a review of analytical separation, concentration and segregation techniques in microbiology

A REVIEW OF ANALYTICAL SEPARATION, CONCENTRATION AND SEGREGATION TECHNIQUES IN

MICROBIOLOGY

PRADIP PATEL'

Leafherhead International Ltd. Randalls Road

Leatherhead, Surrey KT22 7RY. UK

Accepted for Publication December IS, 2000

ABSTRACT

There are several major factors responsible for accentuating the need for real-time detection techniques for jbodborne pathogens. These include: ( I ) the general upward trena3 in the reported statistics of food-poisoning worldwide and emergence of foodborne diseases (e.g. Salmonella enteritidis, Campylobacter jejuni, Escherichia coli OI5 7:H7, Listeria monocytogenes and Yersinia enterocolitica); (2) the strict national and international food safe9 and hygiene regulations, including the US Pathogen reduction Act (e.g. setting zero tolerance classification by the USDA FSIS of E. coli OI57:H7) and implementation of mandatory standards for microorganisms in the recent EEC directives for shell-fish, dairy products and eggs; (3) and the progressive introduction of a range of quality assurance and quality management programs (such as BS 5750, I S 0 9000 and TQM) to enhance the eflciency and streamline modern food production and distribution systems.

Increasing adoption of the HACCP approach to food production together with the recent information derivedfiom a range of mathematical models for prediction of growth and survival offood-poisoning microorganisms can efectively reduce the potential rish by microbial contamination. Microbiological testing should be considered in the context of properly evaluated and implemented safety assurance systems. The role of microbiological testing in relation to HACCP has been discussed and considered in detail elsewhere (Hall 1994).

Over the past ten years, a plethora of new methods for the microbiological examination of food has become available. The peflormance of these methods, outside of the originating laboratory, has to be effective and reliable, and this can only be assessed by proper validation of the methods. Validated methods are used by food control laboratories to analyze commodities to verijj regulatory compliance. There are several international schemes available for validation of

'Correspondence to: E-mail: [email protected]

Journal of Rapid Methods and Automation in Microbiology 8 (2000) 227-248. AIZRights Reserved. OCopyright 2000 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 227

228 P. PATEL

modem microbiological methodr, including AOAC International. IDF procedures, EMMAS and Microval (Anon. 1996). In order to produce consistently reliable data, a laboratory must ensure appropriate quality assurance measures are in place. These include filly documented methodr, proper trained laboratory staff and participation in a proficiency testing scheme, many of which are currently available.

CURRENT STATUS

Modem techniques for the estimation of total viable flora include Direct epifluorescent Filter Technique (DEFT), ATP bioluminescence, impedance and flow cytometry (Pate1 1994b). The techniques for foodborne pathogens are largely based on the following principles: (1) immunological analysis (2) nucleic acid probes (3) polymerase chain reaction (PCR) (4) electrical conductance and impedance; and ( 5 ) biochemical and other identification systems. These techniques are often described as “Rapid”, but this terminology needs to be qualified in terms of relative time points during the standard cultural enrichment procedures that the techniques are applied. In general, for foodborne pathogens, the immunological tests based on enzyme immunoassays and immunomagnetic particles are applied after approximately 24 to 48 h of cultural enrichment, the immunolatex particle-based tests after approximately 96 h, nucleic acid probes are applied after approximately 48 to 72 h, polymerase chain reaction (PCR)based tests after approximately 22 4 electrical techniques after approximately 24 h and the majority of biochemical and enzymic tests after approximately 96 h of cultural enrichment. For a detailed review of these techniques and their performance, the reader isreferred to Robinson et al. (2000) and Swamhathan and Feng (1994).

There is a definite trend towards reduction of the analysis time to approximately 24 h, preferably even to within a single working day. The techniques reported to detect Salmonella reliably in a total time of < 30 h include automated EIAFoss, Tecra Unique Salmonella assay, automated Tecra OPUS Salmonella assay, DuPont BAXTM PCR assay and TaqmanTM LS-SOB PCR detection.

However, although each technique has its advantages, the two most significant limiting factors in current detection technologies are; (1) the time-consuming growth amplification required to increase the low levels of the target organism which results in a minimum period prior to detection dependent on the limit of detection of the detector, and (2) the potential interference of the background food components and the nontarget flora. These problems can be effectively addressed by integrating rapid nongrowth based separation, concentration and segregation techniques with modem (and classical) detection techniques. The following arbitrary definitions are used Separation: gross resolution of microorganisms from crude matrices; Concentration: collection of microorganisms from large volume

TECHNIQUES IN MICROBIOLOGY 229

into smaller volumes; and Segregation: one group, species or strain from another.

Microbial Cell Wall Structure

Microorganisms have complex cell wall structures. Both, Gram-positive and Gram-negative bacteria possess peptidoglycan, the stress-bearing and shape-determining structure. In Gram-positive bacteria, the peptidoglycan is relatively thick, about 20-40 nm, while it is about 5-10 nm thick in the Gram-negative bacteria. The peptidoglycan is exposed to the environment in the case of Gram-positive bacteria whilst in the Gram-negative organisms it is surrounded by an outer membrane. The cell wall of Gram-positive bacteria also has other polymers such as teichoic acid, lipoteichoic acid, teichuronic acid, protein and fmbriae. The Gram-negative wall additionally contains lipoprotein, and within the outer membrane, also porin proteins, lipid A, 0 antigens (polysaccharides) and fhbriae. Both, Gram-positive and Gram-negative bacterial walls may also contain flagella and outer capsule (slimy layer) largely made up of polysaccharides exposed to the environment (Ofek and Doyle 1994). The situation is hrther complicated by the occasional presence of surface capsular material made up of polysaccharides and cell surface appendages like the flagella and fimbriae. In complex matrices such as foodstuff; the microorganisms are generally distributed heterogeneously and are likely to be associated with food components. Different types, size and forms of flora are also likely to be present in a given food type. This, together with the fact that the target organisms (e.g. Salmonella) are generally present in very low numbers, makes it a very difficult task to predict the behavior of the microbial flora when attempting to separate, concentrate and segregate the desired organisms. A range of separation and concentration techniques for foodborne pathogens has been reviewed previously (Sharpe 1997).

Classic Food Sample Preparation for Microbial Analysis

The common procedure for the extraction and analysis of the total viable microbial populations (referred to as TVC or plate count) in foods involve suspension of the food in a buffer or a nutrient medium, followed by homogenization using the “stomacher”. This process helps to desorb organisms from the gross food debris and suspends them in the aqueous medium. It is a nonspecific procedure which allows inefficient extraction of the associated flora from the food particles. The resulting suspension is usually turbid, but free from large lumps of food. The suspension is then serially diluted, the extent being dependent on the microbial levels, and portions applied to a nutrient agar plate. The plate is then incubated for at least 24 h, often longer, at appropriate temperature and then visually observed for microbial colonies which are counted. The small particles associated with the bacteria do not generally interfere during enumeration because the bacterial colonies are larger and of defined characteristics

230 P. PATEL

compared with the particles it may be associated with. The colonies foxmed in agar plates may have resulted from one bacterium or a clump of bacteria. Microbiological specifications are based on colony count techniques, the so-called “cultural” techniques.

In the case of pathogens (e.g. Salmonella), the same procedure of stomaching is used followed by incubation in buffered peptone water or lactose broth. Further time consuming selective cultural enrichment procedures are incorporated in order to increase the levels of salmonellae, against the higher “noise” generated from the background flora, that can then be presumptively detected on selective agars. In contaminated foods, salmonellae is generally present at very low levels and would require at least 3-4 days for presumptive detection and longer for the confimatory procedure based on biochemical identification and serology.

The enhancement of sensitivity, reliability and speed of microbial enumeration from foodstuffs using modem detection systems has been widely attempted, but with varying degrees of success. The following text represents examples of many of the approaches undertaken to date. Dielectrophoresis as a means of separation, concentration and segregation of particles is also an exciting approach in separation technology.

Centrifugation

This was one of the earliest technique that has been successfully exploited to separate and concentrate microorganisms from aqueous broths and foodstuffs. At high rotor speed (say 15,000 x g), the majority of the microorganisms (> lpm) separate out as pellet fiom an aqueous suspension within 15 min. However, at the same settings, most of the food particles and associated microorganisms will be pelleted, thus separation of the microorganisms from the food debris will not be achieved. However, by a combination of gentle and high speed centrifugation (differential centrifugation) microorganisms can be grossly resolved from the large food particles. This approach has been used to rapidly separate microbial populations from raw meat prior to estimation using the ATP bioluminescence technique (Pate1 1984).

The schematic Fig. 1 shows the complexity of a typical meat homogenate, including location of microorganisms, background particulates and soluble components. A combination of enzyme (e.g. protease and collagenase) and detergent (e.g. Tween 80, Triton X- 100 and Tergitol) pretreatments has been used to enhance the detachment and recovery of microbial flora from raw and cooked beef (Rodrigues-Szulc et al. 1996). The resulting extracts were subjected to further differential centrifugation to isolate the intrinsic microbial flora which were subsequently concentrated on a polycarbonate filter, prior to staining and enumeration by Direct Epifluorescent Filter Technique (DEFT). The 1-h separation and concentration procedure was claimed to increase the detection of bacteria by

TECHNIQUES IN MICROBIOLOGY 23 1

Muscle,

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my oglobin water enzymes

soluble sugars, nucleotides, carbohydrate vitamins

Microorganisms

FIG. 1. SCHEMATIC DIAGRAM REPRESENTING A TYPICAL MEAT HOMOGENATE

at least one and a half order of magnitude compared with the classic stomacher procedure

Another centrifugal technique recently introduced is Continuous flow centrifugation (Centrifugem Stratos, Kendro Laboratory Products, Ltd.), which has been used for the separation of suspended matter from river water (Zellmer 1999) on a continuous basis. The table top centrifuge connected through a pipe directly linked to a river was shown to achieve a rotor speed of 17,000 rpm with a degree of separation of just over 90%. The system would be valuable in the brewing industry where continuous microbial isolation and resuspension may be required to improve product yields. Recently, a portable continuous flow centrifuge has been used for the concentration of Cryptosporidium oocysts and Giardia cysts from water with recoveries of 90% (Zuckerman et al. 1999).

Density gradient centrifugation (DGC), based on the Percoll" or BacXtractorm media placed in an Eppendorf tube, has been used in a 1 min bench top

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centrifugation technique, to rapidly separate and concentrate microbial flora from beef matrix whilst also removing the potential PCR inhibitors (Lindqvist 1997). The resulting PCR amplification technique allowed detection of low levels (approximately lo’ -104 cM25 g) of E. coli 0157 from a range of food samples, including beef, shrimp and milk. The automated Bactoscan (BioMerieux) uses DGC for the rapid separation and ATP based estimation of viable microbial flora from raw milk.

Filtration

Like centrifugation, filtration has been used widely for sterilization purposes and for concentration of microorganisms from broth cultures. Although many new varieties of filtration materials (e.g. Cyclopore@ polycarbonate sieve filters, nylon mesh filters, Anapore and Whatman GDIX type filters combining glass fiber prefilter with filter) with varying porosities (0.2 - > 300 pm) have been introduced, duect filtration of sufficient volume of food homogenates is stil l not possible. Their application to food broths have been hampered by the problems of clogging and back pressure due to the heterogeneous particulate nature of foodstuffs.

However, a number of presample treatments have been reported that can clarify food homogenates sufficiently to allow subsequent microbial concentration through 0.45 pm filters. Stannard and Wood (1983) used an ion exchange resin at a defined pH value to separate the gross food debris from the microorganisms. The resulting homogenate was subsequently filtered through 0.45 pm filter to concentrate the bacteria. The filter was washed and the bacteria then estimated using an ATP bioluminescence technique. The BactoFoss is an automated ATP-based technique for the estimation of total viable microorganisms from raw meats and milk within 5 min (Olsen 1991). The sample preparation procedure required includes homogenization using the Stomacher, followed by a brief centrifugation to remove large food particles. The remaining supernatant is then automatically drawn into the instrument, where it is filtered to concentrate the microbial cells whilst removing any soluble nonmicrobial A n . The cells are subsequently extracted and ATP measured. The correlation between the BactoFoss and the plate count method was satisfactory down to approximately los cfu/g, below which the nonmicrobial ATP presented a high background. This is not surprising since the sample preparation procedure used was crude and there is a possibility that large meat cells (somatic cells) and ATP-bound particles may well contribute to the background “noise”. The Cobra automated system (BioMerieux) for the real-time testing of raw milk uses filter cytometry derived from a DEFT. The milk is rendered filtrable through a 0.6 pm polycarbonate filter by pretreatment with trypsin and Triton X-100 (Anon. 1995a; Reybroeck 1996). Bacteria on the filter are then stained and enumerated.


Cross-flow filtration (or tangential flow) is a process in which the test liquid flows tangentially to the membrane inside surface with the result that a small percentage of the flow permeates through the membrane and is collected from the exterior of the membrane as a permeate. This is considered an ideal alternative to centrifugation for applications where pellet formation is undesirable and are particularly suited to separation of analytes from turbid matrices. Most of the applications have been targeted to commercial brewing and water treatment for clarification and cold sterilization (Anon. 1995b; Fillaudeau 1999; Gans and Kunden 1999). The commercial RMQA (Rapid Microbiology Quality Assurance System, GEM Biomedical, Hamden, CT) system uses cross-filtration in combination with “vortex mixing” to rapidly ( 15 min) and gently filter organisms from large volumes of liquid product samples (e.g. water and beverages). The concentrated organisms can then be estimated by techniques such as ATP bioluminescence, ELISA, Flow cytometry, PCR and impedance. Recently, cross-flow modules that are applicable to sample volumes as low as 1 mL have become commercially available (e.g. MicroKros@; Spectrum). Their potential application to microbial separation from food broths will depend on a number of factors, including cost per test, sample through put and speed of analysis.

Expanded bed chromatography (UpFront Chromatography, Denmark) is used for direct recovery of proteins from crude raw materials (e.g. plant extract, milk animal tissue) without prior clarification or concentration (Chase 1994). Thus, it does not require Centrifugation, filtration or precipitation. Available in ion exchange, hydrophobic-hydrophilic and affinity media. The basic idea is to fluidize the chromatographic beads by an upward liquid flow. This avoids the classic problem of traditional packed bed i.e. increased back pressure and clogging of the bed by particulates. In the traditional packed bed impurities accumulate because the bed functions as a depth filter. In the expanded bed the beads are separated and particulates can pass through the bed without accumulating.

The legislation for Cryptosporidium states that “It will be an offence for water from plants that are subject to the Regulations to contain on average more than one (Ciyptosporidium oocyst in ten liters of water” (Anon. 1999a). The Filta-Max (Genera Technologies, Ltd.) system claims to achieve the capture and recovery of Cryptosporidium and other waterborne pathogens by using multiple layers of reticulated open cell foam discs, which are compressed (from 60 cm to around 3 cm) and then used to capture the parasites in the foam pockets as water is forced through. The oocysts, once captured, remain captured until they are released in the washmg process. The system overcomes all the problems of the traditional filters (e.g. clogging and back pressure). The recovery of particles trapped in the foam involves placing the filter module within an enclosed wash station, and washing the foam using expansiodcompression cycles with an elution buffer. The sample is reduced to approximately 25 mL, without the need for centrifugation, and takes less then 10 min. In a recent independent study, the Filta-Max was compared with

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the Envirochek capsule (Gelman) for the recovery of oocysts of Cyptosporidium and Giardia spp. from large volumes (about 20,000 L) of mineral water (Paton et al. 1999). The recoveries from the Filta-Max were similar to the Envirochek system, ranging from 0 to 28.4% and 10.1 to 68% (Filta-Max) and 1.6 to 22.5% and 6.6 to 70.5% (Envirochek) for Cryptosporidium and for Giardiu, respectively.

Chromatography

Chromatography media based on particle size ranging from approximately 40-140 pm have been used for resolving microbial populations from mixed cultures and from raw foods (e.g. meat). The chromatography media included gel filtration materials, anion, cation and mixed bed ion exchangers and hydrophilic and hydrophobic gels (Patel and Wood 1984). A range of formats which include large and mini-columns, and batch chromatography has been applied for this purpose with some interesting fmdings. For example, the Gram-positive organisms (L. pluntarum and Staph. aureus) were found to adsorb significantly to the cation exchange resin Bio-Rex 70 at pH 4.5-5.5 compared with the Gram-negative E. coli and Ps. jluorescens (Fig. 2). This behavior is also reflected in raw pork and milk splked with the mixed cultures of these organisms.

In another investigation, it was demonstrated that E. coli containing hydrophobic K88+ fimbriae showed significant adsorption to the hydrophilic-hydrophobic media compared with the nodibriated E. coli (Patel and Wood 1984). Based on the nature of the microbial cell surfaces, it is not surprising that such differential adsorptive characteristics exist between the chromatographic media and microorganisms. The technology has yet to find commercially exploitable applications. In the previous discussion, the application of a cation exchanger in a batch process to clarify meat extracts prior to filtration and estimation of the total viable organisms using ATP bioluminescence has been demonstrated (Stannard and Wood 1983).

Affinity Media

Affinity media have largely been used for the Segregation of specific microbial species from the background interference. The two most widely exploited technologies are those based on membrane and magnetic solid phases. In the former case, simple, rapid and disposable immunochromatographic tests are available for a range of microbial antigens, including Salmonella (Celsis-Lumac Path stick), Listeria (e.g. Oxoid Clearview) and E. coli 0157 (Diffchamb Transia card E. coli 0157). These tests are not applicable directly to viable cells and require at least 10’ cWmL cell concentration prior to detection.

Immunomagnetic particles (2-5 pm), on the other hand, are established techniques that are available commercially for direct application to segregation of Salmonella, E. coli 0157 and Cryptosporidium, (Dynal) and Listeriu (e.g.


Listertest, Vicam). The basis of the immunomagnetic separation (IMS) is shown in Fig. 3. IMS is a generic technology which can be applied to both soluble and particulate antigens. The resulting “enriched” cells can then be detected by a range of modem detection systems.

An example of the combination of IMS with an electrical conductance technique is shown in Fig. 4. Clearly, the IMS can reduce the time to detection of Salmonella by several hours. For detail of the applications, the reader is referred to the following articles (Patel 1994; Cudjoe 1999). A combination of the IMS with PCR for the detection of Campylobacter in poultry (Docherty et al. 1996) and IMS with flow cytometry for the detection of E. coli 0157:H7 in ground beef (Seo et 01. 1998) has been demonstrated previously.

Use of colloidal ferrofluidic media (50-100 nm) in microbial segregation studies is relatively new and represents an effective alternative to the particulate systems described above. Unlike the particulate systems, the ferrofluidic system does not require shaking, the reaction with the corresponding antigen is rapid ( 10 min) and does not require removal of unbound excess reactants from the bound reagents. Figure 5 shows efficient separation of a range of food-spoilage yeasts from suspensions using a ferrofluidic system. For details of the technique, the reader is referred to Patel et al. (1993). The system has also been applied to the enrichment of salmonellae from mixed cultures. Plate I shows the extent of enrichment of salmonellae from a mixed culture with E. coli as demonstrated on an Xylose Lysine Desoxycholate (XLD) selective plate using a ferrofluidic system in comparison with a control.

Plate I1 shows an electron micrograph of a single cell of Salmonella, specifically coated with ferrofluid.

Genera technologies also markets the Puri-Max system, based on recirculation of immunomagnetic particles, for isolating Cryptosporidium from high volume turbid water samples. The mixture of immunomagnetic particles and sample is circulated for 25 min before being placed in a magnet, and circulated for another 55 min. The beads, once placed in a magnet, attach to a metal mesh, with water constantly circulating past the fured beads. All the bound oocysts are therefore trapped on the phase. This can then be removed, and the beadloocyst complexes eluted. The total “hands-on” time for this operation is 15 min.

Aqueous Two-Phase Systems Aqueous two-phase systems may form on mixing pairs or multiples of polymers

in solution (polymer-polymer system) or between mixtures of polymers and salts (polymer-salt system). For example, when pairs of polymers (e.g. polyethylene glycol, PEG, and dextran) are mixed together above defrned concentrations, a biphasic system is formed in which each phase is preferentially enriched in one or the other of the polymer. This technology has been commonly applied as a manual

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FIG. 2. DIFFERENCES IN THE AFFINITY OF MICROORGANISMS FOR THE CATION EXCHANGE RESIN BIO-REX 70

procedure for the purification of proteins and glycoproteins, usually in combination with other techniques (e.g. ultrafiltration) whilst enhanced purification has been reported using automated countercurrent distribution procedures (Huddleston et al. 1991; Ortin et al. 1992). Delgado et al. (1991) developed an effective biphasic system that used immunoaffity cell partitioning to purify human red blood cells. In their system, the antibodies against the red blood cells were coupled with PEG with the result that the labeled cells partitioned in the top PEG-rich phase in the PEG-dextran system.

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Leji-hand agar plate: untreated mixed suspension Right-hand agar plate: same suspension following immunomagnetic separation

PLATE 1. XLD PLATES SHOWING THE RESULTS OF THE APPLICATION OF SALMONELLA-SPECIFIC IMMUNOFERROFLUID TO A MIXED SUSPENSION CONTAINING

S. ENTERITIDIS (BLACK COLONIES) AND E. CLOACAE (WHITE COLONIES)

PLATE 11. ELECTRON MICROGRAPH SHOWING SALMONELLA-SPECIFIC IMMUNOFERROFLUID BOUND TO THE SURFACE OF A CELL OF S. ENTERITIDIS


Although simple and rapid to implement, the two-phase system does not seem to be widely used on a routine basis for purifications involving complex matrices (e.g. blood and food). This is largely due to many parameters that are known to influence the purification process, including the partition coefficient of the analyte, type and molecular weight of biphasic polymers, nature and concentration of added salts, concentration difference between phases of phase-forming polymers, overall system pH and interference of “background” components (e.g. having similar partitioning behavior to the target analyte and influence on pH and ionic balance).

The application of two phase systems to purification of microorganisms has been previously reported, including biotechnological applications (Magnusson el al. 1985) and fine characterization of cell surface properties such as hydrophobicity and charge (Ascencio et al. 1995). The potential of a biphasic system based on PEG/dextran for the separation of salmonellae from foods was investigated by Bennett et al. (1994). The authors found that microbial strains partitioned to varying extents in both the phases and that the partitioning behavior was influenced by certain ions, indicating their influence on the net cell surface charge. Although it was demonstrated that a strain of S. typhimurium can be “enriched” from a 24 h food preenrichment broth using the biophasic system, the authors found that not all Salmonella serotypes exhibited the same partitioning characteristics.

Field Flow Fractionation

Field flow fractionation (FFE) technique is a family of chromatographic-like elution techniques in which an external field or gradient (e.g. gravitational, centrifugal, electrical, flow and thermal) causes differential retention of biomolecules (e.g. proteins, peptides and polysaccharides) and particles (latex, microbial and parasites) ranging typically from 1 nm to 300 pm (Giddings 1995; Hanselmannet al. 1995).

In practice, FFF takes place in a thin ribbonlike channel. A field applied perpendicular to the channel axis drives components towards one wall (the accumulation wall) of the channel where each forms a steady-state distribution. In most cases, the particles are driven to within 1-10 pm of the accumulation wall (Giddings 1995). The flow of the sample is laminar and parabolic because of the channel dimensions, ranging from 75-250 pm. The particles are driven to the exit port where they can be detected and characterized using an array of detection systems, including hyphenated detectors (e.g. electron microscope, scan cytometer, photon correlation spectroscopy, multiangle light scattering, FT-IR, ICP-MS).

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Four fields have been widely studied. (1) Flow FFF (FFFF) is one of the most universal separation techniques. In

FFFF, a cross flowstream of canier liquid, in which the force orginates from the friction of the cross flow stream across the components results in the separation of the components. The FFFF is rapid (1 min mn time) and can be automated, and has been applied to the fine separation of biomolecules and particles (e.g. single and double stranded DNA, protein dimers) based on differences in the molecule's diffusion coefficient.

(2) Sedimentation FFF (SFFF) is when the force is generated usually by gravity or centrifugation. The sedimentation force acts perpendicular to the flow separation axis and is a prominent method for separation and characterization of colloidal particles.

(3) Temperature gradient or thermal FFF (TFFF) is when the perpendicular force is a result of thermal diffusion. The technique has been primarily used for fractionating polymers of high molecular weight, although recently it has also been applied to particles in both aqueous and nonaqueous media.

(4) Electrical FFF (EFFF) is when the force is due to the electrical field and the separation depends on the polymer or particle charge and mass. Unlike capillary electrophoresis, in the EFFF the field is perpendicular to the flow of carrier liquid, the potential difference required to produce selectivity are far less stringent since it is applied across the thin gap in the FFF channel and EFFF can process larger sample volumes and larger particle sizes (Caldwell and Gao 1993). Unfortunately, the low currents required to keep the electrode polarization at a minimum, and the fields maintained at reasonable levels, limit the choice of carrier to solutions of low ionic strengths (e.g. 150 p M NaCl).

Ultrasonic Standing Waves

When particles in suspension are placed in a stationary acoustic field, they move towards and concentration at half-wavelength intervals where there is minimum acoustic potential energy (Coakley 1997). A number of different configurations have been used in order to separate particles and measure their efficiencies. These include the static banding cell, ultrasonic flow cell and ultrasonic centrifuge (Zamani et al. 1993; Miles et al. 1995). The reported efficiencies for the yeast Rhodotomla glutinans in the flow through design were 49-65% retention rate at a concentration of 1 x lo7 cWmL in aqueous suspensions to 86-96% at 1 x 103 cfu/mL (Zamani et al. 1993). However, this rate was shown to be reduced (>60%) with smaller size bacterial cells at a concentration of (1 O9 - 10'' cWmL; Coakley 1997). Thus, the higher the biomass the greater the efficiency of retention with the implication that ability to form clumps at high biomass is an important aspect of harvesting prokaryotes.


Miles et al. (1995) studied the application of ultrasonic waves for the separation and concentration of microbial cells from aqueous cultures and from dilute milk samples. A frequency range of 1-3 MHz was reported to be suitable for banding and moving vegetative bacterial cells (e.g. Micrococcus luteus and Listeria innocua) from pure cultures. When a frequency of 2.05 MHz was applied to a diluted sample of milk (l%), contaminated with a high level of E. coli K12 (6 x lo7 cMmL,), the authors found that the bacteria formed clumps and organized as bands near the central axis of the field. The authors suggest that further studies, particularly of the engineering nature, were required to develop a system that might be able to cope with low levels of microbial populations. Preliminary studies have also been reported for the noninvasive flocculation and aggregation of brewing yeast from fermentation vessels. The results showed that the separation efficiency was controlled by the size of the particles and the liquid flow rate (Davies 1993).

The immediate application of ultrasonic systems is likely to be in the enhancement of agglutination-based assays available for microorganisms and soluble antigens. In classical latex agglutination techniques it takes from a few minutes to several hours to form visual agglutination depending on the analyte. The ultrasonic system speeds up this process considerably and, indeed, improves the sensitivity of the tests from about 10 to more than a 1000-fold (Coakley 1997). However, it is difficult to decipher from the information given what the difference, if any, would be between the specific agglutination due to the presence of the antigen and the corresponding antibodycoated latex particles compared with control samples containing all the reactants but not the antigen.

Molecular Imprinting

Molecular imprinting involves preparation of a “mold” comprising polymerisable functional monomers around a print molecule (e.g. Penicillin V, Fig. 6). Initially, the monomer is allowed to establish bond formation with the print molecule and the resulting complexes or adducts are then copolymerized with cross linkers into a rigid polymer. The print molecule is extracted to leave specific recognition sites in the polymer that is structurally and functionally complementary to the print molecule. The resulting “plastic antibodies” can be functionally as effective as the biological antibodies, but with added benefits that include mass production in many formats (e.g. films, particles) by simple chemical processes, cost-effective, stable and long shelf-life.

Molecular imprinting is a relatively new technique for generating solid phase molecular imprinted polymer (MI€’)-based affinity systems but already many applications have been demonstrated, particularly involving organically soluble low molecular weight compounds. Examples include drugs, herbicides, sugars, nucleotides, amino acids and proteins (Briiggemann et al. 2000).

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In a recent European-funded project (FAIR CT96-1219), involving partners from University of Lund, University of Rome, Merck Eurolab (Prolabo), UNIR association and Leatherhead Food Research Association (Coordinator), MIP particles were developed for p-lactam antibiotics and p-agonists (Skudar et al. 1999; Crescenzi et al. 1998). The project also demonstrated the application of MIPs to food analysis, together with the development of MIP-based amperometric sensor for clenbuterol (Papers currently being prepared).

Overall, MIPs are not as yet widely used for routine applications in analytical laboratories because of several limitations which include the following: (1) robustness and high sensitivity still not adequate due to inefficient total removal of the print molecules which can gradually leach out; and (2) MIPs are produced at the laboratory level and procedures now need to be developed for scaling up to commercial production. The combination of MIPs with a range of tranducers to produce sensors will be a powerful analytical technology. Already, this has been demonstrated as described previously, but their value will only be realized when the basic limitations are adequately addressed.

Future Prospects

Future considerations in the development of new analytical technologies should take into account the developments already made in a number of scientific areas relating to the separation and concentration fields. Increasing number of companies are now incorporating the techniques mentioned previously into commercial kits and instrumentation for the detection of contaminants. Some examples include:

(1) immunomagnetic particles (Dynal anti-pathogen particles; Organon Teknika ELISA for Salmonella; EIA Foss automated system for pathogen analysis from Foss Electric, Vicam irnmunomagnetic system for Listeria and Genera Technologies Cvptosporidium test); (2) centrifugation (Promega ATP-based test for total viable count; Bactoscan from Foss Electric) and (3) cross-flow filtration (Gem Biomedical’s RMQA automated system for concentration of organisms from aqueous flowing streams).

It appears that a large majority of the “newer” separation and concentration techniques have not yet been used in commercial analytical kits and instrumentation. Further developments in the various fields are required which address the analykal needs of the industry and other end-users, in particular tests and instrumentation that are cost-effective, simple to use, preferably automated, high specificity and speed of analysis, robust analytical protocols, etc. before these developments will reach commercial realization.

246 P. PATEL

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