Impedance Microbiology-A Rapid Change for Microbiologists

Journal of Applied Bacteriology 1996, 80, 233-243

A REVIEW

Impedance microbiology-a rapid change for microbiologists

P. Silley and S. Forsythe’ Don Whitley Scientific Limited, Shipley, West Yorkshire, and ’Department of Life Sciences, The Nottingham Trent University, Nottingham, UK

5377/06/95: received 1 June 1995, revised and accepted 8 September 1995

1 . Introduction, 233 1 . 1 Direct impedance, 234 1.2 Indirect impedance, 234

2. Applications in the food industry 2.1 Total viable counts, 235 2.2 Salmonella detection, 235 2.3 Listeria detection, 236 2.4 Coliform testing, 236 2.5 Clostridia, 237 2.6 Yeast, 237

2.7 Lactic acid bacteria and the dairy industry, 237 2.8 Predictive microbial growth modelling, 238

3. Non-food related applications 3.1 Antibiotic resistance, 238 3.2 Biocide efficacy testing and microbial biofilms, 239 3.3 Detection of plant pathogens, 240 3.4 DNA damage evaluation, 240 3.5 Impedance-based enzyme assays, 240

4. Conclusions, 240 5 . References, 240

1. INTRODUCTION Impedance can be defined simply as the resistance to flow

Impedance microbiology is not new. It was first mentioned at a meeting of the British Medical Association at Edinburgh in July 1898 where Stewart presented a paper later to be published in The Journal of Experimenta 1 Medicine entitled ‘The changes produced by the growth of bacteria in the molecular concentration and electrical conductivity of culture media’ (Stewart 1899). The electrical response curves presented followed the putrefaction of blood and serum and were very similar to those obtained from currently available impedance systems, the significant difference being that today impedance can be considered as a rapid microbiological method whereas Stewart was measuring changes in impedance over periods in excess of 30 d. Further work followed on from Stewart’s initial findings (Oker-Blom 1912 ; Parsons and Sturges 1926; Parsons et al. 1929; Allison et al. 1938; McPhillips and Snow 1958). However, it was not until the mid seventies that the technique began to receive the attention it merited. This coincided with the introduction of dedicated impedance systems and a consequent increase in published work notably by Ur and Brown (1973, 1974, 1975), Cady (1975) and the very important work of Eden and Torry Research Station (Richards et al. 1978 ; Eden and Eden 1984).

Currespondenrr IO : D r S.J. Forsythr, Drpur,mrnr uf Lifr Scirncrs. Thr Nutringhum Trrnr Uniocrsir.y, CIifion Lane, Nottingham BGI I 8NS, UK.

0 1996 The Society for Applied Bacteriology

of an alternating current as it passes through a conducting material. The reader is referred to Eden and Eden (1984) and the more recent discourse by Kell and Davey (1990) for a detailed review of impedance theory. However, it is sufficient for the purposes of this review, however, to consider, as first proposed by Warburg (1899, 1901), that when two metal electrodes are immersed in a conductive medium the test system behaves either as a resistor and capacitor in series or as a conductor and capacitor in parallel (Kell and Davey 1990). Considering the case where the system is treated as a series combination, then application of an alternating sinu- soidal potential will produce a resultant current which is dependent on the impedance (2) of the system which in turn is a function of its resistance (R), capacitance (C) and applied frequency (F) thus :

Any increase in conductance, defined as the reciprocal of resistance or capacitance results in a decrease of impedance and an increase in current. The AC equivalent of conductance is admittance which is defined as the reciprocal of the impedance. The units of impedance measurement are Siemens (S).

Microbial metabolism usually results in an increase in both conductance and capacitance, causing a decrease in impedance and a consequent increase in admittance. Therefore the

234 P. SILLEY AND S. FORSYTHE

concepts of impedance, admittance, conductance, capacitance and resistance are only different ways of monitoring the test system and are all inter-related. In practical terms we need to consider that the electrical signal is frequency dependent, has a conductive and capacitative component and is temperature dependent. The importance of temperature control in any impedance system is critical, as a temperature increase of 1°C will result in an average increase of 0.9% in capacitance and 1.8 O/o in conductance (Eden and Eden 1984).

1.1 Direct impedance

It can be readily appreciated that changes in impedance of the growth medium result directly from the changes taking place in the bulk electrolyte. Substrates in microbiological growth media are generally uncharged or weakly charged but are transformed into highly charged end products as organisms follow normal metabolic pathways, thus increasing the conductivity of the test medium. Simple examples include the conversion of glucose from a non-ionized substrate to two molecules of lactic acid with a corresponding increase in conductivity. Further metabolism will take the lactic acid and three oxygen molecules to carbonic acid, resulting in three ion pairs including the smaller more mobile bicarbonate ion, which is a more effective electrical conductor than the lactate ion. Hydrogen ions are nearly seven times more effective conductors than sodium ions (Eden and Eden 1984), therefore one might predict that a weakly buffered medium would allow a greater impedance change than a more strongly buffered one. For a more detailed appraisal of the effect of medium buffers on conductimetric response the reader is referred to the work of Owens (1985). It is important to stress, however, that the principles of medium design, fundamental to traditional microbiology, are equally if not more important in impedance microbiology. In the first instance, a medium must be chosen which will support and select for the growth of the test organism. Secondly, that medium needs to be optimized for an electrical signal. This is well illustrated by Staphylococcus aureus which will grow in nutrient broth but does not produce a significant electrical response, whereas in Whitley Impedance Broth (WIB, Don Whitley Scientific Ltd, Shipley), not only does it grow well but it produces a strong impedance signal. The growth of some organisms, particularly yeasts and moulds, does not result in large changes in impedance. This is considered to be due in part to the fact that they do not produce strongly ionized metabolites, but non-ionized end-products such as ethanol. Additionally, Suo- malainen and Oura (1971) have shown that yeasts can absorb ions from solution resulting in a net decrease in medium conductivity.

An impedance system can therefore be considered simply as measuring net changes in impedance in the culture medium at regular intervals. When a test is initially set up the user

defines the detection criteria and when the rate of change of impedance exceeds this pre-determined value the system will detect growth. The time required to reach the point of detection is referred to as the 'detection time' (DT) and is a function of the size of the initial microbial population, the growth kinetics of the test organism and the properties of the test medium. For a given test protocol the D T is proportional to the initial microbial loading of the sample. At the point of detection it is generally considered that there will be approxi- mately lo6 cfu ml-' of the test organism present in the system. This will vary depending on organism type and medium, but will be constant for any organism growing under defined test conditions. It is important to differentiate between this detection threshold and the sensitivity of an impedance system which is capable of detecting the presence of organisms at levels as low as < 10 cfu ml-l providing the organisms are viable. It is well established that the electrode construction, stainless steel compared to platinum, will affect sensitivity of the test system. Eden and Eden (1984) showed that electrodes located at the bottom of a test cell resulted in detection thresholds 1 log cycle lower than with the same electrodes located at the top of the test cell. The fact that real time microbial activity is being measured rather than the activity at a single point in time is a significant and powerful feature and one which enables the system to detect the presence of low numbers of organisms. A number of factors will affect time to detection. Firstly D T will correlate only with the initial concentration of test organisms providing the generation time of the test population is more or less constant under the experimental conditions. Therefore not only does incubation temperature need to be kept constant due to physico-electrical properties as discussed earlier, but also because it will have a direct effect on the generation time of micro-organisms.

1.2 Indirect impedance

High salt concentrations are routinely used in many selective media. For example LiCl is incorporated into Baird-Parker staphylococcal medium at 5 g I- ' and into Oxford Listeria medium at 15 g 1-'. Also MgClz (36 g I - ' ) is used in Rap- paport-Vassiliadis broth for salmonella isolation. The resultant high impedance readings of these media are outside the normal working range of the direct impedance technique. However, using the indirect technique these problems can be overcome by monitoring microbial metabolism via the production of CO, (Owens et al. 1989). In this instance potassium hydroxide is added to the impedance tube across the electrodes. The inoculated culture medium is in a separate chamber and not in contact with the electrodes or potassium hydroxide. The unit is tightly sealed such that any C 0 2 produced as a result of normal metabolism is absorbed by the

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IMPEDANCE MICROBIOLOGY 235

potassium hydroxide causing a resultant decrease in impedance.

The dynamics of carbon dioxide absorption and the ratio between the impedance variation and the amount of carbon dioxide produced were investigated by Dezenclos et al. (1994). After injection of carbon dioxide either directly in the potassium hydroxide solution, or above the potassium hydroxide solution, the optimal results were obtained with potassium hydroxide (5-6 g 1 - I ) in a volume of 0.7-1.2 ml. Impedance changes of 280 $3 pmol-’ carbon dioxide was obtained at 27°C with potassium hydroxide concentrations of 0.5-8 g 1 - I . This agrees well with that predicted by Owens (- 278.6 S cm’ mol-’ carbon dioxide absorbed ; Owens et al. 1989). Not surprisingly the results were temperature dependent.

The work of Bolton (1990) has already shown the indirect technique to be a powerful tool for working with strains of Staph. aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aer- omonas hydrophila and Salmonella spp. Furthermore, it now means the medium does not necessarily need to be optimized for electrical response, allowing media previously considered unsuitable to be used for impedance applications. Appli- cations of indirect impedance technology are of considerable potential for anyone with a requirement for a rapid, easily manageable, highly sensitive system for monitoring and quantifying COz production whether in whole cell or isolated enzyme studies.

2. APPLICATIONS IN THE FOOD INDUSTRY

2.1 Total viable counts

Much of the early work on impedance microbiology was in the food and dairy industries. O’Connor (1979), Gnan and Luedecke (1982) and Nieuwenhofand Hoolwerf( 1987) established its use for monitoring total bacterial counts in raw milk. Firstenberg-Eden and Tricarico (1983) extended this to the determination of mesophilic and psychrotrophic counts in raw milk. Monitoring the total microbial loading of a wide range of food products has been evaluated and shown to be successful for frozen vegetables (Hardy et al. 1977), grain products (Sorrells 1981), U H T low-acid foods (Coppola and Firstenberg-Eden 1988), confectionery (Pugh et al. 1988), fish (Ogden 1986) and meat products (Firstenberg-Eden 1983 ; Fletcher et al. 1993; Russell et al. 1994).

2.2 Salmonella detection

Salmonella testing has been a major focus of impedance microbiology. This has culminated in impedance technology being recognized as a recommended methodology for screening of animal feeds under the 1989 Processed Animal

Protein Order (Gibson et al. 1992). The initial Easter- Gibson salmonella detection medium was selenite-cystine/trimethylamine oxide/dulcitol (SC/T/D ; Easter and Gibson 1985). However, subsequent salmonella strains that were negative in SC/T /D due to their inability to ferment dulcitol were reported. Hence a proposed modification of the medium was the replacement of dulcitol by mannitol (Gibson 1987 ; Ogden and Cann 1987). This medium, however, still gave false-positive impedance curves for Citrobacter freundii and E. coli. Greater impedance changes were obtained by Pettipher and Watts (1989a) with mannitol or deoxyribose in place of dulcitol. However, using mannitol more non- salmonella strains also exceeded the detection criteria. Lysine decarboxylation to cadaverine has been used to distinguish between salmonellae and citrobacter (Ogden 1988, 1990a ; Arnott et al. 1988). Similarly Lysine-iron-cystine-neutral red (LICNR) broth has been used to simultaneously test for lysine metabolism and hydrogen sulphide production (Bullock and Frodsham 1989 ; Pettipher and Watts 1989b). False positives due to Cit. freundii and false negatives due to H2S negative salmonellae were notable problems with this medium, but this problem is also found with conventional methods. Modified levels of glucose, sodium chloride and selenite in the lysine medium were used to overcome inhibition of salmonella growth due to selenite inhibition under acid conditions. The modified lysine medium was not as sensitive as S C / T / D for the detection of salmonella from animal feeds (70% of impedance positive samples). However, it was proposed that both SC/T /D and lysine media should be used in impedance procedures (Smith et al. 1990). Davda and Pugh (1991) developed a modified ornithine decarboxylase broth and a selenite cystine trimethylamine oxide deoxyribose medium to improve selection and detection of salmonellae. They were used with LICNR broth (incubated conventionally) in a screen of 80 salmonellae and 32 non-salmonellae which showed the combination of media to be specific and sensitive. Subsequent evaluation with 90 confectionary ingredients and products (spiked and naturally contaminated) resulted in complete agreement by rapid and conventional methods.

Since bacterial reduction of trimethylamine oxide to trimethylamine is repressed under aerobic conditions it was assumed that salmonella impedance media required anaerobic conditions as provided by the use of large volumes with small surface areas. Surprisingly Ogden (1990b) reported that impedance changes increased and time to detection decreased when aerobic impedance conditions were used. Additionally low pre-enriched salmonella numbers (10’ ml-I) were only detected under aerobic conditions. Unfortunately the reason for these observations has not been established but is possibly due to prolonged enzyme induction periods which will be affected by the previous (inoculum) growth conditions.

Mackey and Derrick (1984) studied the lag phase of injured Salm. typhirnurium using impedance measurements. The



advantage of using impedance was that large numbers of experiments could be performed in comparison to time-consuming plate count enumerations. The lag phase varied and in extreme cases ranged from 16 h to 70 h. Alexandrou et al. (1995) used impedance measurement to assess acid-injury to Salm. enteritidis PT4. The prolonged detection time noted with sub-lethally injured cells was due to an extended lag phase and not the result of delayed detection of growth of a small, uninjured sub-population. The results indicated that weak organic acids cause more reversible damage to cellular sites prior to death. These results emphasize the need to recover sublethally injured cells in any isolation procedure.

Parmar et al. (1992) combined the use of immunomagnetic separation to capture and concentrate salmonellae from pre- enriched broth prior to inoculating SC/T /M impedance medium. Salmonella detection was enhanced by reducing the number and type of competing bacteria in skimmed milk powder. Additionally a reduced pre-enrichment period was proposed since salmonellae from a 6 h pre-incubation period were detected in 5-7 h by impedance. Although there was cross-reaction with Ctt. freundii the impedance curves were sufficiently different to distinguish between the two organisms.

Donaghy and Madden (1992) reported a 95 O/O detection rate of Salmonella in raw meat using commercially produced SC/T /M which was equal to that obtained with conventional enrichment methods. However, the rate of false-positive results was high and impedance was less sensitive with processed all animal protein samples. Subsequently they investigated the combined use of indirect impedance with Rappaport-Vassiliadis enrichment broth (Donaghy and Mad- den 1993). Using Lab M medium, the indirect impedance technique could distinguish between Salmonella spp. and the closely related genera Proteus and Citrobacter. The impedance technique showed recoveries of salmonella from processed animal protein and raw meats equivalent to, or better than, those obtained with Rappaport-Vassiliadis used in a conventional salmonella isolation procedure. Dziadkowiec et al. ( 1 995) combined immunomagnetic separation with the indirect RV technique to determine the levels of salmonellae in pre-enrichment broths. Surprisingly the cell density was only lo7 ml-’ after 24 h incubation. A growth curve of Salm. virchow in buffered peptone water was obtained during the pre-enrichment period and demonstrated that non-salmonellae out-numbered the salmonella cells by thousand- fold.

Pless et al. (1994) compared the detection of salmonellae by impedance microbiology with 250 food samples. Food samples were pre-enriched 14-16 h at 37°C in peptone water supplemented with mannitol. The impedance medium con- sisted of magnesium chloride, malachite green oxalate, novo- biocin, phosphate buffer, mannitol, peptone and yeast extract. One hundred and twenty-two salmonella positive samples

were obtained of which 119 were obtained by the impedance method as compared to that obtained by conventional testing with the selenite cystine (106), Rappaport-Vassiliadis soya (95), Rappaport-Vassiliadis (92) and tetrathionate brilliant green medium (64). Six samples gave false-positive results which were due to Enterobacter cloacae. One strain each of Salmonella enteritidis PT8 and Salm. panama were not detected. The impedance method was very suitable as a screening test since negative results were obtained within 38 h.

Quinn et al. (1995) compared a conventional culture technique with three rapid methods (impedance, Gene-Trak and Salmonella-Tek) for the detection of salmonellae in poultry feeds and environmental samples. Salmonellae were isolated from a total of 39.2 O/o samples. However, the percentage positive samples by each method were 38.4% for the impedance method, 25.5 Yo for conventional culture, 28.9 O/O

for the Gene-Trak and 28.5 O/o for the Salmonella-Tek. Since all three rapid methods have AOAC approval and were more sensitive than the conventional procedure, the authors proposed that they warranted further consideration as routine salmonella detection methods and that impedance was the least labour intensive.

The impedance method was accepted by the AOAC as a first action method following a 17 laboratory collaborative trial (Gibson et al. 1992). Samples of coconut, fish meal, prawns, non-fat dried milk, liquid egg and minced beef were artificially contaminated with different Salmonella serotypes at two target levels of 1-5 cells in 25 g and 1 M O cells in 25 g. Each par- ticipating laboratory tested 10 contaminated and five non-contaminated samples per product. Results showed no significant difference between BAM/AOAC and impedance methods.

2.3 Listeria detection

Philips and Griffiths (1989) reported that Listeria spp. could be detected by impedance using a modified growth medium containing acriflavine, ceftazidime, nalidixic acid and aesculin. Although other organisms grew in the medium the shape of the impedance curve could be used for differentiation between Listeria and non-listeria species. Later Hancock et ul. (1993) used a glucose-enriched nutrient broth supplemented with proflavine and moxalactam to detect Listeria species from cheese at spiked levels of lo3 cfu g-’. Neither of these methods could distinguish between L. monocytogenes and other Listeria species. There is only one publication on the use of impedance microbiology to detect Listeria in unspiked foods (Bolton and Gibson 1994).

2.4 Coliform testing

Coliform organisms are frequently used as biological indictors of faecal pollution. The traditional method of ‘most probable number’ is laborious and requires several days before a result is

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IMPEDANCE MICROBIOLOGY 237

obtained. Therefore impedance is an appropriate method since serial dilution of samples is often unnecessary and the results are automatically recorded. Since coliforms and E. coli detection is a routine technique appropriate media have been developed which are suitable for impedance methods. Silverman and Munoz (1979) used an impedance technique to rapidly enumerate faecal coliforms in effluents from sewage treatment plants. Martins and Selby (1980) evaluated an impedance method for quantifying coliforms in meat. Similarly a method for the detection of E. coli in shellfish has been described with coliform broth at 44°C (Dupont et al. 1994).

Druggan et a/. (1993) reported the simultaneous detection of coliforms and E. coli using indirect impedance. The formulation was based on the ability of coliform bacteria to ferment lactose (indicated by a change in broth colour from red to yellow) and a negative impedance change. The presence of E. coli was demonstrated by its ability to cleave the substrate methyl- umbelliferyl-P-D-glucuronide (MUG) to yield fluorescent methylumbelliferone. Fluorescence was determined by exposing the impedance cell to ultraviolet light at 366 nm at the end of the incubation period.

2.5 Clostridia

Gibson (1987) used impedance measurements to detect growth of Clostridzum hotulinum in a selective medium. 'The linear relationship between detection time and log,,, counts (r = 0.77) had such wide confidence limits (log,,, 2.3) that it was not possible to enumerate CI. hotulinum in pork slurries. However, knowledge that a sample contained clostridia allowed a count to be made within 24 h, whereas 48 h would be required for conventional plate methods.

Clostridium tyrohutyricum is of considerable commercial importance in the spoilage of high-pH cheeses, particularly some cheeses from France, Italy, Spain and Switzerland. Since the organism ferments lactic acid to carbon dioxide, hydrogen and butyric acid, the indirect technique was used (Druggan et al. 1993). The growth medium Whitley Anaerobe broth (WAB) was supplemented with 1 YO skimmed milk powder to promote gas production and the potassium hydroxide concentration increased to 1 (Yo (w/v).

2.6 Yeast

Owens et a/. (1992) measured the impedance changes during growth of S. cereririar, Z~~~srrcchiirom),cesn~ices hailii and Rhodotorula ruhra in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source in comparison with a medium containing r,-asparagine as sole nitrogen source. Decreases in impedance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with I,-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for

impedance change in yeast cultures is the uptake of charged ammonium ions as the nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula ruhra cultures with L-asparagine as sole carbon source caused large increases in impedance with growth. Chemical analysis of culture filtrates showed that this increase in impedance was due to use of L- asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic requirement as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in impedance.

Frozen fruit juice concentrates containing an average microbial population of log 1.54 cfu ml-' were examined by traditional plating techniques and direct and indirect impedance (Deak and Beuchat 1993). The initial populations in diluted (1 : 4) concentrates increased to an average of log 3.82 cfu ml-' during incubation at 25°C for 24 h. Pre-incubation before analysis facilitated the resuscitation of cells that may have been freeze-injured. Yeasts were recovered in equal numbers on acidified (pH 3.5) potato dextrose agar and di- chloran rose bengal chloramphenical agar (pH 5.6). Yeasts and bacteria were recovered on orange serum agar. Detection times determined by indirect impedance correlated fairly well ( r = 0.73) with populations detected by conventional media. Populations in diluted concentrates which were not incubated before examination were detected by impedance in an average of 48.9 h, whereas detection times for diluted concentrates incubated for 24 h at 25°C before testing were reduced to an average of 14.1 h. Examination by direct impedance required an additional 10-20 h to reach changes in impedance of 5 pS h-I.

Betts (1993) appraised direct impedance, direct capacitance and indirect impedance measurement. Although these methods can be used to detect and enumerate yeasts, the two direct methods must be linked to the correct growth medium. The optimum medium for direct impedance monitoring was found to be CBAT, whereas either CBAT or Yeast Carbon Base and ammonium sulphate (CBAS) could be used for direct capacitance monitoring. CBAT could also be used in indirect impedance systems. However, in some cases a medium not required in indirect systems as the food sample itself provided growth requirements. Generally, a direct capacitance system was better than a direct impedance system for yeast detection and enumeration, as it detected more yeast species. Indirect impedance was equivalent to direct capacitance. In a survey of samples from European fruit juice manufacturers Druggan et al. (1993) reported the detection of 2. bailii and R. rubra using indirect impedance with CBAT as the growth medium. Samples were incubated at 30°C for between 24 and 72 h.

2.7 Lactic acid bacteria in the dairy industry

The dairy industry produces a range of fermented milk products which require the controlled growth of lactic acid

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bacteria. Additionally dairy products can act as a vector for food poisoning organisms. Therefore the application of impedance microbiology in the dairy industry has been to determine the presence of food pathogens such as salmonellae and to monitor the growth of the lactic acid bacteria starter cultures (Asperger and Pless 1994). Lanzanova et al. (1993) measured the impedance changes due to the growth and metabolic activity of lactic acid bacteria in milk. The activity of pure starter cultures and the stability of mixed cultures could be monitored and controlled. Since instability can be due to the presence of bacteriophage, lactic acid starter cultures are regularly rotated. Detection of phages against two major groups of DL-lactococcal mixed-strain starter cultures have been studied (Svensson 1994). A sensitive assay was obtained that detected phages against acid-producing strains within 3 4 h. Phages caused a delay in detection time and a decrease in the total impedance change. The limit ofdetection with pure strains of acid- or carbon dioxide-producing starter cultures was 1 and 100 pfu respectively.

2.8 Predictive microbial growth modelling

In recent years, modelling for the purpose of predicting microbiological spoilage of foods has gained much interest. Pre- dictive modelling requires several experimental parameters to be varied and the collection of adequate data. The advantage of using impedance is that large numbers of experiments can be designed without the tedious and time-consuming nature of plate count enumerations. An early example is the work of Mackey and Derrick (1984) who studied the lag phase of injured Salm. typhimurium using impedance measurements. Deak and Beuchat (1993, 1994) studied four variables (temperature, water activity, pH and potassium sorbate concentration) at three levels to determine their effects on the growth of six yeasts (Candida glabrata, C . parapsilosis, Debar- yom,yces hansenii, Pichia membranaefaciens, S . cerevisiae and Z . bailii) isolated from spoiled food products. The detection time and the maximum change in impedance were measured by the indirect technique. Temperature and water activity and potassium sorbate concentrations were the most important variables individually and in combination that affected yeast growth. Shelf-life of fruit juice at a, less than or equal to 0.96, pH less than or equal to 3.8 and containing less than or equal to 0.03 O/o potassium sorbate, when stored at less than or equal to lO"C, would be predicted to be greatly extended. Zygosaccharomyces bailii was the most resistant of the yeasts in terms of ability to tolerate stress conditions and was proposed as a test species to develop a predictive model for spoilage.

Borch and Wallentin (1993) used direct impedance to study the effect of growth medium composition, growth conditions and inoculum level with Y. enterocolitica 0 : 3. A polynomial model was developed describing the effect of temperature,

pH and L-lactate concentration on impedance response parameters. The model could be used for predicting growth rates and these corresponded with viable counts of Y. enterocolitica in minced pork. The impedance response curves were fitted to the Gompertz equation 0, = A + C exp( - exp( - B (time - M)))) for microbial growth by Lindberg and Borch (1994). Inoculum levels between log3 and 7 did not affect the B or C parameters. The M parameter was, however, affected; the lower the inoculum level, the higher the M value. Polynomial models for log B and log C were developed describing the effect of temperature (7-23"C), pH (5+6.5), L-lactate (0-1.2 Yo) and their combinations under aerobic conditions. The impedance rate (B.C/e) was of the same magnitude at 23"C, pH 5.4 and 1.2 O/o 1.-lactate as at 7"C, pH 6.5 and 0 O/o 1,-lactate. A high correlation was found between the impedance rates predicted from impedance polynomial models and rates predicted from a published absorbance model. Dengremont and Membre (1994) also used impedance to model the growth of Y. enterocolitica 0: 3 according to various temperature, pH and salt combinations. The predicted rate of growth correlated well with observed values.

3. NON-FOOD RELATED APPLICATIONS

3.1 Antibiotic resistance

The effects of antibacterial compounds on impedance curves when pure cultures are exposed to test drugs can be divided into three principle classes of response. Firstly, there is an increase in the D T as the initial test population size is reduced. The slope of the response curve remains unchanged for those organisms not affected by the drug, indicating an unchanged growth rate. Secondly, increasing the drug concentration results in a decreasing slope due to a drug-related reduction in growth rate of the test organism. Finally, a combination of the two phenomena in which a low drug concentration simply delays onset of growth by virtue of killing the most sensitive organisms in the population and the remaining cells grow normally. Antimicrobials, especially when used in combination, can also affect the overall change in conductance.

The post-antibiotic effect (PAE) is the persistent inhibition of bacterial growth after a brief exposure to an antibiotic (MacKenzie et al. 1994). Most 0-lactams do not induce a PAE for Gram-negative bacteria, but PAEs have been reported for carbapenems and penems. Majcherczyk et al. (1994) studied the effect of sequential doses of imipenem on the PAE for Ps. aeruginosa and E. coli cultures in a chemostat. The PAE for the bacterial population did not change even after six successive doses of imipenem. However, a Ps. aeruginosu mutant was isolated which had a shortened imipenem PAE yet unchanged MIC. Comparison of growth of parent and


I M P E DAN C E MI C ROB I0 LOG Y 239

mutant using impedance microbiology, viable counts, incor- poration of radiolabelled N-acetyl-D-ghcosamine and cell volume changes confirmed the PAE difference. The mutant was found to have a reduced expression of a 52 kDa membrane protein.

Detection of antibiotic-resistant strains can be rapidly determined using impedance microbiology by measuring the detection time in the presence and absence of the test antibiotic. Gibson (1988) used impedance measurements to estimate numbers of antibiotic-resistant salmonella strains in pork slurries. Later Blackburn and Davis (1994) enumerated antibiotic-resistant strains of salmonella, verotoxigenic E. colt 0157 : H7, Y. enterocolitica and Aeromonas in foods. Most antibiotic-resistant strains had slower growth rates at their optimum incubation temperature than the parent strain. This difference was reduced as the incubation temperature was lowered.

3.2 Biocide efficacy testing and microbial biofilms

The use of impedance microbiology is not only related to microbiological analysis of raw materials and finished products, but is being used increasingly as a research tool in the evaluation ofnovel antibacterial agents (Gould et al. 1989) and challenge testing of final products. Impedance microbiology enables the monitoring of real time observations on organism- drug interactions. The ability of a drug at a fixed concentration to inhibit or kill a bacterial population is measured over a fixed time period. This is in contrast with the single time point after overnight incubation used with most conventional methods. Applications of preservative testing for pharmaceuticals and cosmetic products were assessed by Connolly et al. (1994). The test organisms were Staph. aureus, Candida albicans, Aspergillus niger and Ps. aeruginosa. A good correlation was obtained between detection time and plate count after exposure to chlorhexidine, methyl paraben and pheoxyethanol.

Microbes frequently colonize an inert matrix by forming a biofilm composed of extracellular polysaccharides and subsequently entrap other micro-organisms. Microbial biofilms are reported to be 10- to 100-fold more resistant to disinfectants than planktonic cultures. This possibility is due to increased exopolysaccharide synthesis. Pseudomonad growth on surgical catheters can cause localized infections. In a food processing microbial biofilms on metal tubing and rubber surfaces can be a source of persistent food contamination. The efficacy of disinfectants for the removal of biofilms is therefore very important.

Holah et al. (1990) described the use of direct impedance to enumerate niicrobcs on steel discs following exposure to 12 disinfectants. The ‘pass’ criterion was a 5 log reduction in test organism viability after 5 niin exposure. Similarly Drug-

gan et al. (1993) used indirect impedance with 1 cm diameter steel discs which were directly transferred to the impedance tubes. This avoided the inaccuracy of physical removal of the microbial growth using sand agitation. Sodium hypochlorite was used as the model for chlorine-based disinfectants. The European suspension test organisms Ps. aeruginosa NCIB 10421, Proteus mirabifis NCIB 12596, Staph. aureus NCTC 10788 and S. cerevisiae ATCC 9763 were used to test the efficacy of sodium hypochlorite to microbial biofilms. Biofilms were produced by the three bacterial strains but not by the yeast. The detection time of the bacterial biofilms did not correspond with the cell density possibly due to differences in microbial metabolism rates. For example Ps. aeruginosa produced a biofilm of 5.0 x lo6 cfu disc which gave a detection time of 4.0 h in WIB, whereas Staph. aureus biofilm was 2.0 x lo7 cfu per disc with a detection time of 6.5 h. Johnston and Jones (1995) used a Modified Robbins Device (whereby a microbial culture is circulated over steel discs) to produce biofilms of Ps. aeruginosa. Enumeration by indirect impedance showed higher numbers of surviving cells than cell recovery by swabbing or vortexing.

Mosteller and Bishop (1993) showed that Ps. Jluorescens, Y. enterocolitica and L. monocytogenes readily attach to rubber and Teflon@ surfaces. The test organisms attached in slightly higher numbers to the rubber surface than the Teflon@. Plate counts, impedance microbiology and the direct epifluorescent filter technique were compared. Impedance microbiology was the best method of enumeration since it allowed the estimation of both reversibly and irreversibly attached bacteria. Biocides against a bacterial suspension resulted in a greater than or equal to Slog cycle reduction. However, the same concentrations were relatively ineffective against the attached bacteria. The goal reduction (3 log cycles) was achieved on the Teflon@ surface with the iodophor, hypochlorite and fatty acid biocides with a log-cycle reduction in the number of Y. enterocolitica of 3.09, 3.19 and 3.3 1 respectively. Pseudomonas JEuorescens was reduced by 3.16 on both the rubber and Teflon@ surfaces when exposed to the hypochlorite biocide.

Microbially influenced corrosion affects various industries but can be partially controlled by the application of biocides. Copper surfaces exposed to natural seawater were colonized by bacteria within 3 weeks of exposure independent of alloy composition (Mansfeld and Little 1992). Jack et al. (1992) and Nivens et al. (1992) studied the corrosion rates of carbon steel by monocultures and various combinations of Bacillus sp., Hafnia alvei and Desulfovibrio gigas biofilms in an aerobic, continuously flowing freshwater reactor containing 0.4 mmol I - ’ sulphate. Debruyn et al. (1994) correlated the viable count of D. desulfuricans in iron sulphite medium with impedance niicrohiology (r = 0.974). Subscquently the impedancc method was used to assess the efficacy of biocides against D. desuljiuricans. A 56 O/o and a 100 O/o kill was obtained using 60


240 P. SILLEY AND S . FORSYTHE

and 200 mg 1-' quaternary ammonium compounds respectively.

3.3 Detection of plant pathogens

Indirect and direct impedance methods have been used to identify and detect plant pathogens (Franken and Van- derzouwen 1993). Strains of pathovars of Ps. syringae and Xanthomonas campestris, Clavibacter michigunensis, Erwinia carotozwu ssp. atroseptica and Erm. chrysanthemi were tested in a Malthus machine. In a direct cell the erwinias gave lower detection times and higher conductivity changes than the pseudomonads and xanthomonads at 27°C. Clavibacter michi- ganensis was not detectable by direct impedance. In indirect impedance the pseudomonads gave a lower detection time and higher maximum rates of impedance change than the xanthomonads. Erwinia detection was temperature dependent in that Erw. curotovoru 161 detection was more sensitive at 17°C whereas Erw. chr-ysanthemi 502 detection was more sensitive at 27°C. Franken and Vanderzouwen (1993) con- cluded than detection of plant pathogens still requires improvements in incubation conditions.

Pseudomonus syringae pv. pisi is the causal agent of bacterial blight which causes considerable financial loss. Fraaije et al. (1 993) compared impedance assays, immunofluorescence microscopy and ELISA with conventional methods based on dilution-plate assays. Immunofluorescence and dilution-plate assays of ground and 2 h soaked pea samples were less sensitive than detection in suspension water of the 6 h soaked pea seeds. Impedance detection times correlated with the viable count at 17 and 27°C. Confirmation of results by isolation was more successful at 17°C because of the relatively lower activity of saprophytic pseudomonads at this temperature.

3.4 DNA damage evaluation

The most widely used method for assessing mutagenicity is the Salmonella-Ames test. This requires the reversion rate of Salmn. typhirnuriurn histidine auxotrophs to be assessed after exposure to a test substance. The procedure is highly labour intensive because of the preparation of minimal media and time consuming since a 2 d incubation period is required. Forsythe (1990) developed a differential killing assay using E. coli WP2 (wild-type) and WP67 (uvrA, polA) to produce a rapid screening method for direct-acting mutagenic compounds. The assay showed that mitomycin C, N-nitroso- guanidine, potassium dichromate, sodium azide and acridine orange were direct-acting mutagens. With this method results could be obtained within hours, as compared with days for standard tests. The technique has been applied to dem- onstrate the production of direct-acting oxidative genotoxins

following bacterial reduction of common food colourants (Sweeney et al. 1994).

3.5 Impedance-based enzyme assays

An enzyme-linked impedance method for the detection of ethanal has been described (Saad and Wallach 1992). The method detects the oxidation of ethanal in the presence of yeast aldehyde dehydrogenase. A linear relationship was demonstrated between impedance changes and ethanol concentrations up to 25 pmol 1-'. The assay was validated using wines by comparison with a spectrophotometric method. Both methods gave similar results, but impediometry avoided the pre-treatment of coloured samples.

Urease is an indicator of microbial activity in soil and can be assayed using direct impedance (Hard, personal com- munication). This method can be used to measure the affect of heavy metals and ozone on microbial activity in soil and is an alternative to the standard respirometry method.

4. CONCLUSIONS

Impedance microbiology has been used in the food industry to monitor quality and to detect specific food-pathogens. Additionally it is now an accepted AOAC method for the detection of salmonella in foods. More recently the technique has been more widely applied, for example the detection of plant pathogens and DNA-damaging compounds. However, although impedance equipment has been commercially available for many years there is still a number of applications to be exploited such as soil microbiology and enzyme assays.

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Impedance Microbiology-A Rapid Change for Microbiologists

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