Conventional and rapid analytical microbiology 197 1989, Stalker 1984). Thus, when using bioluminescence it is important to consider. the type of microorganism being analysed; generally, vegetative bacteria will contain 1 fg of ATP/cell(Karl 1980), yeasts will contain ten times this value(Stannard 1989), whilst spores will contain no ATP(Sharpe et al. 970) 2. whether the cells have been subjected to stress, such as nutrient depletion, chilling or pH change. In these cases a short resuscitation may be required prior to testing, 3. whether the cells are in a relatively ATP-free environment, such as a growth medium, or are contained within a complex matrix, like food, that will have very high background ATP levels When testing food samples one of the greatest problems is that noted in 3 above All foods will contain ATP and the levels present in the food will generally be much higher than those found in microorganisms within the food. Data from Sharpe et al.(1970)indicated that the ratio of food ATP to bacterial ATPranges from 40000: 1 in ice-cream to 15: 1 in milk. Thus, to be able to use ATP analysis as a rapid test for foodborne microorganisms, methods for the separation of microbial ATP were developed. The techniques that have been investigated fall into two categories: either to physically separate micro from other sources of ATP, or to use specific extractants to remove and destroy non- microbial ATP. Filtration methods have been successfully used to separate microorganisms from drinks(LaRocco et al. 1985, Littel and LaRocco 1986) and brewery samples( Hysert et al. 1976). These methods are, however, difficult to apply to particulate-containing solutions as filters rapidly become blocked. A potential way around this problem has been investigated by some workers and utilise a double filtration system/scheme littel et al. 1986), the first filter removing food debris but allowing microorganisms through, the second filter trapping microorganisms prior to lysis and bioluminescent analysis. Other workers(Baumgart et al. 1980, Stannard and Wood 1983)have utilised ion exchange resins to trap selectively either food debris or microorganisms before bioluminescent tests were done The use of selective chemical extraction to separate microbial and non- microbial ATP has been extensively tested for both milk(Bossuyt 1981)and meat(Billte and Reuter 1985)and found to be successful. In general, this chnique involves the lysis of somatic(food) cells followed by destruction of the released ATP with an apyrase(ATPase)enzyme. A more powerful extraction reagent can then be used to lyse microbial cells, which can then be tested with luciferase, thus enabling the detection of microbial ATP only There are a number of commercially available instruments aimed specifically at the detection of microbial ATP, Lumac(Netherlands), Foss Electric Denmark), Bio Orbit(Finland) and Biotrace (UK)all produce systems cluding separation methods, specifically designed to detect microorganisms in foods. Generally, all of the systems perform well and have similar specifica-
1989, Stalker 1984). Thus, when using bioluminescence it is important to consider: 1. the type of microorganism being analysed; generally, vegetative bacteria will contain 1 fg of ATP/cell (Karl 1980), yeasts will contain ten times this value (Stannard 1989), whilst spores will contain no ATP (Sharpe et al. 1970); 2. whether the cells have been subjected to stress, such as nutrient depletion, chilling or pH change. In these cases a short resuscitation may be required prior to testing; 3. whether the cells are in a relatively ATP-free environment, such as a growth medium, or are contained within a complex matrix, like food, that will have very high background ATP levels. When testing food samples one of the greatest problems is that noted in 3 above. All foods will contain ATP and the levels present in the food will generally be much higher than those found in microorganisms within the food. Data from Sharpe et al. (1970) indicated that the ratio of food ATP to bacterial ATP ranges from 40000:1 in ice-cream to 15:1 in milk. Thus, to be able to use ATP analysis as a rapid test for foodborne microorganisms, methods for the separation of microbial ATP were developed. The techniques that have been investigated fall into two categories: either to physically separate microorganisms from other sources of ATP, or to use specific extractants to remove and destroy nonmicrobial ATP. Filtration methods have been successfully used to separate microorganisms from drinks (LaRocco et al. 1985, Littel and LaRocco 1986) and brewery samples (Hysert et al. 1976). These methods are, however, difficult to apply to particulate-containing solutions as filters rapidly become blocked. A potential way around this problem has been investigated by some workers and utilise a double filtration system/scheme (Littel et al. 1986), the first filter removing food debris but allowing microorganisms through, the second filter trapping microorganisms prior to lysis and bioluminescent analysis. Other workers (Baumgart et al. 1980, Stannard and Wood 1983) have utilised ion exchange resins to trap selectively either food debris or microorganisms before bioluminescent tests were done. The use of selective chemical extraction to separate microbial and nonmicrobial ATP has been extensively tested for both milk (Bossuyt 1981) and meat (Billte and Reuter 1985) and found to be successful. In general, this technique involves the lysis of somatic (food) cells followed by destruction of the released ATP with an apyrase (ATPase) enzyme. A more powerful extraction reagent can then be used to lyse microbial cells, which can then be tested with luciferase, thus enabling the detection of microbial ATP only. There are a number of commercially available instruments aimed specifically at the detection of microbial ATP; Lumac (Netherlands), Foss Electric (Denmark), Bio Orbit (Finland) and Biotrace (UK) all produce systems, including separation methods, specifically designed to detect microorganisms in foods. Generally, all of the systems perform well and have similar specificaConventional and rapid analytical microbiology 197
198 Chilled foods tions, including a minimum detection threshold of 10 bacteria(10 yeasts)and analysis times of under one hour In addition to testing food samples for total viable microorganisms, there have been a number of reports concerning potential alternative uses of ATP bioluminescence within the food industry. The application of ATP analysis to rapid hygiene testing has been considered (Holah 1989), both as a method of rapidly assessing microbiological contamination, and as a procedure for measuring total surface cleanliness. It is in the latter area that ATP measurement can give a unique result. As described earlier, almost all foods contain very high levels of ATP, thus food debris left on a production line could be detected in minutes using a bioluminescence method, allowing a very rapid check of hygienic status to be done. The use of ATP bioluminescence to monitor surface hygiene has now been widely adopted by industry. The availability of relatively expensive, portable, easy to use luminometer has now enabled numerous food producers to implement rapid hygiene testing procedures that are ideal for HACCP monitoring applications where surface hygiene is a critical control point. Reports suggest( Griffiths 1995)that all companies surveyed who egularly use ATP hygiene monitoring techniques note improvements in cleanliness after initiation of the procedure. Such ATP based test systems can be applied to most types of food processing plant, food service and retail establishments and even assessing the cleanliness of transportation vehicles such as tankers One area that atp bioluminescence has not yet been able to address has been the detection of specific microorganisms. It may be possible to use selective enrichment media for particular microorganisms in order to allow selective growth prior to ATP analysis. This approach would, however, considerably increase analysis time and some false high counts would be expected. The use of specific lysis agents that release ATP only from the cells being analysed have been investigated(Stannard 1989)and shown to be successful. The number of these specific reagents is, however, small and thus the method is of only limited use. Perhaps the most promising method developed for the detection of specific organisms is the use of genetically engineered bacteriophages (Ulitzur and Kuhn, 1987, Ulitzur et al. 1989, Schutzbank et al. 1989) Bacteriophages are viruses that infect bacteria. Screening of bacteriophages has shown that some are very specific, infecting only a particular type of bacteria. Workers have shown it is possible to add into the bacteriophage the genetic information that causes the production of bacterial luciferase. Thus, when a bacteriophage infects its specific host bacterium, the latter produces luciferase and becomes luminescent. This method requires careful selection of the bacteriophage in order to ensure false positive or false negative results do ne occur: it does however indicate that in the future. luminescence-based methods could be used for the rapid detection of specific microorganisms(Stewart 1990) In conclusion, the use of ATP bioluminescence in the food industry has been developed to a stage at which it can be reliably used as a rapid test for viable microorganisms, as long as an effective separation technique for microbial ATP
tions, including a minimum detection threshold of 104 bacteria (103 yeasts) and analysis times of under one hour. In addition to testing food samples for total viable microorganisms, there have been a number of reports concerning potential alternative uses of ATP bioluminescence within the food industry. The application of ATP analysis to rapid hygiene testing has been considered (Holah 1989), both as a method of rapidly assessing microbiological contamination, and as a procedure for measuring total surface cleanliness. It is in the latter area that ATP measurement can give a unique result. As described earlier, almost all foods contain very high levels of ATP, thus food debris left on a production line could be detected in minutes using a bioluminescence method, allowing a very rapid check of hygienic status to be done. The use of ATP bioluminescence to monitor surface hygiene has now been widely adopted by industry. The availability of relatively inexpensive, portable, easy to use luminometers has now enabled numerous food producers to implement rapid hygiene testing procedures that are ideal for HACCP monitoring applications where surface hygiene is a critical control point. Reports suggest (Griffiths 1995) that all companies surveyed who regularly use ATP hygiene monitoring techniques note improvements in cleanliness after initiation of the procedure. Such ATP based test systems can be applied to most types of food processing plant, food service and retail establishments and even assessing the cleanliness of transportation vehicles such as tankers. One area that ATP bioluminescence has not yet been able to address has been the detection of specific microorganisms. It may be possible to use selective enrichment media for particular microorganisms in order to allow selective growth prior to ATP analysis. This approach would, however, considerably increase analysis time and some false high counts would be expected. The use of specific lysis agents that release ATP only from the cells being analysed have been investigated (Stannard 1989) and shown to be successful. The number of these specific reagents is, however, small and thus the method is of only limited use. Perhaps the most promising method developed for the detection of specific organisms is the use of genetically engineered bacteriophages (Ulitzur and Kuhn, 1987, Ulitzur et al. 1989, Schutzbank et al. 1989). Bacteriophages are viruses that infect bacteria. Screening of bacteriophages has shown that some are very specific, infecting only a particular type of bacteria. Workers have shown it is possible to add into the bacteriophage the genetic information that causes the production of bacterial luciferase. Thus, when a bacteriophage infects its specific host bacterium, the latter produces luciferase and becomes luminescent. This method requires careful selection of the bacteriophage in order to ensure false positive or false negative results do not occur; it does however indicate that, in the future, luminescence-based methods could be used for the rapid detection of specific microorganisms (Stewart 1990). In conclusion, the use of ATP bioluminescence in the food industry has been developed to a stage at which it can be reliably used as a rapid test for viable microorganisms, as long as an effective separation technique for microbial ATP 198 Chilled foods
Conventional and rapid analytical microbiology 199 is used. Its potential use in rapid hygiene testing has been realised and the technique is being used within the industry. Work has also shown that luminescence can allow the rapid detection of specific microorganisms but such a system would need to be commercialised before widespread use within the food industry 8.4.3 Microscopy methods Microscopy is a well established and simple technique for the enumeration of microorganisms. One of the first descriptions of its use was for rapidly counting bacteria in films of milk stained with the dye methylene blue(Breed and Brew 1916). One of the main advantages of microscope methods is the speed with which individual analyses can be done; however, this must be balanced against the high manual workload and the potential for operator fatigue caused by constant microscopic counting The use of fluorescent stains, instead of conventional coloured compounds, allows cells to be more easily counted and thus these stains have been the subject of considerable research. Microbial ecologists first made use of such compounds to visualise and count microorganisms in natural waters( francisco et al 1973, Jones and Simon 1975). Hobbies et al.(1977) first described the use of Nuclepore polycarbonate membrane filters to capture microorganisms before fluorescent staining, whilst enumeration was considered in depth by Pettipher et al.(1980), the method developed by the latter author being known as the direct epifluorescent filter technique(DEFT) The DEFT is a labour-intensive manual procedure and this has led to research into automated fluorescence microscope methods that offer both automated sample preparation and high sample throughput. The first fully automated instrument based on fluorescence microscopy was the Bactoscan(Foss Electric Denmark), which was developed to count bacteria in milk and urine. Milk samples placed in the instrument are chemically treated to lyse somatic cells and dissolve casein micelles. Bacteria are then separated by continuous centrifuga- tion in a dextran/sucrose gradient. Microorganisms recovered from the gradient are incubated with a protease to remove residual protein, then stained with acridine orange and applied as a thin film to a disc rotating under a microscope The fluorescent light from the microscope image is converted into electrical impulses and recorded. The Bactoscan has been used widely for raw milk testing in continental uro and correlations with conventional methods have reportedly been good(Kaereby and Asmussen 1989). The technique does however, have a poor sensitivity(approximately 5 x 10 cells/ml) and this negates its use on samples with lower bacterial counts An instrument-based fluorescence counting method, in which samples were spread onto a thin plastic tape, was developed for the food industry. The instrument( Autotrak) deposited samples onto the tape, which was then passed through staining and washing solutions, before travelling under a fluorescence microscope. The light pulses from the stained microorganisms were then
is used. Its potential use in rapid hygiene testing has been realised and the technique is being used within the industry. Work has also shown that luminescence can allow the rapid detection of specific microorganisms but such a system would need to be commercialised before widespread use within the food industry. 8.4.3 Microscopy methods Microscopy is a well established and simple technique for the enumeration of microorganisms. One of the first descriptions of its use was for rapidly counting bacteria in films of milk stained with the dye methylene blue (Breed and Brew 1916). One of the main advantages of microscope methods is the speed with which individual analyses can be done; however, this must be balanced against the high manual workload and the potential for operator fatigue caused by constant microscopic counting. The use of fluorescent stains, instead of conventional coloured compounds, allows cells to be more easily counted and thus these stains have been the subject of considerable research. Microbial ecologists first made use of such compounds to visualise and count microorganisms in natural waters (Francisco et al. 1973, Jones and Simon 1975). Hobbies et al. (1977) first described the use of Nuclepore polycarbonate membrane filters to capture microorganisms before fluorescent staining, whilst enumeration was considered in depth by Pettipher et al. (1980), the method developed by the latter author being known as the direct epifluorescent filter technique (DEFT). The DEFT is a labour-intensive manual procedure and this has led to research into automated fluorescence microscope methods that offer both automated sample preparation and high sample throughput. The first fully automated instrument based on fluorescence microscopy was the Bactoscan (Foss Electric, Denmark), which was developed to count bacteria in milk and urine. Milk samples placed in the instrument are chemically treated to lyse somatic cells and dissolve casein micelles. Bacteria are then separated by continuous centrifugation in a dextran/sucrose gradient. Microorganisms recovered from the gradient are incubated with a protease to remove residual protein, then stained with acridline orange and applied as a thin film to a disc rotating under a microscope. The fluorescent light from the microscope image is converted into electrical impulses and recorded. The Bactoscan has been used widely for raw milk testing in continental Europe, and correlations with conventional methods have reportedly been good (Kaereby and Asmussen 1989). The technique does, however, have a poor sensitivity (approximately 5 104 cells/ml) and this negates its use on samples with lower bacterial counts. An instrument-based fluorescence counting method, in which samples were spread onto a thin plastic tape, was developed for the food industry. The instrument (Autotrak) deposited samples onto the tape, which was then passed through staining and washing solutions, before travelling under a fluorescence microscope. The light pulses from the stained microorganisms were then Conventional and rapid analytical microbiology 199