Vol50, No 2 May 1997 International Journal of Dairy Technology

Principles of rapid testing and a look to the future

WILL M WAITES Department of Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK

INTRODUCTION Classical methods of detecting and enumerat- ing microorganisms depend on the recognition of microbial colonies often after growth on selective agar. Such methods are time consum- ing, labour intensive and require well trained staff able, not only to carry out aseptic work, but also to recognize different morphological types of microorganisms. Since most foodborne pathogenic microorganisms generally occur in low numbers, selective enrichment is also usually required in order to allow detection and prevent overgrowth by other microorganisms. In addition, since some cells, particularly from food processing environments, may be dam- aged, a pre-enrichment step is often needed so that cells can recover before coming into contact with the selective agent(s) used in enrichment and enumeration, Hence for detec- tion of Salmonella (Fig. 1) pre-enrichment requires incubation for 16 to 20 hours and selective enrichment for between 24 and 28 hours while growth to form colonies will need a further 24 or even 48 hours. This would provide an indication of the presence of presumptive Salmonella, but for confirmation five character- istic colonies from each plate should be incu- bated for 18 to 24 hours before biochemical and serological confirmation on day 4 or day 6.’ Any method which would reduce this time would have obvious advantages in allowing earlier release of products with a consequent reduction in storage costs.

The rapid methods of testing which will be discussed here include adenosine triphosphate (ATP) detection, impedance, antibody linked probes, phage based assays and DNA/RNA probes, including the use of the polymerase chain reaction.

RAPID TESTING METHODS

ATP detection ATP is found in all living cells. Using the enzyme luciferase (Fig. 2) from fire-flies, as little as lo2 to lo3 femtograms g) (equivalent to lo2 and lo3 bacterial cells) can be detected. Unfortunately, since plant and animal cells generally contain far more ATP than those of bacteria, much non-microbial ATP will be present in foods and on food processing surfaces. To use ATP as a measure of microbial numbers, non-microbial ATP can be selectively removed by disrupting the cells of animal and plant origin with a mild surfac- tant before destroying the ATP released with the enzyme ATPase, subsequently inactivating the ATPase and then extracting the microbial ATP with a more powerful surfactant. How- ever, different microbial cells contain different amounts of ATP, with bacterial spores having no ATP, sublethally stressed cells containing low levels and yeast cells having up to 100 times more ATP than bacterial cells. One alternative to using ATP to give an estimation of microbial numbers is to use it to measure the extent of cleaning of food processing surfaces, since a cleaned surface should be free of animal, plant and microbial cells and hence of ATP. Under such circumstances this test can be carried out in less than 5 minutes and provides a powerful tool in the hands of anyone wishing to give an instant feedback to cleaning oper- atives. In many cases ATP testing is now incorporated into routine Quality Assurance monitoring or Hazard Analysis Critical Control Point (HACCP) programmes and this allows manufacturers to gain an accurate picture of the hygiene status of surfaces and equipment.

Electrical monitoring As microorganisms grow, their metabolism changes the chemical composition of the growth medium. For example, proteins are broken down into amino acids and carbohy-

Day 1 Sample + pre-enrichment medium (16 to 20 h)

.1 Selective enrichment Day 2 or 3

(24 and 48 h)

1 Day 3 or 5(6) Plate out onto selective media

(incubate 20 to 24 or possible 48 h)

-1 Pick 5 characteristic colonies

(incubate on non-selective agar for 18 to 24 h)

Paper presented at Residential Course on Day Or 6(7) ‘Basic Microbiology and HACCP’.

Luciferin + Luciferase + ATP (Substrate) (Enzyme)

.1

1 Luciferin - Luciferase - AMP + PP + 0,

Oxyluciferin* - Luciferase - AMP + H,O

Brackenhurst College, -1 -1 Biochemicalherological Confirmation Oxyluciferin + Luciferase + AMP + h p (560 nm) 4 4 September 1996.

0 1997 Society of Dairy Technology Fig. 1. Isolation of Salmonella from food. Fig. 2. ATP determination. *Electronically excited state.

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drates are converted to organic acids, such as lactic acid. As a result, electrically neutral to weakly ionized molecules are transformed into molecules with a greater charge and electrical motility. By growing microorganisms in cells with two electrodes the electrical properties of the medium can be measured frequently (as often as every 6 minutes) and for a large number of samples (up to 512) (for reviews, see Firstenberg-Eden and Eden2 and Silley and Forsythe3). The results can be stored in a computer which will print out the data and decide on the detection time, which is the time when the first microbially induced changes to the medium can be detected. By comparing viable counts and detection times a calibration curve can be produced, which in future work will allow the number of cells to be calculated from the detection time on an unknown sample. The organisms for which suitable media are available include aerobic mesophilic bacteria, psychrotrophs, thermophiles, Enterobacteria- ceae4, Salmonella5, coliforms, yeast and moulds6 and lactic acid bacteria. Aerobic mesophilic bacterial counts are available within 24 hours, while enumeration of specific organ- isms is at least one day faster than with conventional techniques. The samples are also available for confirmation by biochemical and/ or serological methods in the same way as colonies on agar plates can be examined. One advantage is that a large number of negative samples can be checked quickly. This is especially useful, for example, for ultra high temperature milk, where there should be no growth at 37”C, since only thermophilic spore formers should survive the heat processing. Theoretically growth from one cell can be detected and, of course, non-viable cells are inactive.

Antibody linked probes Since an antibody linked probe can be produced against any material that is or can be made immunogenic, it is possible to manufacture antibodies able to detect both microbial cells and their toxins. A commonly used system is the sandwich ELISA (enzyme-linked immuno- sorbent assay) in which a capture antibody is immobilized on a microtitre plate well. The sample is then added to the well and, after mixing, removed to leave any antigens present attached to the antibodies. A second antibody coupled to an enzyme is then added to produce an antibody sandwich and the extent of binding can be detected by using a chromogenic substrate for the enzyme. Commercial ELISA systems are available for bacteria such as Salmonella, Escherichia coli and Listeria spp, although they require 105-106 cells in order for detection to occur. Staphylococcal toxins can

under identification and enumeration. A growth step is generally used since antibody linked probes will detect non-viable as well as viable cells, but since the presence of the cells of other organisms do not interfere with the activity of the antibody this growth step does not invaria- bly involve the use of selective agents.

In an interesting advance, Dynal have pro- duced a system which allows immunomagnetic separation for Salmonella’, Listeria8 and E coli 0157.9 This method also removes the need for selective enrichment and so can reduce the time required for detection to two days (for a review see Safarik et all0). For Salmonella, for exam- ple, this approach also reduced the number of selective plates required from eight to two. In addition, there are fewer false positives, which reduces the cost of confirmatory tests.

An obvious need for all antibody linked probes is the requirement that the system can detect all the serotypes of the target organisms and no other organisms. Given that there are over 2000 serotypes of Salmonella, this is an obvious problem and it is unclear if commer- cially available antibodies will detect all of the serotypes so far described. However, most food poisoning incidents are related to only a minority of serotypes. With Listeria the re- quirement is really for detection of Listeria monocytogenes and not other species of Lis- teria. However, the antibodies currently availa- ble will detect all Listeria, although it has been said that since their environmental niches are similar, where other species occur, L monocy- togenes will be isolated sooner or later.

Phage based assays Phage based assays are not yet commercially available. However, Gordon Stewart at Not- tingham, has developed a new and promising method of detection based on bacteriophage (bacterial virus) infection of bacteria. Such infection is very specific for a particular target bacterium. The method is to add the target specific bacterial virus to the sample, before waiting until the target bacteria are all infected. The next step is to kill all the free, non- absorbed, bacterial viruses. This can be done with pomegranate extract. Subsequently, after 30 to 40 min of incubation the viruses within the target cells lyse the bacteria and 100 to 200 new virus particles are released from each cell. These viruses are amplified by additions of non-pathogenic variants of the target bacterium and a further incubation step. This method allows detection of one viable bacterial cell within a few hours and shows great promise, particularly for detection of Listeria, following the discovery of a bacterial virus” which is specific for members of this genus.

also be detected and automatic systems which determine the presence of heat killed cells reduce the problem of growth and possible contamination of other products by the bacteria

DNA/RNA probes The specificity of DNA/RNA probes depends simply on the region chosen to be detected. The

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Vol50, No 2 May I997 International Journal of Dairy Technology

nucleic acid is released from the cell by lysis and, if it is double stranded DNA, it is also denatured to the single stranded form by heating. The denatured nucleic acid is fixed on to a membrane by heat or alkali and then hybridized to a probe labelled with an enzyme which can usually react with a chromogenic substrate.

About lo6 copies of a target sequence are needed for detection so that the method can be applied particularly well to a colony on a plate. 1 2 q 1 Alternatively, the polymerase chain reaction (PCR) provides a method for amplify- ing specific fragments of DNA from as little as one The approach utilizes two short oligonucleotide primer sequences which will hybridize to opposite strands of the heat denatured DNA at either end of the region to be probed. The enzyme DNA polymerase is then used to extend the primers to produce two double stranded copies. This process is repeated and 25 cycles will create about lo6 copies in about 4 hours. Computer controlled heating blocks are available which automatically pro- duce the necessary thermal cycling. The disad- vantages of PCR include the fact that it does not distinguish between viable and non-viable cells and that when it is used directly in food systems then components of the food can interfere with the sensitivity of the reaction.

The future It is apparent that many of the methods discussed in this paper have advantages over the approach of conventional microbiologists. However, it should be remembered that about 90% of the tests carried out for detection of microorganisms important to food microbiolo- gists are still those used by classical micro- biologists and involve agar plating techniques

or most probable number counts. While the conservatism of the food industry is partly responsible for this take up and development of new methods, it is apparent that many of the newer and faster methods have disadvantages, as well as advantages (Table 1). In addition, the records of viable counts held by companies often go back a number of years so that any change in the level of counts can be detected quickly and related to previous events or to changes in raw materials or processing methods.

With the introduction of the HACCP con- cept, the number of endproduct tests carried out is likely to decrease, particularly because of the problem of sampling sufficiently to produce a statistically meaningful result. However, in addition to the visual, physical and chemical checks carried out on critical control points it is also becoming apparent that microbiological examination is needed. In addition, simple enumeration of microorganisms is now recog- nized as only supplying the minimum of information, since the numbers of a particular microorganism in a food moving down a processing line may decrease while new strains, perhaps more resistant, more able to produce spoilage, or more virulent, may gain entry through a hitherto unrecognized critical control point. Such new strains can be detected by a number of new techniques involving molecular typing”, the most efficient and quickest of which are often based on the various forms of DNA fingerprinting.18 This new approach has also led to innovative methods, for example, the use of E coli as a monitor for Salmonella. In one study, in poultry processing the carriage rate of Salmonella at the start of processing was found to be less than 1% but despite this, 25% of carcasses carried Salmonella by the end of processing. It is therefore very difficult to discriminate between contaminated and uncon- taminated flocks, although the Advisory Com- mittee for Microbiological Safety have sug- gested that flocks carrying Salmonella should be treated with more care than uncontaminated flocks and should be processed last in order to prevent cross-contamination of other flocks. An additional development of HACCP is to use E coli, which is carried at much higher levels, as an indication of critical control points. The use of molecular biology to detect the appearance of new strains and hence to monitor critical control points has also been used for liquid whole egg processing (Bacillus cereus19), meat spoilage (Brochothrix thermosphacta, Dodd CER, unpublished), contamination of prawns (E coli) and Staphylococcus aureus in poultry processing.20

Such innovative approaches suggest that the future role of the food microbiologist will become more technical (and more interesting) but that, as a result, the food that we consume will not only have a longer shelf-life but will also be safer to eat. Given the media publicity of

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food safety over the last 10 years, this is a future which should be welcomed by all concerned.

REFERENCES 1 Food and Drug Administration (1984) Bacteriological

Analytical Manual, 6th ed. Arlington VA: Association of Analytical Chemists.

2 Firstenberg-Eden R and Eden G (1984) Impedance and Microbiology. Letchworth: Research Studies Press.

3 Silley P and Forsythe S J (1996) Impedance micro- biology-a rapid change for microbiologists. Journal of Applied Bacteriology 80 233-243.

4 Petitt S B (1989) A conductance screen for Enterobac- teriaceae in foods. In Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals pp 13 1-141. Stannard C J, Petitt S B and Skinner F A, eds. Oxford: Blackwell Science.

5 Pless P, Futschik K and Schopf E (1994) Rapid detection of Salmonellae by means of a new impedance-splitting method. Journal of Food Protection 57 369-375.

6 Betts R P (1993) Rapid electrical methods for the detection and enumeration of food spoilage yeasts. International Biodeterioration and Biodegradation 32 19-32.

7 Haines J and Patel P D (1996) The development of the BIAcoreTM based optical biosensor techniques for the rapid detection of foodbome pathogens. Society for Applied Bacteriology 81(2) xxii.

8 Jones K L, MacPhee S, Turner A and Betts R P (1996) An evaluation of the Oxoid Listeria Rapid Test (incorporating Clearview) for the detection of Listeria from foods. Society for Applied Bacteriology 81(2) xxi.

9 Pimbley D W and Patel P D (1996) A brief evaluation of four ELISA-based kits for the detection of E coli 0157 in foods. Society for Applied Bacteriology 81(2) xxii.

10 Safarik I, Safarikovi M and Forsythe S J (1995) The application of magnetic separations in applied

Book review

microbiology. Journal of Applied Bacteriology 78 575-585.

11 Loessner M J, Rees C E D, Stewart G S A B and Scherer S (1996) Construction of luciferase reporter bacter- iophageA51 I - luAB for rapid and sensitive detection of viable Listeria cells. Applied and Environmental Micro- biology 62 1133-1 140.

12 Bennett A R, Greenwood D, Tennant C and Betts R P (1996) The detection of Salmonella in foods by the use of a commercial PCR system. Society for Applied Bacteriol- ogy 81(2) xxi.

13 Lin C K and Tsen H Y (1995) Development and evaluation of two novel oligonucleotide probes based on 16s RNA sequence for the identification of Salmonella in foods. Journal of Applied Bacteriology 78 507-520.

14 Bennett A R, Greenwood D, Tennant C and Betts R P (1996) The detection of Salmonella in foods by the use of a commercial PCR system. Society for Applied Bacteriol- ogy 81(2) xxi.

15 Knight A I, Pimbley D W and Patel P D (1996) Evaluation of the BaxTM system for screening Salmonella. Societyfor Applied Bacteriology 81(2) xxii.

16 Lin C K and Tsen H Y (1996) Use of two 16s DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods. Journal of Applied Bacteriology 80 659-666.

17 Dodd C E R (1994) The application of molecular typing techniques to HACCP. Trends in Food Science and Technology 5 16C164.

18 Bloomfield P L E (1996) Molecular typing of bacterial isolates as a contract service. Microbiology Europe 4(4) 24-26.

19 Ellison A, Dodd C E R and Waites W M (1989) Use of plasmid profiles to differentiate between strains of Bacillus cereus. Food Microbiology 6 93-98.

20 Dodd C E R, Chaffey B J and Waites W M (1988) Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants. Applied and Environmental Microbiology 51 1541-1549.

Dairy India Yearbook, 5th ed. P R Gupta. A- 25 Priyadarshini Vihar, Delhi 110092, India. 1997. US$295, plus US $20 for air postage and handling.

A country with a human population of 953 million (including 70 million dairy farmers), a dairy animal population of 57 million cows and 39 million buffaloes, producing 74.3 million tonnes of milk annually, of which 10% is processed in dairy plants, has a story to tell about its dairy industry. This remarkable com- pilation of over 900 pages of information bears witness to that fact.

By 1998, India is set to become the world’s leading milk producer, with a projected 76 million tonnes, although the per capita avail- ability remains woefully low, at 220 g per day or 80.3 kg per year. Indian dairy development, based on the Village Milk Producers Cooper- ative concept conceived by Dr V Kurien and the staff of the National Dairy Development Board at Anand, represents one of the greatest and

most successful social experiments of our time. This encyclopaedic yearbook tells the full story of all aspects of Indian dairy development in a series of over 70 articles by specialists and 250 statistical tables and graphs. It covers produc- tion, processing, distribution, marketing and research and development, including such top- ical items as the GA’IT agreement and its effect on Indian dairying. The data base section of the directory includes lists of analytical laborato- ries; associations; consultants; cheese manu- facturers; dairy cooperatives; dairy plants; dairy product distributors; equipment manufacturers; feed manufacturers; semen banks; dairy periodicals, to mention but a few. It also includes a useful Who’s Who section.

The Yearbook is recommended as essential reading matter not only for those with a direct interest in the Indian dairy industry, but also for libraries/information departments in univer- sities, colleges and industry.

ERNEST MANN

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