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