Conventional and rapid analytical microbiology R. P. Betts, Campden and Chorleywood Food Research Association 8.1 Introduction The detection and enumeration of microorganisms either in foods or on food contact surfaces forms an integral part of any quality control or quality assurance plan. Microbiological tests done on foods can be divided into two types:(a) quantitative or enumerative, in which a group of microorganisms in the sample are counted and the result is expressed as the number of the organisms present per unit weight of sample; or(b) qualitative or presence/absence, in which the requirement is simply to detect whether a particular organism is present or absent in a known weight of sample The basis of methods used for the testing of microorganisms in foods is very well established, and relies on the incorporation of a food sample into a nutrient medium in which microorganisms can replicate thus resulting in a visual indication of growth. Such methods are simple, adaptable, convenient and generally inexpensive. However, they have two drawbacks: firstly, the tests rely on the growth of organisms in media, which can take many days and result in a long test elapse time; and secondly, the methods are manually oriented and are thus labour intensive Over recent years, there has been considerable research into rapid and automated microbiological methods the aim of this work has been to reduce the st elapse time by using methods other than growth to detect and/or count microorganisms and to decrease the level of manual input into tests by automating methods as much as possible. These rapid and automated methods have gained some acceptance within the food industry and could form an important quality control tool in the chilled foods area. Positive release of chilled foods on the results of a rapid method could increase the shelf-life of a
8.1 Introduction The detection and enumeration of microorganisms either in foods or on food contact surfaces forms an integral part of any quality control or quality assurance plan. Microbiological tests done on foods can be divided into two types: (a) quantitative or enumerative, in which a group of microorganisms in the sample are counted and the result is expressed as the number of the organisms present per unit weight of sample; or (b) qualitative or presence/absence, in which the requirement is simply to detect whether a particular organism is present or absent in a known weight of sample. The basis of methods used for the testing of microorganisms in foods is very well established, and relies on the incorporation of a food sample into a nutrient medium in which microorganisms can replicate thus resulting in a visual indication of growth. Such methods are simple, adaptable, convenient and generally inexpensive. However, they have two drawbacks: firstly, the tests rely on the growth of organisms in media, which can take many days and result in a long test elapse time; and secondly, the methods are manually oriented and are thus labour intensive. Over recent years, there has been considerable research into rapid and automated microbiological methods. The aim of this work has been to reduce the test elapse time by using methods other than growth to detect and/or count microorganisms and to decrease the level of manual input into tests by automating methods as much as possible. These rapid and automated methods have gained some acceptance within the food industry and could form an important quality control tool in the chilled foods area. Positive release of chilled foods on the results of a rapid method could increase the shelf-life of a 8 Conventional and rapid analytical microbiology R. P. Betts, Campden and Chorleywood Food Research Association
188 Chilled foods product by one or two days compared with a conventional microbiological d microbiological tes methods indicates a potential for on-line control and the use of such systems Hazard Analysis Critical Control Point(HACCP) procedures 8.2 Sampling Although this chapter deals with the methodologies employed to test foods, it is important for the microbiologist to consider sampling. No matter how good a method is, if the sample has not been taken correctly and is not representative of the batch of food that it has been taken from then the test result is meaningless It is useful to devise a sampling plan in which results are interpreted from a number of analyses, rather than a single result. It is now common for microbiologists to use two or three class sampling plans, in which the number of individual samples to be tested from one batch are specified, together with the microbiological limits that apply. These type of sampling plans are full described in Anon. 1986 Once a sampling plan has been devised then a representative portion must be taken for analysis. In order to do this the microbiologist must understand the food product and its microbiology in some detail. Many chilled products will not be homogeneous mixtures but will be made up of layers or sections, a good example would be a prepared sandwich. It must be decided if the microbiological result is needed for the whole sandwich (i.e. bread and filling), or just the bread, or just the filling, indeed in some cases one part of a mixed analyses can be taken, using appropriate aseptic technique and sterile sampli filling may need to be tested, when this has been decided then the sample f implements(Kyriakides et al, 1996). The sampling procedure having be developed, the microbiologist will have confidence that samples taken are representative of the foods being tested and test methods can be used with nfidence 8.3 Conventional microbiological technique As outlined in the introduction, conventional microbiological techniques are based on the established method of incorporating food samples into nutrient media and incubating for a period of time to allow the microorganisms to grow The detection or counting method is then a simple visual assessment of growth These methods are thus technically simple and relatively inexpensive, requiring no complex instrumentation. The methods are however very adaptable, allowing the enumeration of different groups of microorganisms Before testing, the food sample must be converted into a liquid form in order to allow mixing with the growth medium. This is usually done by accurately weighing the sample into a sterile container and adding a known volume of
product by one or two days compared with a conventional microbiological technique. In addition, the availability of very rapid microbiological test methods indicates a potential for on-line control and the use of such systems in Hazard Analysis Critical Control Point (HACCP) procedures. 8.2 Sampling Although this chapter deals with the methodologies employed to test foods, it is important for the microbiologist to consider sampling. No matter how good a method is, if the sample has not been taken correctly and is not representative of the batch of food that it has been taken from, then the test result is meaningless. It is useful to devise a sampling plan in which results are interpreted from a number of analyses, rather than a single result. It is now common for microbiologists to use two or three class sampling plans, in which the number of individual samples to be tested from one batch are specified, together with the microbiological limits that apply. These type of sampling plans are fully described in Anon. 1986. Once a sampling plan has been devised then a representative portion must be taken for analysis. In order to do this the microbiologist must understand the food product and its microbiology in some detail. Many chilled products will not be homogeneous mixtures but will be made up of layers or sections, a good example would be a prepared sandwich. It must be decided if the microbiological result is needed for the whole sandwich (i.e. bread and filling), or just the bread, or just the filling, indeed in some cases one part of a mixed filling may need to be tested, when this has been decided then the sample for analyses can be taken, using appropriate aseptic technique and sterile sampling implements (Kyriakides et al., 1996). The sampling procedure having been developed, the microbiologist will have confidence that samples taken are representative of the foods being tested and test methods can be used with confidence. 8.3 Conventional microbiological techniques As outlined in the introduction, conventional microbiological techniques are based on the established method of incorporating food samples into nutrient media and incubating for a period of time to allow the microorganisms to grow. The detection or counting method is then a simple visual assessment of growth. These methods are thus technically simple and relatively inexpensive, requiring no complex instrumentation. The methods are however very adaptable, allowing the enumeration of different groups of microorganisms. Before testing, the food sample must be converted into a liquid form in order to allow mixing with the growth medium. This is usually done by accurately weighing the sample into a sterile container and adding a known volume of 188 Chilled foods
Conventional and rapid analytical microbiology 189 sterile diluent(the sample to diluent ratio is usually 1: 10); this mixture is then homogenised using a homogeniser (e.g. stomacher or pulsifier)that breaks the ample apart, releasing any organisms into the diluent. The correct choice of diluent is important. If the organisms in the sample are stressed by incorrect pH or low osmotic strength, then they could be injured or killed, thus affecting the final result obtained from the microbiological test the diluent must be well buffered at a pH suitable for the food being tested and be osmotically balanced When testing some foods (e.g. dried products) which may contain highly stressed microorganisms, then a suitable recovery period may be required before the test commences, in order to ensure cells are not killed during the initial phase of the test procedure(Davis and Jones 1997) 8.3.1 Conventional quantitative procedures The enumeration of organisms in samples is generally done by using plate count, or most probable number(MPN)methods. The former are the most widely used whilst the latter tend to be used only for certain organisms(e.g. Escherichia coll) or groups(e.g. coliforms) Plate count method The plate count method is based on the deposition of the sample, in or on an agar ayer in a Petri dish. Individual organisms or small groups of organisms will occupy a discrete site in the agar, and on incubation will grow to form discrete colonies that are counted visually. Various types of agar media can be used in this form to enumerate different types of microorganisms. The use of a non- selective nutrient medium that is incubated at 30oC aerobically will result in a total viable count or mesophilic aerobic count. By changing the conditions of incubation to anaerobic. a total anaerobe count will be obtained. altering the incubation temperature will result in changes in the type of organism capable of growth, thus showing some of the flexibility in the conventional agar approach If there is a requirement to enumerate a specific type of organism from the ample, then in most cases the composition of the medium will need to be djusted to allow only that particular organism to grow. There are three approaches used in media design that allow a specific medium to be produced he elective, selective and differential procedures Elective procedures refer to the inclusion in the medium of reagents, or the e of growth conditions, that encourage the development of the target organisms, but do not inhibit the growth of other microorganisms. Such reagents may be sugars, amino acids or other growth factors. Selective procedures refer to he inclusion of reagents or the use of growth conditions that inhibit the development of non-target microorganisms. It should be noted that, in many cases, selective agents will also have a negative effect on the growth of the target microorganism, but this will be less great than the effect on non-target cells. Examples of selective procedures would be the inclusion of antibiotics in a medium or the use of anaerobic growth conditions. Finally, differential
sterile diluent (the sample to diluent ratio is usually 1:10); this mixture is then homogenised using a homogeniser (e.g. stomacher or pulsifier) that breaks the sample apart, releasing any organisms into the diluent. The correct choice of diluent is important. If the organisms in the sample are stressed by incorrect pH or low osmotic strength, then they could be injured or killed, thus affecting the final result obtained from the microbiological test. The diluent must be well buffered at a pH suitable for the food being tested and be osmotically balanced. When testing some foods (e.g. dried products) which may contain highly stressed microorganisms, then a suitable recovery period may be required before the test commences, in order to ensure cells are not killed during the initial phase of the test procedure (Davis and Jones 1997). 8.3.1 Conventional quantitative procedures The enumeration of organisms in samples is generally done by using plate count, or most probable number (MPN) methods. The former are the most widely used, whilst the latter tend to be used only for certain organisms (e.g. Escherichia coli) or groups (e.g. coliforms). Plate count method The plate count method is based on the deposition of the sample, in or on an agar layer in a Petri dish. Individual organisms or small groups of organisms will occupy a discrete site in the agar, and on incubation will grow to form discrete colonies that are counted visually. Various types of agar media can be used in this form to enumerate different types of microorganisms. The use of a nonselective nutrient medium that is incubated at 30ºC aerobically will result in a total viable count or mesophilic aerobic count. By changing the conditions of incubation to anaerobic, a total anaerobe count will be obtained. Altering the incubation temperature will result in changes in the type of organism capable of growth, thus showing some of the flexibility in the conventional agar approach. If there is a requirement to enumerate a specific type of organism from the sample, then in most cases the composition of the medium will need to be adjusted to allow only that particular organism to grow. There are three approaches used in media design that allow a specific medium to be produced: the elective, selective and differential procedures. Elective procedures refer to the inclusion in the medium of reagents, or the use of growth conditions, that encourage the development of the target organisms, but do not inhibit the growth of other microorganisms. Such reagents may be sugars, amino acids or other growth factors. Selective procedures refer to the inclusion of reagents or the use of growth conditions that inhibit the development of non-target microorganisms. It should be noted that, in many cases, selective agents will also have a negative effect on the growth of the target microorganism, but this will be less great than the effect on non-target cells. Examples of selective procedures would be the inclusion of antibiotics in a medium or the use of anaerobic growth conditions. Finally, differential Conventional and rapid analytical microbiology 189
190 Chilled foods procedures allow organisms to be distinguished from each other by the reactions that their colonies cause in the medium. An example would be the inclusion of a pH indicator in a medium to differentiate acid-producing organisms. In most cases, media will utilise a multiple approach system, containing elective selective and differential components in order to ensure that the user can identify and count the target organism The number of types of agar currently available are far too numerous to list For details of these, the manuals of media manufacturing companies(e.g Oxoid LabM, difco, Merck) should be consulted MPN method The second enumerative procedure mentioned earlier was the MPN method. This procedure allows the estimation of the number of viable organisms in a sample based on probability statistics. The estimate is obtained by preparing decimal tenfold) dilutions of a sample, and transferring sub-samples of each dilution to (usually) three tubes of a broth medium. These tubes are incubated, and those that how any growth(turbidity) are recorded and compared to a standard table of esults(Anon. 1986)that indicate the contamination level of the product. As indicated earlier, this method is used only for particular types of test tends to be more labour and materials intensive than plate count methods ddition, the confidence limits are large even if many replicates are studied at each dilution level. Thus the method tends to be less accurate than plate counting methods 8.3.2 Conventional qualitative procedures Qualitative procedures are used when a count of the number of organisms in a sample is not required and only their presence or absence needs to be determined. Generally such methods are used to test for potentially pathogenic microorganisms such as Salmonella spp, Listeria spp, Yersinia spp. and campylobacter spp. The technique requires an accurately weighed sample (usually 25g)to be homogenised in a primary enrichment broth and incubated for a stated time at a known temperature. In some cases, a sample of the primary enrichment may require transfer to a secondary enrichment broth and further incubation. The final enrichment is usually then streaked out onto a selective agar plate that allows the growth of the organisms under test. The long enrichment procedure is used because the sample may contain very low levels of the test organism in the presence of high numbers of background microorgan- isms. Also, in processed foods the target organisms themselves may be in an injured state. Thus the enrichment methods allow the resuscitation of injured cells followed by their selective growth in the presence of high numbers of competing organisms The organism under test is usually indistinguishable in a broth culture, so broth must be streaked onto a selective/differential agar plate. The microorg isms can then be identified by their colonial appearance. The formation
procedures allow organisms to be distinguished from each other by the reactions that their colonies cause in the medium. An example would be the inclusion of a pH indicator in a medium to differentiate acid-producing organisms. In most cases, media will utilise a multiple approach system, containing elective, selective and differential components in order to ensure that the user can identify and count the target organism. The number of types of agar currently available are far too numerous to list. For details of these, the manuals of media manufacturing companies (e.g. Oxoid, LabM, Difco, Merck) should be consulted. MPN method The second enumerative procedure mentioned earlier was the MPN method. This procedure allows the estimation of the number of viable organisms in a sample based on probability statistics. The estimate is obtained by preparing decimal (tenfold) dilutions of a sample, and transferring sub-samples of each dilution to (usually) three tubes of a broth medium. These tubes are incubated, and those that show any growth (turbidity) are recorded and compared to a standard table of results (Anon. 1986) that indicate the contamination level of the product. As indicated earlier, this method is used only for particular types of test and tends to be more labour and materials intensive than plate count methods. In addition, the confidence limits are large even if many replicates are studied at each dilution level. Thus the method tends to be less accurate than plate counting methods. 8.3.2 Conventional qualitative procedures Qualitative procedures are used when a count of the number of organisms in a sample is not required and only their presence or absence needs to be determined. Generally such methods are used to test for potentially pathogenic microorganisms such as Salmonella spp., Listeria spp., Yersinia spp. and Campylobacter spp. The technique requires an accurately weighed sample (usually 25g) to be homogenised in a primary enrichment broth and incubated for a stated time at a known temperature. In some cases, a sample of the primary enrichment may require transfer to a secondary enrichment broth and further incubation. The final enrichment is usually then streaked out onto a selective agar plate that allows the growth of the organisms under test. The long enrichment procedure is used because the sample may contain very low levels of the test organism in the presence of high numbers of background microorganisms. Also, in processed foods the target organisms themselves may be in an injured state. Thus the enrichment methods allow the resuscitation of injured cells followed by their selective growth in the presence of high numbers of competing organisms. The organism under test is usually indistinguishable in a broth culture, so the broth must be streaked onto a selective/differential agar plate. The microorganisms can then be identified by their colonial appearance. The formation of 190 Chilled foods
Conventional and rapid analytical microbiology 191 colonies on the agar that are typical of the microorganism under test are described as presumptive colonies. In order to confirm that the colonies are composed of the test organism, further biochemical and serological tests are sually performed on pure cultures of the organism. This usually requires colonies from primary isolation plates being restreaked to ensure purity. The purified colonies are then tested biochemically by culturing in media that will indicate whether the organism produces particular enzymes or utilises certain At present a number of companies market miniaturised biochemical test systems that allow rapid or automated biochemical tests to be quickly and easily set up by microbiologists. Serological tests are done on pure cultures of some isolated organisms, e.g. Salmonella using commercially available antisera 8.4 Rapid and automated methods The general interest in alternative microbiological methods has been stimulated in part by the increased output of food production sites. This has resulted Greater numbers of samples being stored prior to positive release reduction in analysis time would reduce storage and warehousing costs a greater sample throughput being required in laboratories- the only way that this can be achieved is by increased laboratory size and staff levels, or by using more rapid and automated methods 3. A requirement for a longer shelf-life in the chilled foods sector-a reduction in analysis time could expedite product release thus increasing the shelf-life of the product 4. The increased application of HACCP procedures- rapid methods can be used in HACCP verification procedures There are a number of different techniques referred to as rapid methods and lost have little in common either with each other or with the conventional procedures that they replace. The methods can generally be divided into quantitative and qualitative tests, the former giving a measurement of the number of organisms in a sample, the latter indicating only presence or absence Laboratories considering the use of rapid methods for routine testing must carefully consider their own requirements before purchasing such a system Every new method will be unique, giving a slightly different result, in a different timescale with varying levels of automation and sample throughput. In addition, ome methods may work poorly with certain types of food or may not be able to detect the specific organism or group that is required. All of these points must be considered before a method is adopted by a laboratory. It is also of importance to ensure that staff using new methods are aware of the principles of operation of the techniques and thus have the ability to troubleshoot if the method clearly hows erroneous results
colonies on the agar that are typical of the microorganism under test are described as presumptive colonies. In order to confirm that the colonies are composed of the test organism, further biochemical and serological tests are usually performed on pure cultures of the organism. This usually requires colonies from primary isolation plates being restreaked to ensure purity. The purified colonies are then tested biochemically by culturing in media that will indicate whether the organism produces particular enzymes or utilises certain sugars. At present a number of companies market miniaturised biochemical test systems that allow rapid or automated biochemical tests to be quickly and easily set up by microbiologists. Serological tests are done on pure cultures of some isolated organisms, e.g. Salmonella using commercially available antisera. 8.4 Rapid and automated methods The general interest in alternative microbiological methods has been stimulated in part by the increased output of food production sites. This has resulted in 1. Greater numbers of samples being stored prior to positive release – a reduction in analysis time would reduce storage and warehousing costs. 2. A greater sample throughput being required in laboratories – the only way that this can be achieved is by increased laboratory size and staff levels, or by using more rapid and automated methods. 3. A requirement for a longer shelf-life in the chilled foods sector – a reduction in analysis time could expedite product release thus increasing the shelf-life of the product. 4. The increased application of HACCP procedures – rapid methods can be used in HACCP verification procedures. There are a number of different techniques referred to as rapid methods and most have little in common either with each other or with the conventional procedures that they replace. The methods can generally be divided into quantitative and qualitative tests, the former giving a measurement of the number of organisms in a sample, the latter indicating only presence or absence. Laboratories considering the use of rapid methods for routine testing must carefully consider their own requirements before purchasing such a system. Every new method will be unique, giving a slightly different result, in a different timescale with varying levels of automation and sample throughput. In addition, some methods may work poorly with certain types of food or may not be able to detect the specific organism or group that is required. All of these points must be considered before a method is adopted by a laboratory. It is also of importance to ensure that staff using new methods are aware of the principles of operation of the techniques and thus have the ability to troubleshoot if the method clearly shows erroneous results. Conventional and rapid analytical microbiology 191
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
