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BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES | Food-Poisoning Microorganisms
Food-Poisoning Microorganisms T Sandle, Bio Products Laboratory Ltd, Elstree, UK Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Daniel Y.C. Fung, volume 1, pp 237–244, Ó 1999, Elsevier Ltd.
Introduction Identification methods can be divided into two groups: phenotypic and genotypic. The genotype–phenotype distinction is drawn in genetics. ‘Genotype’ is an organism’s full hereditary information, even if not expressed. ‘Phenotype’ is an organism’s actual observed properties, such as morphology, development, or behavior (Sutton and Cundell, 2004). Phenotypic methods are the most widespread due to their relatively lower costs for many laboratories. It should be recognized, however, that expressions of the microbial phenotype – that is, cell size and shape, sporulation, cellular composition, antigenicity, biochemical activity, sensitivity to antimicrobial agents, and so on – frequently depend on the media and growth conditions that have been used. These conditions will include variables such as temperature, pH, redox potential, and osmolality and possibly lesser-known variables such as nutrient depletion, vitamin and mineral availability, growth cycle, water activity of solid media, static or rotatory liquid culture, and solid versus liquid media culture, as well as colony density on the plate. Therefore, some care is required in the interpretation of microbiological identification test results and the trending of data. A further limitation with phenotypic methods is the size and type of the Phenetic Classification Database. With the type of database, many databases are orientated toward clinical applications and do not necessarily serve industrial application well. In terms of size, databases are limited based on the relatively low number of microorganisms that have been characterized (Stager and Davis, 1992). The classical scheme of identification of bacteria by biochemical methods depends on whether a pure culture of the microorganism of interest can grow in an agar plate, an agar slant, a broth, a paper strip, or other supportive material containing specialized growth promoters or inhibitors in the presence of a fermentable or degradable compound, resulting in the medium changing color, development of gas, development of fluorescent compound, and other manifestation of metabolic activities. If the behavior of known cultures in these media is known, an unknown culture can be matched with these characteristics, and based on the closest match to a database, an analyst can make an identification of the unknown culture. This process is tedious, is time-consuming, and requires a lot of labor, materials, time, and energy to perform the tests. In addition, the skill of the analyst in interpreting the reactions and arriving at a correct judgment makes this process subjective and often unreliable (Kalamaki et al., 1997). Genotypic methods are not reliant on the isolation medium or growth characteristics of the microorganism. Genotypic methods have considerably enhanced databases of different types of microorganisms. Before the advent of genotypic methods, microbiologists speculated that a number of taxa
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were present and unculturable (so-termed viable-but-nonculturable strains). Genotypic methods have opened up a whole new set of species and subspecies, as well as reclassifying species and related species (thus, taxa that often are grouped similarly by phenotypic methods actually are polyphyletic groups – that is, they contain organisms with different evolutionary histories that are homologously dissimilar organisms that have been grouped together). Another advantage with genotypic methods is their accuracy and faster time to result (as microorganisms do not need to be grown on culture media). They are, however, relatively expensive. To make a complete identification, a great many tests can be done, as shown in Table 1.
Phenotypic Identification Methods Phenotypic reactions typically incorporate reactions to different chemicals or different biochemical markers. These rely on the more subjective determinations. The reliance on biochemical reactions and carbon utilization patterns introduces some disadvantages to the achievement of consistent
Table 1 Information needed for the identification of foodborne pathogens (Fung, 1995). Phenotypic characteristics Macroscopic morphology on agar plates Morphology under microscopic magnification Gram reaction (positive, negative, or variable) and special staining properties Biochemical activity profile and special enzyme systems Pigment production, bioluminescence, chemiluminescence, and fluorescent compound production Nutritional and growth factor requirements Temperature and pH requirements and tolerance Fermentation products, metabolites, and toxin production Antibiotic sensitivity pattern (antibiogram) Gas requirements and tolerance Cell wall, cell membrane, and cellular components Growth rate constant and generation time Motility and spore formation Resistance to organic dyes and special compounds Impedance, conductance, and capacitance characteristics Genotypic characteristics Genetic profile: DNA/RNA sequences and fingerprinting Extracellular and intracellular products Information relating to the microorganism Pathogenicity to animals and humans Serology and phage typing Ecological niche and survival ability Response to electromagnetic fields, light, sound, and radiation
Encyclopedia of Food Microbiology, Volume 1
http://dx.doi.org/10.1016/B978-0-12-384730-0.00036-7
BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food-Poisoning Microorganisms (repeatable and reproducible) identification. To improve on the classical methods of biochemical identification, several developments have been made and refined in recent years. Collectively, these methods are considered to be modern biochemical identification techniques. Although it is possible to prepared militarized biochemical tests within the laboratory, the purchase of commercial test kits is preferable as these can be closely aligned to a database. Commercial diagnostic test kits consist of miniaturized and multitest units. The two main types of diagnostic kits are agar based and dehydrated media based. In these systems, the pure cultures grow in a variety of solid or liquid media, changing color or gas formation, or utilizing their enzymes to change the color of the substrates. Diagnostic charts can be used to identify unknown cultures or the numerical manuals of computerassisted systems. Most of these systems were first designed to identify the family Enterobacteriaceae, and the databases remain largely orientated toward clinical microbiology rather than toward food microbiology. Later, some systems branched out to identify other microorganisms, such as the nonfermentors, lactics, yeast, and so on. The following are synopses of how diagnostic kits operate and the range of microorganisms tested (Russell et al., 1997). As far as possible, information concerning comparative analysis of these kits with conventional methods will be made (also see the Further Reading section). For many diagnostic kits, accuracy ratings are provided in cases in which kits are compared with conventional methods. Most comparative analyses of diagnostic kits were done many years ago and not repeated. It is difficult to compare one kit with another as the databases often vary. The consensus of opinion is that, to be acceptable, a kit should have a 90–95% accuracy correlated with the conventional method. When the value drops to 85% or below, the system is marginally acceptable and any value below that is not acceptable (Thippareddi and Fung, 1998).
Agar-Based Diagnostic Kits Several agar-based multimedia diagnostic kits are available, such as the Enterotube system (Roche Diagnostic, Nutley, NJ). The Enterotube II is a self-contained, compartmented plastic tube containing 12 different conventional media and an enclosed inoculating wire, which is threaded through the entire unit (Farmer, 2003). This system permits 15 standard biochemical tests to be inoculated and performed from a single bacterial colony. Reagents are added to the indole test and Voges–Proskauer (VP) test before color reactions and gas formation are read. Table 2 shows the color reactions of the tests. Because such a table exists for every diagnostic kit described in this article, this table will serve as a model for other kits. Similar tables will not be repeated. After reading the reactions, each result is given a score according to the system. After all the scores are added, an identification (ID) value in the form of a five-digit number will be generated. Other systems (to be described) may have 7- or 10-digit numbers. From the code book, the microorganism can be identified. This procedure is repeated for most other systems and will not be described again. The Enterotube II was developed to identify Enterobacteriaceae only; it has developed to be
Table 2
Reactions of biochemical tests for Enterotube II
Test GLU Gas LYS ORN H2S IND ADON LAC ARAB SORB VP DUL PA UREA CIT
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Glucose utilization Gas production Lysine decarboxylase Ornithine decarboxylase H2S production Indole formation Adonitol fermentation Lactose fermentation Arabinose fermentation Sorbitol fermentation Voges–Proskauer Dulcitol fermentation Phenylalanine deaminase Urease Citrate utilization
Positive reaction
Negative reaction
Yellow Wax lifted Purple Purple Black Pink-red Yellow Yellow Yellow Yellow Red in 20 min Yellow or pale yellow Black to smoky gray Red-purple Deep blue
Red Wax not lifted Yellow Yellow Beige Colorless Red Red Red Red Green Green Yellow Green
able to identify a variety of other oxidase-negative Gramnegative rods. A similar unit, the Oxi/Ferm Tube, was designed for Gramnegative nonfermenters. Advantages of Enterotube II include rapidity and ease of inoculation, that inoculum suspension is not required, and a single colony can be used for identification. Disadvantages include that it is only useful for Enterobacteriaceae, it is difficult to stack in the incubator, and it has a short shelf life.
Dehydrated Media Diagnostic Kits Dehydrated media diagnostic kits are another type of miniaturized microbiological method. Dehydrated media kits have the advantage of much longer shelf life than agar-based media (18 months versus a few months). Those currently used in clinical, environmental, industrial, and food microbiology will be discussed in the following sections. Of course, many similar systems are available worldwide: The systems discussed here have been well tested and used in the United States and Europe.
Analytical Profile Index The Analytical Profile Index (API; bioMérieux, Hazelwood, MO) is arguably the most popular system for diagnostic bacteriology in the world, especially for Enterobacteriaceae. The API 20E system is a miniaturized microtube system that has 20 small wells designed to perform 23 standard biochemical tests from isolated colonies of bacteria on plating medium. The system has procedures for same-day and 18–24 h identification of Enterobacteriaceae. It consists of microtubes containing dehydrated substrates. The substrates are reconstituted by adding a bacterial suspension into each of the 20 wells; some of the wells are filled with mineral oil to create anaerobic conditions. The unit then is incubated so that the microorganisms react with the contents of the tubes and are read when the indicator systems are affected by the metabolites or added reagents – generally after 18–24 h incubation at 35–37 C.
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After all the reactions are read and recorded in a data sheet, a number code is generated and the code can be matched with the code book for identification. The strip also can be read in a reader and the results interpreted by a computer (Woolfrey et al., 1984). Using similar formats, other microorganisms can be identified, such as API Gram-negative Identification: API Rapid 20E (4 h identification of Enterobacteriaceae); API 20NE (24–48 h identification of Gram-negative non-Enterobacteriaceae); API Ò Campy (24 h identification of Campylobacter species); API Gram-positive Identification: API Staph (identification of clinical staphylococci and micrococci); API 20 Strep (identification of streptococci and enterococci); API Coryne (identification of Corynebacteria and corynelike organisms); API Ò Listeria (24 h identification of all Listeria species); API Anaerobe Identification: API 20A (identification of anaerobes); Rapid ID 32 A (identification of anaerobes); and API Yeast Identification: API 20C AUX (48–72 h identification of yeasts). Advantages include being the most complete system commercially available for the identification of Enterobacteriaceae and an excellent database. Disadvantages include that it is difficult and time-consuming to inoculate, problems in handling and stacking of tray and lids due to flexible plastic materials, and that a competent microbiologist is needed to read and interpret the color changes.
MicroID The MicroID (Organon Teknika, Durham, NC) system provides results in 4 h. The system measures enzyme activities and not growth of the culture. It consists of a molded polystyrene tray containing 15 reaction chambers and a hinged cover. The first five reaction chambers contain a single combination substrate or detection disc with upper and lower discs in the same trough. The remaining 10 reaction chambers each contain a single combination substrate or detection disc. Discs contain all substrate and detection reagents required to perform the indicated biochemical tests (except for the Voges–Proskauer test). The surface of the tray is covered with clear polypropylene tape to prevent spillage and also for reading the reactions (Appelbaum and Olmstead, 1982). A few cultures from a Gram-negative isolation agar plate are first mixed into a liquid form and 0.2 ml of the liquid is then introduced into each of the 15 wells. The unit is then incubated at 35 C for 4 h, after which time two drops of 20% KOH are added into the VP well. The unit is then rotated 90 so that the liquid from the lower part of the first five wells comes into contact with the upper discs of the same chamber for final reactions. The reactions of these five tests are read from the upper discs. Then the reactions from the remaining 10 discs are read. Again, a number is generated in the data sheet and the code number is matched, with codes in the code book for identification. A similar format with different substrates was available to identify Listeria spp. (Goosh and Hill, 1982). Advantages of the system include high accuracy, speed of reaction (4 h), and convenience: It is self-contained, easy to use, requires only one reagent addition, and has a long shelf life. A disadvantage is that a competent microbiologist is needed to read the color reaction.
Minitek Minitek (Becton Dickinson Microbiology Systems, Cockeyville, MD) is a flexible system. The unit contains 10 wells. Two units (20 wells) can be used to identify one culture. The system supplies 36 different substrates, and thus the user can choose which test to perform. First, paper discs containing individual substrates are applied to the wells, one disc per well. A liquid culture is prepared and applied to each well using an automatic application gun (about 0.2 ml per well). Some wells will be filled with mineral oil to create an anaerobic environment. The unit is then incubated overnight at 35 C. After incubation, the color reactions are read and identification is made with the aid of a code book (Holloway et al., 1979). On the one hand, the advantage of the system is versatility and flexibility, but this may be a disadvantage when no code book is available for microorganisms other than Enterobacteriaceae. The construction of the unit is sturdy. Disadvantages include that the various components of the total system are handled excessively in preparation and operation. Again skill is needed to read borderline reactions in the discs.
BBL Crystal The BBL Crystal (Becton Dickinson Microbiology Systems, Cockeysville, MD) system requires relatively little manipulation. In one system (Enteric/Nonfermentor ID Kit), both enteric and nonfermenters can be identified. It is important to ensure that the unit is marked correctly as to whether an oxidase-positive (nonfermenter) or oxidase-negative (fermenter) pure culture is to be analyzed (Knapp et al., 1994). The system is easy to use. On one panel, 30 dried biochemical substrates are housed and a companion unit (base) is used for the liquid sample. The liquid culture (approximately 2 ml) is poured carefully into the trough of the base. Then the upper unit containing the 30 tests is simply snapped into the base such that the culture interacts with the 30 substrates. The unit is then incubated at 35 C overnight. After incubation, the unit is introduced into a Crystal light box to record reactions and for identification using a 10-digit system. Identification can be made using a computer. In addition, there is also a Rapid Stool/Enteric ID kit for stool samples. Advantages of the system include sturdy panels, ease of operation, and computer-assisted identification. Very few disadvantages are noted.
RapID One System RapID One (Remel, Lenexa, KS) is a miniaturized unit housed in an ingenious chamber. On one side of the chamber, there is a trough where a liquid culture can be introduced. Then the unit can be tilted slowly at a 45-degree angle forward: The liquid will flow into individual wells, each containing a separate substrate. Thus, inoculation into 20 wells can be made in one motion. This is more convenient than the API system where the analyst needs to insert 20 drops of liquids into 20 miniaturized wells. After incubation, the color reactions can be read after 4 h incubation and the cultures identified. The forerunner of the RapID enteric system is the Spectrum 10 system. The Spectrum 10 is rated as 91% accurate. Using the basic design, Remel markets strips for Enterobacteriaceae,
BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food-Poisoning Microorganisms nonfermenters, yeasts, anaerobes, streptococci, Leuconostoc, Pediococcus, Listeria, Neisseria, Haemophilus, and urinary tract bacteria (Stager et al., 1983). Identification of various anaerobes to the genus level using the anaerobic RapID system ranges from 83% to 97% accuracy and to the genus level from 76% to 97% (Celig and Schreckenberger, 1991). Advantages include results in 4 h, clear chromogenic reactions, and one-step inoculation. A disadvantage is the skill required to read the color changes.
ATB ATB (bioMérieux Vitek, Inc., Hazelwood, MO) is a 32-carbon assimilation test system. The culture is first made into a solution and then the liquid is introduced to the unit. After incubation (4–24 h depending on the culture), the tests can be read manually or automatically. Test strips are available for anaerobes, staphylococci, micrococci, yeast, Enterobacteriaceae, streptococci, and Gram-negative bacilli. The automatic reader also can read API 20 and API 50 test strips.
Omnilog System Omnilog (Biolog, Hayward, CA) is a miniaturized system utilizing the microtiter plate format for growth of bacteria in various liquid media. Some 95 different carbon sources are used in the microtiter plate; one well, containing a rich growth medium, is used as the positive control. An unknown culture is suspended in a liquid medium and the liquid aliquots are injected into the 96 wells. The plate then is incubated overnight at 35 C, and after incubation, the color of the wells is examined. The advantage of this system is that the color is either clear (no reaction) or blue (as a result of reduction of the dye in the medium). The pattern of blue wells will indicate the identity of the unknown culture. Using the human eye to interpret these data would be tedious and unreliable. For this, an automatic reader is used to provide instant identification of the unknown culture by matching the profile of the known cultures in the data bank against the profiles of the unknown cultures (Sellyei et al., 2011). This is indeed a simple system to use and interpretation of the results is easy. The system is reliant upon its database to match an unknown microorganism against a probable species. Sometimes nontypical isolates are not identifiable and the system is less accurate at identifying anaerobes.
Vitek System Vitek (bioMérieux Vitek, Hazelwood, MO) is a similar automated identification system to Omnilog. The system has its origin in the Viking Mission during the early stages of the US space program during the 1980s. The heart of the system is a plastic cord with 30 tiny wells containing selective media and specialized substrates designed to discriminate bacterial taxa by the growth pattern and kinetics of the unknown culture in media in the 30 wells. A pure culture is first suspended in a liquid, and liquid is introduced into the card (the size of an ordinary credit card) by pneumatic pressure, such that all 30 wells will be filled with an aliquot of the culture. The card then is placed into the incubator. Up to 240 cards can be
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inserted into one large unit. More units can be tested at the same time if more incubators are connected to the system. The instrument periodically scans each card and determines the kinetics of the growth of the microorganism in each well and then determines the identity of the unknown culture. For typical cultures, the identification can be completed in 2 h. Other bacterial cultures can be identified in about 18 h (Crowley et al., 2012). Overall, the performance is exceptionally good and can identify Gram-negative, Gram-positive, yeast, Bacillus, anaerobes, nonfermenters, Neisseria/Haemophilus, and other classes of microorganisms. It constantly receives high correlations with conventional methods of identification of unknown microbial cultures.
Fatty Acid Analysis of cellular fatty acids by using gas chromatography (where patterns of fatty acid esters are determined by gas chromatography) has been available for a number of years, but until recently this was not in a format easy for laboratories to adopt. The technology works by screening for different fatty acids and then comparing the fatty acid profile to a library of different bacterial species. An example of fatty acid analysis is the Sherlock system (MIDI Inc.) (Osterhout et al., 1991).
Mass Spectrometry Mass spectrometry can be orientated toward the identification and classification of microorganisms by using protein ‘fingerprints’ (characteristic protein expression patterns that are stored and used as specific biomarker proteins for crossmatching). The utilization of long-standing technology is based on the measurement of high-abundance proteins, including many ribosomal proteins. As ribosomal proteins are part of the cellular translational machinery, they are present in all living cells. As a result, the mass spectrometry protein fingerprints are less influenced by variability in environmental or growth conditions than other ‘phenotypic’ methods. An example is the matrix-assisted laser desorption ionization time-of-flight BioTyper system from Bruker Daltonics (Hsieh et al., 2008).
Flow Cytometry Flow cytometry is a technique that can employ serological methods (although it does not in all cases) that analyzes cells suspended in a liquid medium by light, electrical conductivity, or fluorescence as the cells individually pass through a small orifice. Most pharmaceutical microbiology laboratories are not equipped to use flow cytometric methods (Muller and Davey, 2009).
Genotypic Methods Identification Methods In contrast to the phenotypic methods, genotypic techniques are more accurate. This is because the microbial genotype is highly conserved and is independent of the culture conditions,
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so the identifications may be conducted on uncultured test material-primary enrichments that increase the amount of nucleic acid available for analysis. Genotypic microbial identification methods based on nucleic acid analyses are less subjective, less dependent on the culture method, and theoretically more reliable because nucleic acid sequences are highly conserved by microbial species. These methods would include DNA–DNA hybridization, polymerase chain reaction (PCR), 16s and 23s rRNA gene sequencing (the 16S rRNA gene is used most commonly), and analytical ribotyping (Olsen et al., 1994). An example is the RiboPrinter (manufactured by Dupont Qualicon), an automated Southern Blot device that uses a labeled ssDNA probe from the 16sRNA codon. The RiboPrinter uses a restriction enzyme and strains can be identified or characterized by analyzing the ribosomal DNA banding pattern (Kolbert and Persing, 1999). Another rapid method is a PCR system that uses a form of ‘bacterial barcodes’ in which the amplified genetic sequence is separated by gel electrophoresis and visualized to give a ‘barcode’ specific to that strain. PCR is a technique that uses a DNA polymerase enzyme to make a huge number of copies of virtually any given piece of DNA or gene. It facilitates a short stretch of DNA (usually fewer than 3000 ‘base pairs’) to be amplified by about a millionfold. In practical terms, it amplifies enough specific copies to be able to carry out any number of other molecular biology applications. Thus, the PCR technique utilizes small amounts of samples to produce a high yield of the targeted DNA material. With this comparative test, differences in the DNA base sequences between different organisms can be determined quantitatively, such that a phylogenetic tree can be constructed to illustrate probable evolutionary relatedness between the microorganisms. An example of such a system is the MicroSeq manufactured by Applied Biosystems (Fontana et al., 2005). The genotypic methods are more technically challenging for the food microbiologist and are more expensive in terms of both equipment and current testing costs. The methods often are used for more critical identifications, such as suspect recall issues, rather than for the routine characterization of the microbial population within a given food sample.
Range of Food Applications The methods and systems described previously are designed for the identification of pure cultures obtained from clinical, food, industrial, and environmental samples. Almost all foods are potential sources of contamination of pathogenic microorganisms. Thus, all microbiological methods are designed to enrich, isolate, enumerate, characterize, and identify the unknown culture in question. The results of the diagnostic tests are only as valuable as the purity of the culture. If there is a mixed culture in the primary isolation, all the valuable identification capabilities of these systems will be meaningless. Thus, for food microbiologists, it is essential that all food samples be properly prepared before either directly plating the sample on selective agars or enriching the foods in preenrichment and enrichment
liquid media and isolating pathogens on appropriate agar plates. The continuing development of excellent primary isolation agars for selectively isolating the target microorganism has assisted in selecting the isolates for further identification by one or more of the diagnostic systems described (Fung, 1998). Another related development of identification of foodpoisoning microorganisms by modern biochemical techniques is the variety of screening tests on the market. Tests for pathogens such as enzyme-linked immunosorbent assay tests, DNA probes, PCR tests, dipstick techniques, and motility tests for pathogens are considered to be screening tests. Negative screening tests would allow the food processors to ship their products to the market but a positive screening test will necessitate an embargo of the product and a confirmation test to be done on the suspected food. This procedure involves conventional methods as well as some of the diagnostic kits mentioned in this article. What are the bacterial food pathogens facing us these days? The list is long but worth reiterating: Salmonella spp., Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Campylobacter jejuni, Escherichia coli O157:H7, Yersinia enterocolitica, Shigella spp., Vibrio, Aeromonas and Plesiomonas, Bacillus cereus, Listeria monocytogenes, and others. The biochemical techniques can identify most of these microorganisms in a laboratory setting, but most commercial diagnostic kits are designed for specific groups of microorganisms. Thus, knowledge of the basic principles of diagnostic microbiology is essential for all food microbiologists, regardless of whether one uses the diagnostic kits described. Which diagnostic system is best for the identification of a particular food-poisoning microorganism is the subject of much debate.
Conclusion Modern biochemical identification techniques, together with genotypic methods, are more convenient modes of improving the conventional biochemical techniques. They make sample operation, inoculation, incubation, reading, data collection, and interpretation of data for diagnostic purposes more convenient than the conventional methods. To make a quantum jump in the identification of food-poisoning microorganisms, one has to look to PCR and biosensor technologies to obtain real-time rapid identification.
See also: Biochemical and Modern Identification Techniques: Introduction; Biochemical and Modern Identification Techniques: Enterobacteriaceae, Coliforms, and Escherichia Coli.
References Appelbaum, P.C., Olmstead, C.C., 1982. Evaluation of Gram-stain screen and Micro-ID methods for direct identification of Enterobacteriaceae from urines. Medical Microbiology and Immunology 170 (3), 173–184. Celig, D.M., Schreckenberger, P.C., 1991. Clinical evaluation of the RapID-ANA II panel for identification of anaerobic bacteria. Journal of Clinical Microbiology 29, 457–462.
BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food-Poisoning Microorganisms Crowley, E., Bird, P., Fisher, K., Goetz, K., Boyle, M., Benzinger Jr., M.J., Juenger, M., Agin, J., Goins, D., Johnson, R., 2012. Evaluation of the VITEK 2 Gram-negative (GN) microbial identification test card: collaborative study. Journal of AOAC International 95 (3), 778–785. Farmer III, J.J., 2003. Enterobacteriaceae: introduction and identification. In: Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A., Yolken, R.H. (Eds.), Manual of Clinical Microbiology, eighth ed. American Society for Microbiology, Washington, D.C. Fung, D.Y.C., 1995. What’s needed in rapid detection of foodborne pathogens. Food Technology 49, 64–67. Fung, D.Y.C., 1998. Handbook for Rapid Methods and Automation in Microbiology in Microbiology Workshop. Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS. Fontana, C., et al., 2005. Use of the MicroSeq 500 16S rRNA Gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. Journal of Clinical Microbiology 43 (2), 615–619. Goosh III, W.M., Hill, G.A., 1982. Comparison of Micro-ID and API 20E in rapid identification of Enterobacteriaceae. Journal of Clinical Microbiology 15, 885–890. Hsieh, S.-Y., Tseng, C.-L., Lee, Y.-S., Kuo, A.-J., Sun, C.-F., Lin, Y.-H., Chen, J.-K., 2008. Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS. Molecular and Cellular Proteomics 7, 448–456. Holloway, Y., Schaareman, M., Dankert, J., 1979. Identification of viridans streptococci on the Minitek Miniaturised Differentiation System. Journal of Clinical Pathology 32 (11), 1168–1173. Kalamaki, M., Price, R.J., Fung, D.Y.C., 1997. Rapid methods for identifying seafood microbial pathogens and toxins. Journal of Rapid Methods and Automation in Microbiology 5, 87–137. Knapp, C.C., Ludwig, M.D., Washington, J.A., 1994. Evaluation of BBL crystal MRSA ID system. Journal of Clinical Microbiology 32 (10), 2588–2589.
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Kolbert, C.P., Persing, D.H., 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Current Opinion in Microbiology 2, 299–305. Muller, M., Davey, H., 2009. Recent advances in the analysis of individual microbial cells. Cytometry Part A: The Journal of the International Society for Analytical Cytology 75, 83–85. Olsen, G.J., Woese, C.R., Overbeek, L.V., 1994. The winds of (evolutionary) change: breathing new life into microbiology. Journal of Bacteriology 176, 1–6. Osterhout, G.J., Shull, V.H., Dick, J.D., 1991. Identification of clinical isolates of Gramnegative nonfermentative bacteria by an automated cellular fatty acid identification system. Journal of Clinical Microbiology 29, 1822–1830. Russell, S.M., Cox, N.A., Bailey, J.S., Fung, D.Y.C., 1997. Miniaturized biochemical procedures for identification of bacteria. Journal of Rapid Methods and Automation in Microbiology 5, 169–178. Sellyei, B., Wehmann, E., Makrai, L., Magyar, T., 2011. Evaluation of the Biolog system for the identification of certain closely related Pasteurella species. Diagnostic Microbiology and Infectious Disease 71 (1), 6–11. Stager, C.E., Erikson, E., Davis, J.R., 1983. Rapid method for detection, identification, and susceptibility testing of enteric pathogens. Journal of Clinical Microbiology 17 (1), 79–84. Stager, C.E., Davis, J.R., 1992. Automated systems for identification of microorganisms. Clinical Microbiology Review 5, 302–327. Sutton, S.V.W., Cundell, A.M., 2004. Microbial identification in the pharmaceutical industry. Pharmacopeial Forum 35 (5), 1884–1894. Thippareddi, H., Fung, D.Y.C., 1998. Laboratory manual section. In: Fung, D.Y.C. (Ed.), Handbook for Rapid Methods and Automation in Microbiology Workshop. Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS. Woolfrey, B.F., Lally, R.T., Ederer, M.N., Quall, C.O., 1984. Evaluation of the AutoMicrobic system for identification and susceptibility testing of gram-negative bacilli. Journal of Clinical Microbiology 20 (6), 1053–1059.
Introduction Identification methods can be divided into two groups: phenotypic and genotypic. The genotype–phenotype distinction is drawn in genetics. ‘Genotype’ is an organism’s full hereditary information, even if not expressed. ‘Phenotype’ is an organism’s actual observed properties, such as morphology, development, or behavior (Sutton and Cundell, 2004). Phenotypic methods are the most widespread due to their relatively lower costs for many laboratories. It should be recognized, however, that expressions of the microbial phenotype – that is, cell size and shape, sporulation, cellular composition, antigenicity, biochemical activity, sensitivity to antimicrobial agents, and so on – frequently depend on the media and growth conditions that have been used. These conditions will include variables such as temperature, pH, redox potential, and osmolality and possibly lesser-known variables such as nutrient depletion, vitamin and mineral availability, growth cycle, water activity of solid media, static or rotatory liquid culture, and solid versus liquid media culture, as well as colony density on the plate. Therefore, some care is required in the interpretation of microbiological identification test results and the trending of data. A further limitation with phenotypic methods is the size and type of the Phenetic Classification Database. With the type of database, many databases are orientated toward clinical applications and do not necessarily serve industrial application well. In terms of size, databases are limited based on the relatively low number of microorganisms that have been characterized (Stager and Davis, 1992). The classical scheme of identification of bacteria by biochemical methods depends on whether a pure culture of the microorganism of interest can grow in an agar plate, an agar slant, a broth, a paper strip, or other supportive material containing specialized growth promoters or inhibitors in the presence of a fermentable or degradable compound, resulting in the medium changing color, development of gas, development of fluorescent compound, and other manifestation of metabolic activities. If the behavior of known cultures in these media is known, an unknown culture can be matched with these characteristics, and based on the closest match to a database, an analyst can make an identification of the unknown culture. This process is tedious, is time-consuming, and requires a lot of labor, materials, time, and energy to perform the tests. In addition, the skill of the analyst in interpreting the reactions and arriving at a correct judgment makes this process subjective and often unreliable (Kalamaki et al., 1997). Genotypic methods are not reliant on the isolation medium or growth characteristics of the microorganism. Genotypic methods have considerably enhanced databases of different types of microorganisms. Before the advent of genotypic methods, microbiologists speculated that a number of taxa
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were present and unculturable (so-termed viable-but-nonculturable strains). Genotypic methods have opened up a whole new set of species and subspecies, as well as reclassifying species and related species (thus, taxa that often are grouped similarly by phenotypic methods actually are polyphyletic groups – that is, they contain organisms with different evolutionary histories that are homologously dissimilar organisms that have been grouped together). Another advantage with genotypic methods is their accuracy and faster time to result (as microorganisms do not need to be grown on culture media). They are, however, relatively expensive. To make a complete identification, a great many tests can be done, as shown in Table 1.
Phenotypic Identification Methods Phenotypic reactions typically incorporate reactions to different chemicals or different biochemical markers. These rely on the more subjective determinations. The reliance on biochemical reactions and carbon utilization patterns introduces some disadvantages to the achievement of consistent
Table 1 Information needed for the identification of foodborne pathogens (Fung, 1995). Phenotypic characteristics Macroscopic morphology on agar plates Morphology under microscopic magnification Gram reaction (positive, negative, or variable) and special staining properties Biochemical activity profile and special enzyme systems Pigment production, bioluminescence, chemiluminescence, and fluorescent compound production Nutritional and growth factor requirements Temperature and pH requirements and tolerance Fermentation products, metabolites, and toxin production Antibiotic sensitivity pattern (antibiogram) Gas requirements and tolerance Cell wall, cell membrane, and cellular components Growth rate constant and generation time Motility and spore formation Resistance to organic dyes and special compounds Impedance, conductance, and capacitance characteristics Genotypic characteristics Genetic profile: DNA/RNA sequences and fingerprinting Extracellular and intracellular products Information relating to the microorganism Pathogenicity to animals and humans Serology and phage typing Ecological niche and survival ability Response to electromagnetic fields, light, sound, and radiation
Encyclopedia of Food Microbiology, Volume 1
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BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food-Poisoning Microorganisms (repeatable and reproducible) identification. To improve on the classical methods of biochemical identification, several developments have been made and refined in recent years. Collectively, these methods are considered to be modern biochemical identification techniques. Although it is possible to prepared militarized biochemical tests within the laboratory, the purchase of commercial test kits is preferable as these can be closely aligned to a database. Commercial diagnostic test kits consist of miniaturized and multitest units. The two main types of diagnostic kits are agar based and dehydrated media based. In these systems, the pure cultures grow in a variety of solid or liquid media, changing color or gas formation, or utilizing their enzymes to change the color of the substrates. Diagnostic charts can be used to identify unknown cultures or the numerical manuals of computerassisted systems. Most of these systems were first designed to identify the family Enterobacteriaceae, and the databases remain largely orientated toward clinical microbiology rather than toward food microbiology. Later, some systems branched out to identify other microorganisms, such as the nonfermentors, lactics, yeast, and so on. The following are synopses of how diagnostic kits operate and the range of microorganisms tested (Russell et al., 1997). As far as possible, information concerning comparative analysis of these kits with conventional methods will be made (also see the Further Reading section). For many diagnostic kits, accuracy ratings are provided in cases in which kits are compared with conventional methods. Most comparative analyses of diagnostic kits were done many years ago and not repeated. It is difficult to compare one kit with another as the databases often vary. The consensus of opinion is that, to be acceptable, a kit should have a 90–95% accuracy correlated with the conventional method. When the value drops to 85% or below, the system is marginally acceptable and any value below that is not acceptable (Thippareddi and Fung, 1998).
Agar-Based Diagnostic Kits Several agar-based multimedia diagnostic kits are available, such as the Enterotube system (Roche Diagnostic, Nutley, NJ). The Enterotube II is a self-contained, compartmented plastic tube containing 12 different conventional media and an enclosed inoculating wire, which is threaded through the entire unit (Farmer, 2003). This system permits 15 standard biochemical tests to be inoculated and performed from a single bacterial colony. Reagents are added to the indole test and Voges–Proskauer (VP) test before color reactions and gas formation are read. Table 2 shows the color reactions of the tests. Because such a table exists for every diagnostic kit described in this article, this table will serve as a model for other kits. Similar tables will not be repeated. After reading the reactions, each result is given a score according to the system. After all the scores are added, an identification (ID) value in the form of a five-digit number will be generated. Other systems (to be described) may have 7- or 10-digit numbers. From the code book, the microorganism can be identified. This procedure is repeated for most other systems and will not be described again. The Enterotube II was developed to identify Enterobacteriaceae only; it has developed to be
Table 2
Reactions of biochemical tests for Enterotube II
Test GLU Gas LYS ORN H2S IND ADON LAC ARAB SORB VP DUL PA UREA CIT
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Glucose utilization Gas production Lysine decarboxylase Ornithine decarboxylase H2S production Indole formation Adonitol fermentation Lactose fermentation Arabinose fermentation Sorbitol fermentation Voges–Proskauer Dulcitol fermentation Phenylalanine deaminase Urease Citrate utilization
Positive reaction
Negative reaction
Yellow Wax lifted Purple Purple Black Pink-red Yellow Yellow Yellow Yellow Red in 20 min Yellow or pale yellow Black to smoky gray Red-purple Deep blue
Red Wax not lifted Yellow Yellow Beige Colorless Red Red Red Red Green Green Yellow Green
able to identify a variety of other oxidase-negative Gramnegative rods. A similar unit, the Oxi/Ferm Tube, was designed for Gramnegative nonfermenters. Advantages of Enterotube II include rapidity and ease of inoculation, that inoculum suspension is not required, and a single colony can be used for identification. Disadvantages include that it is only useful for Enterobacteriaceae, it is difficult to stack in the incubator, and it has a short shelf life.
Dehydrated Media Diagnostic Kits Dehydrated media diagnostic kits are another type of miniaturized microbiological method. Dehydrated media kits have the advantage of much longer shelf life than agar-based media (18 months versus a few months). Those currently used in clinical, environmental, industrial, and food microbiology will be discussed in the following sections. Of course, many similar systems are available worldwide: The systems discussed here have been well tested and used in the United States and Europe.
Analytical Profile Index The Analytical Profile Index (API; bioMérieux, Hazelwood, MO) is arguably the most popular system for diagnostic bacteriology in the world, especially for Enterobacteriaceae. The API 20E system is a miniaturized microtube system that has 20 small wells designed to perform 23 standard biochemical tests from isolated colonies of bacteria on plating medium. The system has procedures for same-day and 18–24 h identification of Enterobacteriaceae. It consists of microtubes containing dehydrated substrates. The substrates are reconstituted by adding a bacterial suspension into each of the 20 wells; some of the wells are filled with mineral oil to create anaerobic conditions. The unit then is incubated so that the microorganisms react with the contents of the tubes and are read when the indicator systems are affected by the metabolites or added reagents – generally after 18–24 h incubation at 35–37 C.
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After all the reactions are read and recorded in a data sheet, a number code is generated and the code can be matched with the code book for identification. The strip also can be read in a reader and the results interpreted by a computer (Woolfrey et al., 1984). Using similar formats, other microorganisms can be identified, such as API Gram-negative Identification: API Rapid 20E (4 h identification of Enterobacteriaceae); API 20NE (24–48 h identification of Gram-negative non-Enterobacteriaceae); API Ò Campy (24 h identification of Campylobacter species); API Gram-positive Identification: API Staph (identification of clinical staphylococci and micrococci); API 20 Strep (identification of streptococci and enterococci); API Coryne (identification of Corynebacteria and corynelike organisms); API Ò Listeria (24 h identification of all Listeria species); API Anaerobe Identification: API 20A (identification of anaerobes); Rapid ID 32 A (identification of anaerobes); and API Yeast Identification: API 20C AUX (48–72 h identification of yeasts). Advantages include being the most complete system commercially available for the identification of Enterobacteriaceae and an excellent database. Disadvantages include that it is difficult and time-consuming to inoculate, problems in handling and stacking of tray and lids due to flexible plastic materials, and that a competent microbiologist is needed to read and interpret the color changes.
MicroID The MicroID (Organon Teknika, Durham, NC) system provides results in 4 h. The system measures enzyme activities and not growth of the culture. It consists of a molded polystyrene tray containing 15 reaction chambers and a hinged cover. The first five reaction chambers contain a single combination substrate or detection disc with upper and lower discs in the same trough. The remaining 10 reaction chambers each contain a single combination substrate or detection disc. Discs contain all substrate and detection reagents required to perform the indicated biochemical tests (except for the Voges–Proskauer test). The surface of the tray is covered with clear polypropylene tape to prevent spillage and also for reading the reactions (Appelbaum and Olmstead, 1982). A few cultures from a Gram-negative isolation agar plate are first mixed into a liquid form and 0.2 ml of the liquid is then introduced into each of the 15 wells. The unit is then incubated at 35 C for 4 h, after which time two drops of 20% KOH are added into the VP well. The unit is then rotated 90 so that the liquid from the lower part of the first five wells comes into contact with the upper discs of the same chamber for final reactions. The reactions of these five tests are read from the upper discs. Then the reactions from the remaining 10 discs are read. Again, a number is generated in the data sheet and the code number is matched, with codes in the code book for identification. A similar format with different substrates was available to identify Listeria spp. (Goosh and Hill, 1982). Advantages of the system include high accuracy, speed of reaction (4 h), and convenience: It is self-contained, easy to use, requires only one reagent addition, and has a long shelf life. A disadvantage is that a competent microbiologist is needed to read the color reaction.
Minitek Minitek (Becton Dickinson Microbiology Systems, Cockeyville, MD) is a flexible system. The unit contains 10 wells. Two units (20 wells) can be used to identify one culture. The system supplies 36 different substrates, and thus the user can choose which test to perform. First, paper discs containing individual substrates are applied to the wells, one disc per well. A liquid culture is prepared and applied to each well using an automatic application gun (about 0.2 ml per well). Some wells will be filled with mineral oil to create an anaerobic environment. The unit is then incubated overnight at 35 C. After incubation, the color reactions are read and identification is made with the aid of a code book (Holloway et al., 1979). On the one hand, the advantage of the system is versatility and flexibility, but this may be a disadvantage when no code book is available for microorganisms other than Enterobacteriaceae. The construction of the unit is sturdy. Disadvantages include that the various components of the total system are handled excessively in preparation and operation. Again skill is needed to read borderline reactions in the discs.
BBL Crystal The BBL Crystal (Becton Dickinson Microbiology Systems, Cockeysville, MD) system requires relatively little manipulation. In one system (Enteric/Nonfermentor ID Kit), both enteric and nonfermenters can be identified. It is important to ensure that the unit is marked correctly as to whether an oxidase-positive (nonfermenter) or oxidase-negative (fermenter) pure culture is to be analyzed (Knapp et al., 1994). The system is easy to use. On one panel, 30 dried biochemical substrates are housed and a companion unit (base) is used for the liquid sample. The liquid culture (approximately 2 ml) is poured carefully into the trough of the base. Then the upper unit containing the 30 tests is simply snapped into the base such that the culture interacts with the 30 substrates. The unit is then incubated at 35 C overnight. After incubation, the unit is introduced into a Crystal light box to record reactions and for identification using a 10-digit system. Identification can be made using a computer. In addition, there is also a Rapid Stool/Enteric ID kit for stool samples. Advantages of the system include sturdy panels, ease of operation, and computer-assisted identification. Very few disadvantages are noted.
RapID One System RapID One (Remel, Lenexa, KS) is a miniaturized unit housed in an ingenious chamber. On one side of the chamber, there is a trough where a liquid culture can be introduced. Then the unit can be tilted slowly at a 45-degree angle forward: The liquid will flow into individual wells, each containing a separate substrate. Thus, inoculation into 20 wells can be made in one motion. This is more convenient than the API system where the analyst needs to insert 20 drops of liquids into 20 miniaturized wells. After incubation, the color reactions can be read after 4 h incubation and the cultures identified. The forerunner of the RapID enteric system is the Spectrum 10 system. The Spectrum 10 is rated as 91% accurate. Using the basic design, Remel markets strips for Enterobacteriaceae,
BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food-Poisoning Microorganisms nonfermenters, yeasts, anaerobes, streptococci, Leuconostoc, Pediococcus, Listeria, Neisseria, Haemophilus, and urinary tract bacteria (Stager et al., 1983). Identification of various anaerobes to the genus level using the anaerobic RapID system ranges from 83% to 97% accuracy and to the genus level from 76% to 97% (Celig and Schreckenberger, 1991). Advantages include results in 4 h, clear chromogenic reactions, and one-step inoculation. A disadvantage is the skill required to read the color changes.
ATB ATB (bioMérieux Vitek, Inc., Hazelwood, MO) is a 32-carbon assimilation test system. The culture is first made into a solution and then the liquid is introduced to the unit. After incubation (4–24 h depending on the culture), the tests can be read manually or automatically. Test strips are available for anaerobes, staphylococci, micrococci, yeast, Enterobacteriaceae, streptococci, and Gram-negative bacilli. The automatic reader also can read API 20 and API 50 test strips.
Omnilog System Omnilog (Biolog, Hayward, CA) is a miniaturized system utilizing the microtiter plate format for growth of bacteria in various liquid media. Some 95 different carbon sources are used in the microtiter plate; one well, containing a rich growth medium, is used as the positive control. An unknown culture is suspended in a liquid medium and the liquid aliquots are injected into the 96 wells. The plate then is incubated overnight at 35 C, and after incubation, the color of the wells is examined. The advantage of this system is that the color is either clear (no reaction) or blue (as a result of reduction of the dye in the medium). The pattern of blue wells will indicate the identity of the unknown culture. Using the human eye to interpret these data would be tedious and unreliable. For this, an automatic reader is used to provide instant identification of the unknown culture by matching the profile of the known cultures in the data bank against the profiles of the unknown cultures (Sellyei et al., 2011). This is indeed a simple system to use and interpretation of the results is easy. The system is reliant upon its database to match an unknown microorganism against a probable species. Sometimes nontypical isolates are not identifiable and the system is less accurate at identifying anaerobes.
Vitek System Vitek (bioMérieux Vitek, Hazelwood, MO) is a similar automated identification system to Omnilog. The system has its origin in the Viking Mission during the early stages of the US space program during the 1980s. The heart of the system is a plastic cord with 30 tiny wells containing selective media and specialized substrates designed to discriminate bacterial taxa by the growth pattern and kinetics of the unknown culture in media in the 30 wells. A pure culture is first suspended in a liquid, and liquid is introduced into the card (the size of an ordinary credit card) by pneumatic pressure, such that all 30 wells will be filled with an aliquot of the culture. The card then is placed into the incubator. Up to 240 cards can be
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inserted into one large unit. More units can be tested at the same time if more incubators are connected to the system. The instrument periodically scans each card and determines the kinetics of the growth of the microorganism in each well and then determines the identity of the unknown culture. For typical cultures, the identification can be completed in 2 h. Other bacterial cultures can be identified in about 18 h (Crowley et al., 2012). Overall, the performance is exceptionally good and can identify Gram-negative, Gram-positive, yeast, Bacillus, anaerobes, nonfermenters, Neisseria/Haemophilus, and other classes of microorganisms. It constantly receives high correlations with conventional methods of identification of unknown microbial cultures.
Fatty Acid Analysis of cellular fatty acids by using gas chromatography (where patterns of fatty acid esters are determined by gas chromatography) has been available for a number of years, but until recently this was not in a format easy for laboratories to adopt. The technology works by screening for different fatty acids and then comparing the fatty acid profile to a library of different bacterial species. An example of fatty acid analysis is the Sherlock system (MIDI Inc.) (Osterhout et al., 1991).
Mass Spectrometry Mass spectrometry can be orientated toward the identification and classification of microorganisms by using protein ‘fingerprints’ (characteristic protein expression patterns that are stored and used as specific biomarker proteins for crossmatching). The utilization of long-standing technology is based on the measurement of high-abundance proteins, including many ribosomal proteins. As ribosomal proteins are part of the cellular translational machinery, they are present in all living cells. As a result, the mass spectrometry protein fingerprints are less influenced by variability in environmental or growth conditions than other ‘phenotypic’ methods. An example is the matrix-assisted laser desorption ionization time-of-flight BioTyper system from Bruker Daltonics (Hsieh et al., 2008).
Flow Cytometry Flow cytometry is a technique that can employ serological methods (although it does not in all cases) that analyzes cells suspended in a liquid medium by light, electrical conductivity, or fluorescence as the cells individually pass through a small orifice. Most pharmaceutical microbiology laboratories are not equipped to use flow cytometric methods (Muller and Davey, 2009).
Genotypic Methods Identification Methods In contrast to the phenotypic methods, genotypic techniques are more accurate. This is because the microbial genotype is highly conserved and is independent of the culture conditions,
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so the identifications may be conducted on uncultured test material-primary enrichments that increase the amount of nucleic acid available for analysis. Genotypic microbial identification methods based on nucleic acid analyses are less subjective, less dependent on the culture method, and theoretically more reliable because nucleic acid sequences are highly conserved by microbial species. These methods would include DNA–DNA hybridization, polymerase chain reaction (PCR), 16s and 23s rRNA gene sequencing (the 16S rRNA gene is used most commonly), and analytical ribotyping (Olsen et al., 1994). An example is the RiboPrinter (manufactured by Dupont Qualicon), an automated Southern Blot device that uses a labeled ssDNA probe from the 16sRNA codon. The RiboPrinter uses a restriction enzyme and strains can be identified or characterized by analyzing the ribosomal DNA banding pattern (Kolbert and Persing, 1999). Another rapid method is a PCR system that uses a form of ‘bacterial barcodes’ in which the amplified genetic sequence is separated by gel electrophoresis and visualized to give a ‘barcode’ specific to that strain. PCR is a technique that uses a DNA polymerase enzyme to make a huge number of copies of virtually any given piece of DNA or gene. It facilitates a short stretch of DNA (usually fewer than 3000 ‘base pairs’) to be amplified by about a millionfold. In practical terms, it amplifies enough specific copies to be able to carry out any number of other molecular biology applications. Thus, the PCR technique utilizes small amounts of samples to produce a high yield of the targeted DNA material. With this comparative test, differences in the DNA base sequences between different organisms can be determined quantitatively, such that a phylogenetic tree can be constructed to illustrate probable evolutionary relatedness between the microorganisms. An example of such a system is the MicroSeq manufactured by Applied Biosystems (Fontana et al., 2005). The genotypic methods are more technically challenging for the food microbiologist and are more expensive in terms of both equipment and current testing costs. The methods often are used for more critical identifications, such as suspect recall issues, rather than for the routine characterization of the microbial population within a given food sample.
Range of Food Applications The methods and systems described previously are designed for the identification of pure cultures obtained from clinical, food, industrial, and environmental samples. Almost all foods are potential sources of contamination of pathogenic microorganisms. Thus, all microbiological methods are designed to enrich, isolate, enumerate, characterize, and identify the unknown culture in question. The results of the diagnostic tests are only as valuable as the purity of the culture. If there is a mixed culture in the primary isolation, all the valuable identification capabilities of these systems will be meaningless. Thus, for food microbiologists, it is essential that all food samples be properly prepared before either directly plating the sample on selective agars or enriching the foods in preenrichment and enrichment
liquid media and isolating pathogens on appropriate agar plates. The continuing development of excellent primary isolation agars for selectively isolating the target microorganism has assisted in selecting the isolates for further identification by one or more of the diagnostic systems described (Fung, 1998). Another related development of identification of foodpoisoning microorganisms by modern biochemical techniques is the variety of screening tests on the market. Tests for pathogens such as enzyme-linked immunosorbent assay tests, DNA probes, PCR tests, dipstick techniques, and motility tests for pathogens are considered to be screening tests. Negative screening tests would allow the food processors to ship their products to the market but a positive screening test will necessitate an embargo of the product and a confirmation test to be done on the suspected food. This procedure involves conventional methods as well as some of the diagnostic kits mentioned in this article. What are the bacterial food pathogens facing us these days? The list is long but worth reiterating: Salmonella spp., Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Campylobacter jejuni, Escherichia coli O157:H7, Yersinia enterocolitica, Shigella spp., Vibrio, Aeromonas and Plesiomonas, Bacillus cereus, Listeria monocytogenes, and others. The biochemical techniques can identify most of these microorganisms in a laboratory setting, but most commercial diagnostic kits are designed for specific groups of microorganisms. Thus, knowledge of the basic principles of diagnostic microbiology is essential for all food microbiologists, regardless of whether one uses the diagnostic kits described. Which diagnostic system is best for the identification of a particular food-poisoning microorganism is the subject of much debate.
Conclusion Modern biochemical identification techniques, together with genotypic methods, are more convenient modes of improving the conventional biochemical techniques. They make sample operation, inoculation, incubation, reading, data collection, and interpretation of data for diagnostic purposes more convenient than the conventional methods. To make a quantum jump in the identification of food-poisoning microorganisms, one has to look to PCR and biosensor technologies to obtain real-time rapid identification.
See also: Biochemical and Modern Identification Techniques: Introduction; Biochemical and Modern Identification Techniques: Enterobacteriaceae, Coliforms, and Escherichia Coli.
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