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

1. INTRODUCTION

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 Experimenta1 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 Stewarts 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

2.7 Lactic acid bacteria a n d 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

Impedance can be defined simply as the resistance to flow

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 sinusoidal 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. T h e AC equivalent of conductance
is admittance which is defined as the reciprocal of the impedance. T h e 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 1C 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, Suomalainen 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 approximately lo6cfu 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. T h e 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 Rappaport-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. T h e unit is tightly sealed such that any C 0 2
produced as a result of normal metabolism is absorbed by the

0 1996 The Society for Applied Bacteriology,Journal of Applied Bacteriology 80, 233-243

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 27C 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, Aeromonas 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. Applications 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. OConnor (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). T h e initial EasterGibson salmonella detection medium was selenite-cystine/trimethylamine oxide/dulcitol ( S C / T / D ; Easter and
Gibson 1985). However, subsequent salmonella strains that
were negative in S C / 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 nonsalmonella 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. T h e
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 S C / 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. T h e

0 1996 The Society for Applied Bacteriology, Journal of Applied Bacteriology 80, 233-243

236 P. SILLEY AND S. FORSYTHE

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 preenriched broth prior to inoculating S C / 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
S C / 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 Madden 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 thousandfold.
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 37C in peptone water
supplemented with mannitol. The impedance medium consisted of magnesium chloride, malachite green oxalate, novobiocin, 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 participating 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 lo3cfu 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

0 1996 The Society for Applied Bacteriology, Journal of Applied Bacteriology80, 233-243

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 44C (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 methylumbelliferyl-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 YOskimmed 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 p H 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 Lasparagine 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). T h e initial populations in
diluted (1 : 4) concentrates increased to an average of log 3.82
cfu ml-' during incubation at 25C 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 dichloran 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 25C 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 30C 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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238

P. SILLEY AND S.FORSYTHE

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. Predictive modelling requires several experimental parameters
to be varied and the collection of adequate data. T h e 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, Debaryom,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. T h e model could be used for predicting growth rates
and these corresponded with viable counts of Y. enterocolitica
in minced pork. T h e 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, p H 5.4 and 1.2O/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

T h e 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. T h e 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.
T h e 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

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IM P E DANC E MI C ROB I0LOG Y 239

mutant using impedance microbiology, viable counts, incorporation of radiolabelled N-acetyl-D-ghcosamine and cell
volume changes confirmed the PAE difference. T h e 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 organismdrug 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 N C T C
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. T h e 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 lo6cfu 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

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240 P. SILLEY AND S . FORSYTHE

and 200 mg 1-' quaternary ammonium compounds respectively.

following bacterial reduction of common food colourants

(Sweeney et al. 1994).

3.3 Detection of plant pathogens

3.5 Impedance-based enzyme assays

Indirect and direct impedance methods have been used to

identify and detect plant pathogens (Franken and Vanderzouwen 1993). Strains of pathovars of Ps. syringae and

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 communication). 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.

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 27C. Clavibacter michiganensis 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 17C whereas Erw. chr-ysanthemi 502 detection was more
sensitive at 27C. Franken and Vanderzouwen (1993) concluded 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 27C. Confirmation of results by
isolation was more successful at 17C 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-nitrosoguanidine, 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 demonstrate the production of direct-acting oxidative genotoxins

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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