells that detached from a biofilm no longer expressed IacZ [30]. These results Spectrochemical techniques obtained using a flowcell system were the first report of transcriptional control at the single-cell level. Light can be absorbed, scattered, and/or emitted by matter. For nearly a decade, confocal laser microscopy (CLM) These types of interactions contain qualitative and quanti- has been a tool in the biological sciences, primarily in tative information on biological systems. Absorption of neurological studies. The instrument uses a physical barrier high-energy radiation (gamma-rays, X-rays, and ultraviolet) system (confocal apertures or 'pinholes') to create a thin can lead to photodecomposition of biomolecules and cell (0.4-/xm) plane-of-focus in which out-of-focus light has death, but radiation in the visible, near-infrared, mid-infra- been eliminated. A laser light source provides the intense, red, and radio frequencies have been used in absorption highly coherent, collimated light necessary to penetrate spectrometry to detect living microorganisms in aqueous deep into a thick specimen. The laser light is used to excite environments. Using a 50-/xm diameter fiber optic probe, fluorophores, either intrinsic biomolecules such as chloro- profiles of visible and near-infrared light (400-900 nm) phyll or chemicals added to the sample such as fluorescein. penetration were measured at different depths within The resulting fluorescence is detected by photomultiplier microbial mat and cores [72,74]. Near-infrared tubes, and a digital image is obtained. Images of the x-y radiation (700-1100 nm) has been used to monitor cell den- plane (parallel to the surface) are collected automatically as sity in bioreactor fluid [49]. Absorption of radio frequencies the z dimension (depth) is altered by a computer-controlled in the presence of magnetic fields (nuclear magnetic reson- stepping motor. A series of optical sections through the ance spectrometry) was employed to study substrate con- specimen iis thus obtained. Sections in the x-z plane sumption and intracellular metabolites in dense suspensions (saggital sections) can also be displayed. The individual of bacteria [119]. NMR has also been used to measure the optical secr can be processed using standard image- effect of biofilms on flow velocity profiles [84]. These as analysis software, and the entire set of images can be com- well as other spectrometric techniques such as Raman scat- puter-processed to create a three-dimensional reconstruc- tering [28] and membrane-inlet mass spectrometry [90] tion. The ability of CLM to resolve three-dimensional struc- may also have attributes that allow continuous monitoring, tures in the micrometer range gives microbiologists a tool but have not yet been directly or extensively applied to for exploration of biofilm architecture in the native state: biofilm analysis. The following discussion pertains to light- hydrated, living cells within an exopolymer matrix. Fluor- sensing technologies that have been directly applied to escent probes can be used to localize and measure intra- biofilm research. and extracellular conditions (pH, ionic concentration, mem- Continuous nondestructive monitoring of biofilms DE Nivens et al 266 Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021

Figure ! Confocal laser microscope images of a fluorescein-stained biofihn on a glass coverslip. (a) Top view, in the plane of the substratum. Composite of twelve 0.3-/zm x-y planes collected at 1-/xm intervals. The effective depth-of-field is thus 12/xm. Four distinct bacterial morphologies can be discerned: large bright rods, dark cocci, tightly-packed bright cocci, and loosely-packed bright cocci. The position at which image b was acquired is indicated by the thin black line across the image. (b) Side view through the biofilm (saggital section, in the x-z plane). The white arrows point to the interface of the biofilm with the glass substratum (black area). Black arrows indicate where the large bright rod-shaped bacterium appears in each image. The scale bar = 10/xm, and it applies to both parts of the figure

Infrared absorption spectrometry this range at 1732 and at 1740 cm -1, respectively [46,101]. In condensed phases (solids and liquids), infrared (IR) spec- Thus, differences in molecular structure may be identified trometry is used to study the interaction between IR radi- as well as quantified in a complex sample, making IR ation and vibrational modes of . In general, IR absorption spectrometry an attractive method to study radiation is absorbed by a when the energy of the biofilms. radiation is equal to that required to promote the molecule Fourier transform-infrared (FT-IR) spectrometers to an excited vibrational state. Absorption occurs only at became affordable in the 1980s and offered numerous discrete frequencies when a molecule is exposed to a con- advantages over older dispersive instruments. Dispersive tinuum of IR radiation. The amount of radiation absorbed instruments used monochromators to scan narrow bands of is proportional to the number of molecules present. This frequencies sequentially, whereas FT-IR spectrometers use frequency-dependent absorption results in a unique interferometers to measure all frequencies simultaneously absorbance pattern or spectrum that is defined by the struc- with the signal modulated over time [56]. The advantages ture of the molecule. For complex systems such as biofilms, of FT-IR over dispersive instruments include increased the spectrum is the sum of the spectral signature of each analysis speed, improved signal to noise ratios (because biomolecule in the sample. The frequency or wavenumber spectra are easily added and averaged with time), better at which a molecule absorbs radiation is mainly determined wavenumber accuracy, and greater signal throughput at by specific groups of atoms (functional groups) within the similar resolution. Although many types of commercially molecule and is less dependent on the remaining structure. available FT-IR instruments have been used to study aque- The individual wavenumber range at which a specific group ous biofilms, a FT-IR spectrometer has been specifically of atoms absorbs radiation is referred to as the character- designed for biofilm studies that incorporates three optical istic, or group, frequency. Tables of characteristic fre- channels, a mercury-cadium-teluride detector for sensi- quencies have been compiled to aid in the identification tivity, and a refractively scanning interferometer for stab- of IR absorbance bands [87,100]. For example, the group- ility [102]. This multichannel instrument can be used to frequency range for the carbonyl stretch of esters is from measure either the spectra of biofilms exposed to three dif- 1750 to 1715 cm 1 [87]. The ester groups in alginic acid ferent treatments or the spectra of triplicate biofilms over and in poly-[3-hydroxyalkanoates absorb radiation within time within a single experiment. Continuous nondestructive monitoring of biofilms DE Nivens et al 267 Attenuated total reflection (ATR) spectroscopy is a Conditioning films are layers of organic-rich material sampling technique used in FT-IR spectrometry to exam- adsorbed to a substratum and are important in the formation ine aqueous environments near the surface of a special of biofilms [7,52]. Conditioning film formation has been substratum called the internal reflection element (IRE, monitored in flow-through systems using ATR/FT-IR spec- Figure 2) ]E58]. The IRE must reflect internally as well trometry. In an early study, the adsorption of substances as be transparent to the IR radiation and, for biofilm stud- (possibly phosphate) from filtered sea water was monitored ies, must be insoluble in water. Materials such as zinc using germanium IREs [10l]. More recently, the technique selenide and germanium meet these requirements. Trans= was used to monitor the adsorption/desorption of alginate parency is a necessity because the IR radiation must and dextran to uncoated and to protein-coated germanium reach the detector. With total internal reflection, a stand- IREs at different pH levels [64]. Three different proteins ing wave of radiation penetrates out from the IRE into (albumin, t3-1actoglobulin and myoglobin) were coated sep-

the water, and the intensity of the radiation decays expo- arately on germanium IREs. Alginate, an acidic polysacch- Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 nentially with distance from the IRE [58]. The decaying aride, adsorbed in greater amounts to protein-coated IREs wave, termed an evanescent wave, consists of the same than to uncoated IREs. In contrast, dextran, a neutral poly- frequencies as the reflected light, and can be absorbed saccharide, adsorbed in greater amounts to uncoated IREs by molecules near the outer surface of the IRE. The dis- than to the protein-coated IREs. Because attachment of bac- tance that the evanescent wave penetrates into the out- teria to solid surfaces is thought to be facilitated by extra- side water (dependent on the refractive index of the IRE, cellular polymers, these results support the hypothesis that the wavelength of the light, and the angle of incidence) the attachment of a bacterium to a conditioned surface will can be estimated using the parameter depth-of-pen- be different than to an unconditioned surface. etration [58] which is defined as the distance from the A study monitoring living cells elucidated the chemical IRE surface at which the intensity of the wave decreases nature of an extracellular polymer that facilitates adhesion to e -1 (approximately 37%) of the initial level. Depth- [131]. Suspensions of Catenaria anguillulae zoospores in of-penetration is 0.4/xm for water in contact with a ger- high-purity water were pumped through flowcells contain- manium crystal, at an incident angle of 45 ~, and with ing a germanium IRE. Infrared spectra showed a time- radiation at 1550 cm -1 (amide II band). Only water mol- dependent increase of bands at 1645 (amide I), 1550 (amide ecules and biofilm components within the evanescent- II), 1450 (CH2 deformation), and 1400 (carboxylate ion) wave region contribute to the absorption spectrum. cm 1. The bands, indicative of proteins, suggested that the

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Figure 2 Schematic diagram of attenuated total reflectance (ATR) flowcell. Infrared radiation from the source is reflected on the inside surfaces of the internal reflection element (IRE). At each reflection, some of the radiation penetrates out from the IRE and into the biofilm (see expanded view). This radiation is the evanescent wave and is depicted as the shaded region. Biofilm material (eg cells, extracellular polymer) as well as interfacial water absorbs radiation and is detected by an infrared detector Continuous nondestructive monitoring of biofllms DE Nivens et al 268 extracellular polymers contacting the surface were protein- nosa biofilms, but also to follow the penetration of an anti- aeeous. biotic (100 ~g m1-1 ciprofloxacin) into the evanescent-wave Bremer and Geesey [15] monitored the development of region located near the base of the biofilm [130]. Transport a biofilm with ATR/FT-IR. Pure cultures of bacteria orig- of the antibiotic was monitored using the area of IR bands inally isolated from corroded copper tubing were inoculated at 1303 and 1270 cm-1. This study demonstrated that the into flowcells containing enriched growth medium. In these biofilm impeded transport of the antibiotic through the experiments, sterile and inoculated flowcells were moni- biofilm and to the IRE. Also noteworthy is the appearance tored with time. The bacteria formed a biofllm on the sur- of bands at 1729, 1400, 1250, 1059 cm -1 after the exposure face of the cylindrical germanium IRE and the resultant IR of the biofilm to the antibiotics. It was suggested that the spectra contained bands that increased in intensity with bands may be associated with changes in RNA and DNA time. vibrational modes. However, these bands are also present

The contributions of bacterial cells to the IR spectrum in the spectrum of alginate which is produced by P. aerugi- Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 have been distinguished from those resulting from medium nosa [46]. components [103]. In this study, Caulobacter crescentus Some investigators have expanded the applications of cells suspended in high-purity water, in minimal medium, ATR/FT-IR spectrometry by coating IREs with thin films or in bioreactor fluid were analyzed with the ATR/FT-IR of industrially relevant materials. A prerequisite of such technique. Examination of the sterile medium showed a research is deposition of a continuous film on the IRE and, time-dependent increase in the intensity of a single at the same time, transmission of the IR radiation to the absorbance band at 1080 cm-1. The band originated from aqueous environment. Using copper-coated germanium the P-O stretch mode of inorganic phosphate, which was IREs, ATR/FT-IR spectrometry was used to monitor probably precipitated on the surface of the IRE as the cal- adsorption of biomolecules in a stagnant cell [51,66] and cium and/or iron salt. Although this band overlapped the biofilm-mediated corrosion of copper in a flow-through sys- biofilm C-O and P-O stretch bands, the amide I and amide tem [14,50]. Corrosion or dissolution of the copper film II (as well as other biofilm bands) were unaffected, and was monitored as an increase in absorbance of the water therefore amide I and II were used to follow the develop- band at 1640 cm-I. In control experiments, the absorbance ment of the biofilm. At the end of each bioreactor exper- of the water band remained nearly constant in a sterile iment, parallelogram IREs (less efficient at capturing the medium indicating that the copper films were stable. IR beam than cylindrical IREs, but more practical for Recently, Ishida and Griffiths [65] showed that copper- microscopic examinations) were removed from the flow- coated IREs (8 to 12-nm nominal thickness) with three cells, and the number of attached cells was determined by reflections could be used to obtain adequate signal-to-noise light microscopy using acridine orange staining. A plot of ratios (IREs with ten reflections are typically used). How- attached cells per unit area versus amide II absorbance ever, when these continuous films were exposed to saline values was generated to estimate a limit of detection of solutions (0.15 M NaC1 at a pH of 7.0 or 4.8), a shift in the 5 x 105 bacteria cm-L water band was discovered. This shift, which could cause The development of biofilms with C. crescentus or spectral artifacts in the water-substracted spectra of Pseudomonas cepacia, and their subsequent production of biofilms, was attributed to the dissolution and reorganiza- poly-/3-hydroxybutyrate, has been monitored (Nivens et al, tion of the copper film (formation of metal islands). In in preparation). Two types of IREs were used to obtain addition to copper, 316-stainless-steel films have also been spectra from biofilms at different depths-of-penetration. successfuly deposited on germanium IREs [109], and were Because the refractive index of zinc selenide is lower than also used in a preliminary microbial influenced corrosion that of germanium, evanescent waves from the zinc selen- study [129]. ide IREs penetrate twice as far as those from germanium Attenuated total reflection/Fourier transform-infrared IREs. Production of poly-/3-hydroxybutyrate was monitored spectrometry has demonstrated efficacy as a technique in by increases in the intensities of bands at 1739 (carbonyl understanding biofilm composition and the effects of stretch of the ester band), and 1188 and 1059 (C-O biofilms on substrata. ATR/FT-IR spectrometry provides a stretches) cm-l(Figure 3). The spectra of P. cepacia unique 'inside out' view of biofilms. However, limitations biofilms on the zinc selenide IREs were similar to but more to the technique exist and should be recognized. The tech- intense than the spectra obtained with the germanium IREs. nique analyzes only the base layer (approximately 1/xm) However, for C. crescentus cells, the poly-/3-hydroxybuty- of biofilms and provides an average picture of the chemistry rate (1739 cm-~) to amide II (1550 cm ~) ratio increased transpiring over the entire area exposed to the aqueous with penetration distance of the evanescent wave. environment. In addition, the technique produces spectra Assuming that C. crescentus biofilms on germanium are containing vibrational information from all the molecules similar to those on zinc selenide, the results indicated that within the evanescent-wave region; the data are coinciden- P. cepacia probably grows proximal to the surface, whereas tal and convoluted. Even with this enormous amount of much of the biomass of the C. crescentus cells, that are information, determinations such as distinguishing dead tethered to the surface with a stalk, is located away from biomass from living biomass from a single spectrum cannot the surface of the IRE. These results support theoretical be done. In order to realize the full potential of the tech- calculations that the depth-of-penetration is less than one nique, further progress is needed in data interpretation, in micrometer for germanium IREs. instrumentation (stability and sensitivity), and in the devel- In a medical application, the ATR/FT-IR technique has opment of new, relevant surfaces. been used to monitor not only the formation of P. aerugi- Continuousnondestructive monitoring of biofilms DE Nivens et al 269

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2000 15'00 o'0o Wavenumber (r 1)

Figure 3 A three-dimensional plot of time-dependent ATR-FT/IR spectra showing poly-/3-hydroxybutyrate production by a Pseudomonas cepacia biofilm. The biofilm, inoculated at time zero, developed on a germanium crystal over 76 h. The first IR spectrum contains bands (eg amide I at 1650, amide II at 1548, carboxylate ion at 1400, the P-O, C-O stretches at 1080 cm-1) representative of a biofilm grown on a balanced medium. A carbon- rich medium GLower ammonium ion concentration) was pumped through the flowcell to stimulate the production of poly-/3-hydroxybutyrate, an intracellular carbon storage product. In this series of spectra, the labeled bands indicative of poly-/3-hydroxybutyrate increased with time, while the bands associated with the biofilm remained constant

Fluorescence with fluorescence measurements have been discussed Absorption of ultraviolet or visible radiation by a molecule [85,125,133] and should be considered when using fluor- causes the transition of an electron from a ground electronic escence to study biofilms. state to an excited electronic state. Fluorescence is the Instrumentation used for fluorescence measurements is emission of a photon during the return to the ground state. composed of a radiation source, an excitation wavelength Typically this photon has less energy (longer wavelength) selector, focusing optics, sample apparatus, collection than the excitation radiation because the molecule experi- optics, an emission wavelength selector, and a detector. A ences a loss; of vibrational energy in the excited state. Sev- fluorometer using a xenon lamp source, double mono- eral biomolecules (including tryptophan, phenylalanine, chromators for both excitation and emission, a bifurcated tyrosine, photosynthetic pigments, riboflavin, the archaeal quartz fiber-optic bundle, and a photomultiplier tube for coenzyme F420, and reduced and oxidized NAD) display detection has been used to monitor the tryptophan fluor- fluorescent quantum efficiencies that are favorable for escence of Vibrio harveyi biofilms generated on stainless detection. Fluorescence spectra can contain quantitative steel substrata in a flow-through system [3]. Flowcells were information as well as some qualitative structural infor- designed to contain five sample substrata and to operate mation. Fluorescence measurements have been used to under laminar flow conditions at a flow rate of 5 ml min -1 monitor on-line microbial changes in bioreactor fluids. The [97]. Flush-mounted quartz windows were sealed into the subject has been reviewed [92] and a table of applications flowcells directly above the substrata. Fiber optic probes has been reported by Li and Humphrey [85]. Although were used to detect fluorescence from the biofilms through numerous studies have demonstrated the relationships of quartz windows. Tryptophan fluorescence correlated lin- fluorescence to biomass (tryptophan fluorescence) and early with the biomass of the biofilm (from approximately activity (NADH fluorescence), the intensity of the fluor- 5 x 105 to 4x 106 cells cm-2) as determined by direct escence signal can be affected by many factors. These counts using acridine orange staining. Also with this test include photodegradation of the excited molecule, quench- system, tryptophan fluorescence was used to monitor the ing of the excited state, absorption of the fluorescence (the formation of V. harveyi biofilms on antifouling coatings [6]. cascade effe,ct), or a loss of intensity of the source radiation The coatings included vinyl co-polymers that released a resulting from a high concentration of absorbing molecules biocide at different constant fluxes. Biocide release rates (the inner filter effect). These and other problems associated correlated with decreased tryptophan fluorescence Continuous nondestructive monitoring of bionlrns DE Nivens et al 270 (normalized to a stainless steel substratum located upstream measurements were used to monitor from the antifouling coatings) as well as with a reduction biofilm formation on glass plates by Pseudomonas fluor- in the number of attached microorganisms as determined at escens containing a lux reporter plasmid [96]. Microbial the end of the experiment by acridine orange direct counts. biomass and lipid biosynthesis both correlated with bio- Application of fluorescence spectrometry to biofilm luminescence intensity. Additionally, an environmental research can be expanded by the use of fluorescent gene- bacterium harboring a transcriptional fusion between lux reporter technology. Transcriptional fusions of promoters and the promoter for algD, the gene for alginate biosynth- for microbial functional genes with the gene coding for a esis, was utilized in studies monitoring bioluminescence fluorescent molecule are created and incorporated into a test during biofilm formation [115]. or probe . For example, green-fluorescent-protein (GFP, cloned from the jellyfish Aequorea victoria) has been

expressed in Escherichia coli [22]. With excitation at 380-- Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 490 rim, fluorescence of GFP can be detected at 509 nm. One advantage of the GFP reporter system is additional Electrochemical techniques substrates or cofactors are not required, in contrast to other reporter methods (such as lacZ and bioluminescence). Large-area metal electrodes--electrode as Development of reporter systems having fluorescence at substratum different wavelengths will result in the ability to simul- One strategy for the nondestructive, continuous monitoring taneously monitor activity of different genes. of biofilms involves the measurement of time-dependent changes in the potential associated with large metal elec- trodes (typically greater than 1 crn in diameter). Potential Bioluminescence (sometimes referred to as the redox, open circuit, or cor- Bioluminescence, light production by organisms such as rosion potential) is measured as a voltage difference fireflies and certain bacteria, is a special case of light-emis- between the electrode of interest (working electrode) and a sion spectrometry that, unlike fluorescence, does not require reference electrode. Both the interracial and the electrode source radiation. Instead, in vivo biochemical reactions chemistries determine the potential of the system. Biofilm induce the excitation of biomolecules and the subsequent activity can alter the interfacial chemistry, and thereby emission of photons. Light production requires the enzyme change the potential of the system. The magnitude and luciferase, oxygen, a long-chain fatty aldehyde, and a direction of the change in potential is dependent on the type reduced flavin mononucleotide [128]. Bacterial biolumin- of metal, the consortia of microorganisms present, and escence can be used to detect bacterial biomass (assuming other environmental conditions (eg light, temperature, constant light flux per cell), cellular activity (at a given salinity). For example, biofilms have been reported to biomass), or gene expression (in genetically engineered increase (referred to as enoblement), decrease, or have little bacteria containing the lux gene fusion) [128]. An on-line effect on the potential of stainless steel and platinum elec- biosensor using genetically engineered bioluminescent trodes [39,41,42,88,93,98]. Under a defined set of con- Pseudomonas fluorescens has been used to detect nap- ditions, a change in potential can be used to indicate thalene and salicalate in mixed waste streams [60]. microbial activity and may correlate with the presence of Both the bioluminescence and the tryptophan fluor- biomass. However, in general, potential is not a direct or escence of V. harveyi biofilms in flow cells were used to quantitative measurement of microbial activity or biomass. evaluate the effectiveness of antifouling and fouling-release Other types of electrochemical analysis of large elec- coatings [6]. Bioluminescence and fluorescence of biofilms trodes measure variables such as current (rate of movement on the coated surfaces were normalized to signals obtained of charge carriers) and impedance (frequency-dependent from uncoated stainless steel surfaces. A bioluminescence resistance to the flow of current). With many types of metal ratio significantly greater than the tryptophan ratio was used electrodes, changes in electrochemical variables can be as an indicator of increased photon flux per cell and greater detected as biofilms form on the electrode and, in many overall cellular activity. It was suggested that the biocide instances, these changes are indicators of surface alterations released from the coating disrupted cellular and induced by the biofilm. For example, microbiologically that the method could be used to rank relative toxicity of influenced corrosion (MIC) is a well-known industrial prob- the anti-fouling coatings. lem and electrochemical techniques have been employed to Bacteria harboring bioluminescence reporter plasmids, monitor variables that assess damage to the metal, and help transcriptional fusions containing the lux genes [67], were determine the mechanism of attack [93]. However, many also utilized in biofilm studies. The attachment to poly- variables (eg polarization resistance, double-layer capaci- styrene surfaces of E. coli K12 (containing the lux tance, corrosion current density) can only be determined by expression plasmid PSB 100) was monitored with biolumin- techniques that apply current to the system and thereby escence measurements [38]. In these experiments, the sub- change the biofilm. Small electrochemical perturbations, strata were exposed for a given time, separated from the such as those applied in electrochemical impedance spec- suspensions, and the bioluminescence determined. In troscopy (316 stainless steel, +5mV at various addition to time-dependent attachment curves obtained at frequencies), do not affect the activity and number of different cell concentrations, adherent bacteria were chal- attached bacteria within a biofilm [47]. Although these non- lenged with biocide to determine biocide efficacy. In a steady-state techniques have been used to provide quantitat- flow-through system designed to create a shear gradient, ive information on corrosion phenomena [93], a relation- Continuous nondestructive monitoring of biofilms DE Nivenset al 271 ship between the variables and the physical properties of have been used to measure oxygen gradients within a the biofilm can only be inferred. microbially colonized alginate bead 1 mm in diameter [10]. Dissolved oxygen and pH have been measured at 100-/xm intervals through a 2.5-mm-thick mixed species biofilm on Analyte-selective microelectrodes--electrode as corroding steel [80,81]. The open system was under flow probe and additional measurements (open circuit potential, Analyte-selective microelectrodes (typically, less than cathodic and anodic polarization, AC impedance) were 20/xm tip diameter) measure the activities (concentrations) made on the stainless steel to describe the corrosion pro- of various chemical species as changes in current or poten- cess. Recently, a J-shaped oxygen microelectrode was used tial within the electrode system (reference and sensing with an open flow system under microscopic examination electrode) [2]. Microelectrodes have been prepared which at depth intervals of 10/xm [31]. The location of the elec-

detect oxygen [8,113], pH [111,134], sulfide [111], nitrate trode tip was confirmed using confocal laser microscopy Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 [32], ammonium [33], nitrous oxide [114], chlorine [35], thereby yielding very accurate information on the depth of and [27]. Ideally, the microelectrode tip should be the electrode within the biofilm and on electrode position small (approximately the size of a bacterium), stable, sensi- relative to pores in the biofilm matrix. Local gradients of tive, selective for only the species of interest, reproducible, oxygen were measured within cell clusters and pores. An accurate, and have a fast response time. In practice, example of the use of the concentration data obtained with microelectrodes respond to interferences and the signal these microelectrodes and the explanatory power which is drifts, they are fragile, must be calibrated, and are difficult gained by performing complementary analyses in parallel is to manufacture. Insertion or adjustment of a microelectrode a study of an aerobic trickling-filter biofilm from a sewage can be destructive to the biofilm, and larger electrodes will treatment [71]. Microelectrodes for oxygen, sulfide, cause more, damage. These problems aside, the use of and pH were applied in parallel to derive depth profiles of microelectrodes in on-line systems has greatly enhanced the oxygen and sulfate respiration rates from concentration understanding of several physiological processes within data. biofilms by measuring spatial and temporal concentration Aggregates are a special case in which the biofilm is gradients. attached to itself rather than to a solid substrate, and floats The size of the electrode limits the spatial precision with free in the liquid medium. Examples of the use of which measurements can be made. Sulfide electrodes have microelectrodes to probe the metabolic activities within large tip di~aneters (generally ->50/xm) and thus the inter- aggregates include Bacillus laevolacticus in pure culture vals over which concentration measurements can be [36], ethanol-producing aggregates from potato starch obtained are large compared to the scale of bacteria [112]. wastewater treatment [82], and nitrifying aggregates grown The dental literature has examples of pH measurement in a fluidized bed reactor [34]. The detailed concentration within dental plaque using large (>1 mm tip diameter) gradients obtained under different regimes of nutrient sup- electrodes. Some of these studies were conducted in flow- ply allowed the localization of the various biotransforma- ing in vitro systems [62,118,121]. tions involved. For example, microelectrodes for Microelectrodes have been constructed that have a tip ammonium, oxygen, nitrate, and pH provided concentration diameter --< 1[0/xm which measure concentrations on a scale profiles within the nitrifying aggregates, which showed that significant for microorganisms in a biofilm. In an early ammonium consumption and nitrate production were study [136], dissolved oxygen was measured at 25-/xm localized in the outer 100-120/~m of the 1 to 3-mm par- intervals with a 'microbial slime system' under flowing ticles. conditions, although the technique used for vertification of the electrode position (attachment to a microscope objective) was crude. These authors reported calculation of Scanning vibrating electrode technique oxygen diffusivities and microbial respiration rates [16]. Recently, small platinum microelectrodes have been Revsbech, JCrgensen, and colleagues applied microelec- used to scan the surface of metal coupons with attached trodes to measure oxygen concentrations and pH at 100- biofllm in order to map the potential field. The scanning /xm intervals through microbial mat cores [112]. This study vibrating electrode technique (SVET), vibrates a plati- demonstrated that such electrodes, while fragile, could be num electrode vertically 100/~m above the surface to used under harsh circumstances. The construction and cali- create an alternating potential signal that can be filtered bration of electrolyte-filled pH microelectrodes is not triv- to increase the signal to noise. The rnicroelectrode is ial, but can be accomplished without a large investment in scanned at intervals 200/zm in the x-y plane to map the special equipment [111]. A less fragile solid-state iridium potential field. The map shows anodic and cathodic sites oxide microelectrode has been used for measurements of on the surface, and the anodic sites have been shown pH at intervals of 50 ~m within biofilms [134]. This prom- to correspond to the location of pitting corrosion. The ising ion-selecfive electrode has advantages over electro- technique was used to show that biofilms facilitated pit lyte-filled electrodes with respect to rigidity, range, cali- propagation on carbon steel electrodes [48]. Expanding bration, and reproducibility. The iridium oxide electrode is, the technique, a SVET system has been coupled to a however, not useful in situations where sulfide is present. microscope to provide both a potential map and a visual Polarographic oxygen microelectrodes can be constructed image of the biofilm [4]. This application demonstrates that have a tip diameter of less than 1 /xm [8], but construc- that many of the techniques presented in this review are tion and use of these electrodes is very complicated. They complementary in nature, and when coupled can enhance Continuousnondestructive monitoring of biofilms DE Nivenset ol 272 our understanding of complex problems such as properties of biofilms (due to production of extracellular microbiologically influenced corrosion. polymers, pili, fimbria) are probably time-dependent. Fur- thermore, the formation of biofilms on substrata involves attachment of microoganisms and development of the attached microorganisms into three-dimensional microcol- Piezoelectric techniques onies. Mature biofilms contain cells within the microcolon- Application of electric fields to some materials (eg some ies that do not contact the surface, and the percentage of crystals, ceramics, polymers, and plastics) causes realign- cells that contact the substratum probably decreases during ment of the atoms resulting in macroscale deformation-- biofilm formation. In liquid and viscoelastic films, mol- twists, bends, contractions, and expansions. This phenom- ecules at the surface of the substratum move with the sur- enon is referred to as the converse piezoelectric effect [18]. face, but the shear wave produced by the vibrational motion

An alternating electric field causes a back-and-forth defor- is dampened with distance from the surface. For example, Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 mation or vibrational motion in the bulk material. This the shear wave has been calculated to penetrate only vibrational motion is central to the function of all piezo- 250 nm into pure water [53]. Thus, cells not directly electric sensors. attached to the substratum will be less affected by the shear A quartz crystal microbalance (QCM) is a piezoelectric wave and might not be detected. sensor that measures frequency shifts due to changes in the Piezoelectric devices actually belong to a broader class mass of material on its surface. QCMs typically use a thin of devices termed acoustic wave microsensors [54]. To our circular plate (~0.3 mm oscillates at 5 MHz) from a speci- knowledge, only quartz crystal microbalances have been ally cut crystal [91]. An alternating electric field is applied applied to microbial biofilm research. However, acoustic to the metal electrodes to stimulate a mechanical oscil- wave microsensors are presently being used in liquids [55] lation. Mass deposited on the surface of the electrode will and may have similar potential for on-line sensors in cause a decrease in the frequency of the vibration, moni- biofilm research. Problems associated with long-term tored with a simple frequency counter. If the deposited industrial or environmental monitoring of biofilms with materials have similar acoustic properties (similar rigidity) acoustic wave microsensors are not insurmountable. If to those of quartz, the change in mass is directly pro- methods of pressure and temperature compensation are portional to the frequency shift [120]. The technique was devised, the QCM technology could be employed as a low originally employed in vacuum deposition systems to meas- cost detector to indicate the formation of biofilms. Since ure the thickness of deposited films [91]. In the 1980s, it the frequency change is not linear with the mass of the was discovered that a QCM can function when one side of attached biofilm, a QCM could be used to study the viscoel- the crystal is exposed to liquid [68,105]. In aqueous astic properties of biofilms. As with the ATR/FT-IR tech- environments, QCMs have been applied to investigate elec- nique, the active surface of the QCM can be coated with trochemical processes [17,37] and have been used as trans- industrially relevant surfaces and formation of biotilms ducers for biosensors [43,44,99] such as for the attachment could be compared. As advancements are made in the of African green monkey kidney cells to a metal surface theory of nonrigid films and in transducer technology, [571. piezoelectric sensors should find more application in QCMs have been used to monitor biofilm formation biofilm research. [104], and the study revealed a number of experimental complications. Changes in either hydraulic pressure or tem- perature resulted in signal drift that could be misinterpreted Conclusion as mass changes. Using a flow-through system designed to maintain constant temperature and pressure, the frequency The techniques reviewed here can be used to measure the of the oscillations was monitored both for sterile oligo- biomass, metabolic activities, or the chemical composition trophic medium and for P. cepacia biofilms over 48 h of of biofilms---continuously and nondestructively. The avail- development. The frequency decreased as the biomass ability of such techniques will allow engineers and increased on the surface of the QCM. Formation of the microbial ecologists to: 1)make on-line measurements of biofilm was terminated at different times, and the number other important biofilm systems; 2) couple complementary of attached cells per unit area was determined by light techniques to obtain more detailed understanding of biofilm microscopy with acridine orange staining. A plot of the fre- processes; and 3)modify or develop devices for routine quency shift versus the numbers of microbial cells per unit industrial and ecological on-line or in-line monitoring. area was used to estimate the limits of detection (3 • 105 The vast majority of biofilms are unexplored, offering cells cm2). Using data near the origin of the resultant curve many research opportunities. Selection of techniques that and a frequency-to-mass conversion equation developed for answer relevant questions and that function within the con- rigid films, the average wet mass of a single attached P. straints of the environment are critical. The interpretation cepacia cell was calculated to be 0.8 pg, which is similar of the resulting data requires consideration of both the to an estimate of 1 pg for E. coli [63]. Nonlinearity in the biofilm and the instrumental system. However, a single ana- curve was related to the viscoelastic (non-rigid) behavior of lytical technique may not provide sufficient information to the biofilm during maturation. Surface roughness, chemical understand the system. adsorption, non-uniform mass sensitivity, and longitudinal Coupling of an on-line technology with another method standing waves affect the frequency response of the QCM may synergistically increase explanatory power. For exam- [17,86,91]. Biofilms are non-rigid; and the viscoelastic ple, in microbially influenced corrosion of steel, it is known Continuous nondestructive monitoring of biofilms DE Nivens et al 273 that bacterial biomass correlates with pit formation. How- 12 Bos R, HC van der Mei, JM Meinders and HJ Busscher. 1994. A ever, it has not been proven whether bacteria induce pit quantitative method to study co-adhesion of microorganisms in a paral- lel plate flow chamber: basic principles of the analysis. J Microbiol formation or incipient pits accumulate bacteria. Coupling Meth 20: 289-305. microscopy with the scanning vibrating electrode would 13 Brelje TC, MW Wessendorf and RL Sorenson. 1993. Multicolor laser provide a :means of simultaneously monitoring bacterial scanning confocal immunofluorescence microscopy: practical appli- accumulation and pit formation, thus observing the process. cation and limitations. Methods Cell Biol 38: 97-181. 14 Bremer PJ and GG Geesey. 1991a. Laboratory-based model of The of these techniques into new devices with microbiologically induced corrosion of copper. Appl Environ improved raggedness, sensitivity, and stability will require Microbiol 57: 1956-1962. more interdisciplinary communication. Analytical chemists 15 Bremer PJ and GG Geesey. 1991b. An evaluation of biofilm develop- and engineers need to understand the complexity of biofilm ment utilizing non-destructive attenuated total reflectance Fourier systems; microbial ecologists need to appreciate the power transform infrared spectroscopy. Biofouling 3: 89-100. 16 Bungay HR. 1969. Microprobe techniques for determining diffusivities and limitations of the techniques. We hope that this review and respiration rates in microbial slime systems. Biotechnol Bioengin Downloaded from https://academic.oup.com/jimb/article/15/4/263/5988582 by guest on 28 September 2021 has helped to extend this dialog. 11: 765-772. 17 Buttry DA and MD Ward. 1992. Measurement of interracial processes at electrode surfaces with the electrochemical quartz microbalance. Acknowledgements Chem Rev 92: 1355-1379. 18 Cady WG 1964. Piezoelectricity, Volume 1, pp 1-9, Dover Publi- The authors wish to thank the Department of Energy, Office cations, New York, NY. of Health and Environmental Research, Subsurface Science 19 Caldwell DE, DR Korber and JR Lawrence. 1992. Confocal laser microscopy and digital image analysis in microbial . Adv Program, grant DE-FG05-90ER60988; the Office of Naval Microbial Ecol 12: 1-67. Research, grants N00014-93-1-1317, N00014-94-1-0441, 20 Caldwell DE, DR Korber and JR Lawrence. 1992. Imaging of bacterial and N000-94-1-0961; and the National Water Research cells by fluorescence exclusion using scanning confocal microscopy. Institute, grant WQI 699-524-94 for partial support of this J Microbiol Meth 15: 249-261. project. DCW is partially supported by Department of 21 Caldwell DE and JR Lawrence. 1988. Study of attached cells in con- tinuous-flow slide culture. 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