192 Chilled foods 8. 4.1 Electrical methods e enumeration of microorganisms in solution can be achieved by one of two electrical methods, one measuring particle numbers and size, the other monitoring metabolic activity Particle counting e counting and sizing of particles can be done with the Coulter principle using instruments such as the Coulter Counter( Coulter Electrics, Luton). The method is based on passing a current between two electrodes placed on either de of a small aperture. As particles or cells suspended in an electrolyte are drawn through the aperture they displace their own volume of electrolyte solution, causing a drop in d. c conductance that is dependent on cell size. These changes in conductance are detected by the instrument and can be presented as a series of voltage pulses, the height of each pulse being proportional to the volume of the particle, and the number of pulses equivalent to the number of particles The technique has been used extensively in research laboratories for experiments that require the determination of cell sizes or distribution. It has found use in the area of clinical microbiology where screening for bacteria is required(Alexander et al. 1981). In food microbiology however, little use has been made of the method. There are reports of the detection of cell numbers in milk(Dijkman et al, 1969)and yeast estimation in beer(MaCrae 1964), but little other work has been published. Any use of particle counting for food microbiology would probably be restricted to non-viscous liquid samples or particle-free fluids, since very small amounts of sample debris could cause significant interference, and cause aperture blockage Metabolic activity Stewart (1899) first reported the use of electrical measurement to monitor microbial growth. This author used conductivity measurements to monitor the putrefaction of blood, and concluded that the electrical changes were caused by ions formed by the bacterial decomposition of blood constituents. After this initial report a number of workers examined the use of electrical measurement to monitor the growth of microorganisms. Most of the work was successful however, the technique was not widely adopted until reliable instrumentation capable of monitoring the electrical changes in microbial cultures became There are currently four instruments commercially available for the detection of organisms by electrical measurement. The Malthus System (IDG, Bury, UK) based on the work of Richards et al.(1978) monitors conductance changes occurring in growth media as does the Rabit System(Don Whitley Scientific Yorkshire), whilst the Bactometer(bioMerieux, Basingstoke, UK), and the Batrac (SyLab, Purkersdorf, Austria)(Bankes 1991) can monitor both conductance and capacitance signals. All of the instruments have similar basic components:(a) an incubator system to hold samples at a constant temperature
8.4.1 Electrical methods The enumeration of microorganisms in solution can be achieved by one of two electrical methods, one measuring particle numbers and size, the other monitoring metabolic activity. Particle counting The counting and sizing of particles can be done with the ‘Coulter’ principle, using instruments such as the Coulter Counter (Coulter Electrics, Luton). The method is based on passing a current between two electrodes placed on either side of a small aperture. As particles or cells suspended in an electrolyte are drawn through the aperture they displace their own volume of electrolyte solution, causing a drop in d.c. conductance that is dependent on cell size. These changes in conductance are detected by the instrument and can be presented as a series of voltage pulses, the height of each pulse being proportional to the volume of the particle, and the number of pulses equivalent to the number of particles. The technique has been used extensively in research laboratories for experiments that require the determination of cell sizes or distribution. It has found use in the area of clinical microbiology where screening for bacteria is required (Alexander et al. 1981). In food microbiology however, little use has been made of the method. There are reports of the detection of cell numbers in milk (Dijkman et al., 1969) and yeast estimation in beer (MaCrae 1964), but little other work has been published. Any use of particle counting for food microbiology would probably be restricted to non-viscous liquid samples or particle-free fluids, since very small amounts of sample debris could cause significant interference, and cause aperture blockage. Metabolic activity Stewart (1899) first reported the use of electrical measurement to monitor microbial growth. This author used conductivity measurements to monitor the putrefaction of blood, and concluded that the electrical changes were caused by ions formed by the bacterial decomposition of blood constituents. After this initial report a number of workers examined the use of electrical measurement to monitor the growth of microorganisms. Most of the work was successful; however, the technique was not widely adopted until reliable instrumentation capable of monitoring the electrical changes in microbial cultures became available. There are currently four instruments commercially available for the detection of organisms by electrical measurement. The Malthus System (IDG, Bury, UK) based on the work of Richards et al. (1978) monitors conductance changes occurring in growth media as does the Rabit System (Don Whitley Scientific, Yorkshire), whilst the Bactometer (bioMerieux, Basingstoke, UK), and the Batrac (SyLab, Purkersdorf, Austria) (Bankes 1991) can monitor both conductance and capacitance signals. All of the instruments have similar basic components: (a) an incubator system to hold samples at a constant temperature 192 Chilled foods
Conventional and rapid analytical microbiology 193 during the test;(b)a monitoring unit that measures the conductance and/or capacitance of every cell at regular frequent intervals(usually every 6 minutes); and (c)a computer-based data handling system that presents the results in usable format The detection of microbial growth using electrical systems is based on the measurement of ionic changes occurring in media, caused by the metabolism of microorganisms. The changes caused by microbial metabolism and the detailed electrochemistry that is involved in these systems has been previously described in some depth(Eden and Eden 1984, Easter and Gibson 1989, Bolton and Gibson 1994). The principle underlying the system is that as bacteria grow and metabolise in a medium, the conductivity of that medium will increase. The electrical changes caused by low numbers of bacteria are impossible to detect using currently available instrumentation, approximately 106 organisms/ml must be present before a detectable change is registered. This is known as the threshold of detection, and the time taken to reach this point is the detection time In order to use electrical systems to enumerate organisms in foods, the sample must initially be homogenised. The growth well or tube of the instrument ontaining medium is inoculated with the homogenised sample and connected to the monitoring unit within the incubation chamber or bath. The electrical properties of the growth medium are recorded throughout the incubation period The sample container is usually in the form of a glass or plastic tube or cell, in which a pair of electrodes is sited. The tube is filled with a suitable microbial growth medium, and a homogenised food sample is added. The electrical changes occurring in the growth medium during microbial metabolism are monitored via the electrodes and recorded by the instrument As microorganisms grow and metabolise they create new end-products in the medium. In general, uncharged or weekly charged substrates are transformed into highly charged end-products(Eden and Eden 1984), and thus the onductance of the medium increases. The growth of some organisms such as yeasts does not result in large increases in conductance. This is possibly due to the fact that these organisms do not produce ionised metabolites and this can result in a decrease in conductivity during growth When an impedance instrument is in use, the electrical resistance of the growth medium is recorded automatically at regular intervals(e.g. 6 minutes) hroughout the incubation period. When a change in the electrical parameter being monitored is detected, then the elapsed time since the test was started is calculated by a computer; this is usually displayed as the detection time. The complete curve of electrical parameter changes with time( Fig. 8. 1) is similar to a bacterial growth curve, being sigmoidal and having three stages: (a) the active stage, where any electrical changes are below the threshold limit of detection of the instrument;(b) the active stage, where rapid electrical changes occur;and(c)the stationary or decline stage, that occurs at the end of the active tage and indicates a deceleration in electrical changes The electrical response curve should not be interpreted as being similar to a microbial growth curve. It is accepted(Easter and Gibson 1989)that the lag and
during the test; (b) a monitoring unit that measures the conductance and/or capacitance of every cell at regular frequent intervals (usually every 6 minutes); and (c) a computer-based data handling system that presents the results in usable format. The detection of microbial growth using electrical systems is based on the measurement of ionic changes occurring in media, caused by the metabolism of microorganisms. The changes caused by microbial metabolism and the detailed electrochemistry that is involved in these systems has been previously described in some depth (Eden and Eden 1984, Easter and Gibson 1989, Bolton and Gibson 1994). The principle underlying the system is that as bacteria grow and metabolise in a medium, the conductivity of that medium will increase. The electrical changes caused by low numbers of bacteria are impossible to detect using currently available instrumentation, approximately 106 organisms/ml must be present before a detectable change is registered. This is known as the threshold of detection, and the time taken to reach this point is the detection time. In order to use electrical systems to enumerate organisms in foods, the sample must initially be homogenised. The growth well or tube of the instrument containing medium is inoculated with the homogenised sample and connected to the monitoring unit within the incubation chamber or bath. The electrical properties of the growth medium are recorded throughout the incubation period. The sample container is usually in the form of a glass or plastic tube or cell, in which a pair of electrodes is sited. The tube is filled with a suitable microbial growth medium, and a homogenised food sample is added. The electrical changes occurring in the growth medium during microbial metabolism are monitored via the electrodes and recorded by the instrument. As microorganisms grow and metabolise they create new end-products in the medium. In general, uncharged or weekly charged substrates are transformed into highly charged end-products (Eden and Eden 1984), and thus the conductance of the medium increases. The growth of some organisms such as yeasts does not result in large increases in conductance. This is possibly due to the fact that these organisms do not produce ionised metabolites and this can result in a decrease in conductivity during growth. When an impedance instrument is in use, the electrical resistance of the growth medium is recorded automatically at regular intervals (e.g. 6 minutes) throughout the incubation period. When a change in the electrical parameter being monitored is detected, then the elapsed time since the test was started is calculated by a computer; this is usually displayed as the detection time. The complete curve of electrical parameter changes with time (Fig. 8.1) is similar to a bacterial growth curve, being sigmoidal and having three stages: (a) the inactive stage, where any electrical changes are below the threshold limit of detection of the instrument; (b) the active stage, where rapid electrical changes occur; and (c) the stationary or decline stage, that occurs at the end of the active stage and indicates a deceleration in electrical changes. The electrical response curve should not be interpreted as being similar to a microbial growth curve. It is accepted (Easter and Gibson 1989) that the lag and Conventional and rapid analytical microbiology 193
194 Chilled foods Time (h Fig. 8.1 A conductance curve generated by the growth of bacteria in a suitable medium logarithmic phases of microbial growth occur in the inactive and active stages of the electrical response curve, up to and beyond the detection threshold of the instrument. The logarithmic and stationary phases of bacterial growth occur during the active and decline stages of electrical response curves In order to use detection time data generated from electrical instruments to assess the microbiological quality of a food sample, calibrations must be done The calibration consists of testing samples using both a conventional plating test and an electrical test. The results are presented graphically with the conventional result on the y-axis and the detection time on the x-axis(Fig. 8.2). The result is a negative line with data covering 4 to 5 log cycles of organisms and a correlation coefficient greater than 0.85(Easter and Gibson, 1989), Calibrations must be done for every sample type to be tested using electrical methods, different samples will contain varying types of microbial flora with differing rates of growth. This can greatly affect electrical detection time and lead to incorrect results unless correct calibrations have been done So far. the use of electrical instruments for total microbial assessment has een described. These systems, however, are based on the use of a growth medium and it is thus possible, using media engineering, to develop methods for the enumeration or detection of specific organisms or groups of organisms Many examples of the use of electrical measurement for the detection/ enumeration of specific organisms have been published; these include
logarithmic phases of microbial growth occur in the inactive and active stages of the electrical response curve, up to and beyond the detection threshold of the instrument. The logarithmic and stationary phases of bacterial growth occur during the active and decline stages of electrical response curves. In order to use detection time data generated from electrical instruments to assess the microbiological quality of a food sample, calibrations must be done. The calibration consists of testing samples using both a conventional plating test and an electrical test. The results are presented graphically with the conventional result on the y-axis and the detection time on the x-axis (Fig. 8.2). The result is a negative line with data covering 4 to 5 log cycles of organisms and a correlation coefficient greater than 0.85 (Easter and Gibson, 1989), Calibrations must be done for every sample type to be tested using electrical methods; different samples will contain varying types of microbial flora with differing rates of growth. This can greatly affect electrical detection time and lead to incorrect results unless correct calibrations have been done. So far, the use of electrical instruments for total microbial assessment has been described. These systems, however, are based on the use of a growth medium and it is thus possible, using media engineering, to develop methods for the enumeration or detection of specific organisms or groups of organisms. Many examples of the use of electrical measurement for the detection/ enumeration of specific organisms have been published; these include: Fig. 8.1 A conductance curve generated by the growth of bacteria in a suitable medium. 194 Chilled foods
Conventional and rapid analytical microbiology 195 2 Fig 8.2 Calibration curve showing changes in conductance detection time with bacterial total viable count(Tvc) Enterobacteriaceae Cousins and marlatt 1990, Petitt 1989), Pseudomonas (Banks et al. 1989), Yersinia enterocolitica(Walker 1989)and yeasts( Connolly et al., 1988), E coli(Druggan et al. 1993), Campylobacter(Bolton and Powell 993). In the future, the number of types of organism capable of being detected will undoubtedly increase. Considerable research is currently being done on media for the detection of Listeria and media for other organisms will follow Most of the electrical methods described above involve the use of direct measurement,i.e. the electrical changes are monitored by electrodes immersed in the culture medium. Some authors have indicated the potential for indirect conductance measurement(Owens et al. 1989)for the detection of microorgan- isms. This method involves the growth medium being in a separate compartment to the electrode within the culture cell. The liquid surrounding the electrode is a gas absorbent, e.g. potassium hydroxide for carbon dioxide. The growth medium is inoculated with the sample and, as the microorganisms grow, gas is released This is absorbed by the liquid surrounding the electrode, causing a change in conductivity, which can be detected This technique may solve the problem caused by microorganisms that oduce only small conductance changes in conventional direct conductance cells. These organisms, e.g. many yeast species, are very difficult to detect using
Enterobacteriaceae (Cousins and Marlatt 1990, Petitt 1989), Pseudomonas (Banks et al. 1989), Yersinia enterocolitica (Walker 1989) and yeasts (Connolly et al., 1988), E.coli (Druggan et al. 1993), Campylobacter (Bolton and Powell 1993). In the future, the number of types of organism capable of being detected will undoubtedly increase. Considerable research is currently being done on media for the detection of Listeria, and media for other organisms will follow. Most of the electrical methods described above involve the use of direct measurement, i.e. the electrical changes are monitored by electrodes immersed in the culture medium. Some authors have indicated the potential for indirect conductance measurement (Owens et al. 1989) for the detection of microorganisms. This method involves the growth medium being in a separate compartment to the electrode within the culture cell. The liquid surrounding the electrode is a gas absorbent, e.g. potassium hydroxide for carbon dioxide. The growth medium is inoculated with the sample and, as the microorganisms grow, gas is released. This is absorbed by the liquid surrounding the electrode, causing a change in conductivity, which can be detected. This technique may solve the problem caused by microorganisms that produce only small conductance changes in conventional direct conductance cells. These organisms, e.g. many yeast species, are very difficult to detect using Fig. 8.2 Calibration curve showing changes in conductance detection time with bacterial total viable count (TVC). Conventional and rapid analytical microbiology 195
196 Chilled foods conventional direct conductance methods, but detection is made easy by the use of indirect conductance monitoring(Betts 1993). The increased use of indirect methods in the future could considerably enhance the ability of electrical systems to detect microorganisms that produce little electrical change in direct ystems, thus increasing the number of applications of the technique within the food 8.4.2 Adenosine triphosphate(ATP) bioluminescence The non-biological synthesis of ATP in the extracellular environment has been demonstrated(Ponnamperuma et al, 1963), but it is universally accepted that such sources of ATP are very rare(Huernnekens and Whiteley 1960). ATP is a high-energy compound found in all living cells(Huernnekens and Whiteley 1960), and it is an essential component in the initial biochemical steps of substrate utilisation and in the synthesis of cell material McElroy (1947)first demonstrated that the emission of light in the bioluminescent reaction of the firefly, Photinus pyralis, was stimulated by ATP. The procedure for the determination of ATP concentrations utilising crude firefly extracts was described by McElroy and Streffier(1949) and has since been used in many fields as a sensitive and accurate measure of ATP. The light yielding reaction is catalysed by the enzyme luciferase, this being the enzyme found in fireflies causing luminescence. Luciferase takes part in the following reaction: 1. Luciferase Luciferin+ ATP-Mg- Luciferase- Luciferin- AMP+ PP The complex is then oxidised 2. Luciferase-Luciferin- AMP O2- Luciferase-Luciferin- AMP= The oxidised complex is in an excited stage, and as it returns to its ground stage a photon of light is released 3. Luciferase-Luciferin- AMP=0-(Luciferase- Luciferin- AMP=0) light The light-yielding reaction is efficient, producing a single photon of light for every luciferin molecule oxidised and thus every ATP molecule used (Seliger and McElroy 1960) Levin et al.(1964)first described the use of the firefly bioluminescence assay of ATP for detecting the presence of viable microorganisms. Since this initial report considerable work has been done on the detection of viable organisms in environmental samples using a bioluminescence technique(Stalker 1984). As all viable organisms contain ATP, it could be considered simple to use a bioluminescence method to rapidly enumerate microorganisms. Research, however. has shown that the amount of atP in different microbial cells varies depending on species, nutrient level, stress level and stage of growth(Stannard
conventional direct conductance methods, but detection is made easy by the use of indirect conductance monitoring (Betts 1993). The increased use of indirect methods in the future could considerably enhance the ability of electrical systems to detect microorganisms that produce little electrical change in direct systems, thus increasing the number of applications of the technique within the food industry. 8.4.2 Adenosine triphosphate (ATP) bioluminescence The non-biological synthesis of ATP in the extracellular environment has been demonstrated (Ponnamperuma et al., 1963), but it is universally accepted that such sources of ATP are very rare (Huernnekens and Whiteley 1960). ATP is a high-energy compound found in all living cells (Huernnekens and Whiteley 1960), and it is an essential component in the initial biochemical steps of substrate utilisation and in the synthesis of cell material. McElroy (1947) first demonstrated that the emission of light in the bioluminescent reaction of the firefly, Photinus pyralis, was stimulated by ATP. The procedure for the determination of ATP concentrations utilising crude firefly extracts was described by McElroy and Streffier (1949) and has since been used in many fields as a sensitive and accurate measure of ATP. The lightyielding reaction is catalysed by the enzyme luciferase, this being the enzyme found in fireflies causing luminescence. Luciferase takes part in the following reaction: 1. Luciferase + Luciferin + ATP Mg2+ Luciferase Luciferin AMP + PP The complex is then oxidised: 2. Luciferase Luciferin AMP + O2 (Luciferase Luciferin AMP = O) + H2O The oxidised complex is in an excited stage, and as it returns to its ground stage a photon of light is released: 3. Luciferase Luciferin AMP = 0 (Luciferase Luciferin AMP = 0) + Light The light-yielding reaction is efficient, producing a single photon of light for every luciferin molecule oxidised and thus every ATP molecule used (Seliger and McElroy 1960). Levin et al. (1964) first described the use of the firefly bioluminescence assay of ATP for detecting the presence of viable microorganisms. Since this initial report considerable work has been done on the detection of viable organisms in environmental samples using a bioluminescence technique (Stalker 1984). As all viable organisms contain ATP, it could be considered simple to use a bioluminescence method to rapidly enumerate microorganisms. Research, however, has shown that the amount of ATP in different microbial cells varies depending on species, nutrient level, stress level and stage of growth (Stannard 196 Chilled foods