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The conventional methods in microbiology involve the analysis of bacteria, yeasts or moulds by the use of plating, testing biochemically or microscopy. But with progress of time, new methods in food processing, quickening pace of the food trade and consumer demand for safer food products caused an ultimate shift towards methods which were more accurate and faster which finally led to the development of Rapid Microbiological Methods(Baumgartner, 1995). The automation and development of rapid methods in the field of microbiology has proved it to be a very dynamic area which makes use of immunological, biochemical, biophysical and microbiological methods to facilitate early detection of microorganisms, the byproducts produced by them and their enumeration, characterization in clinical, food, industrial and environmental samples(Fung, 1992). In the past twenty years the field of rapid microbiological methods has advanced to great levels so much so that speed, ease of use of operation and repeatability can be achieved in a very accurate and easy way through automation. In present time there are many companies which specialize in the manufacture and production of instruments to provide assistance in rapid methods of detection(Robinson, 1995). A rapid microbiological method to be of any significant value must be proven to have defined parameters or attributes of accuracy, precision, specificity, sensitivity, selectivity and must be applicable in a variety of laboratory applications, this is one of the principle ways to identify the viability of a method(Archer, 1996).
RAPID MICROBIOLOGICAL METHODS
Rapid methods have become an inseparable part of the food industry as the trade conventions convened over the years have passed a resolution that the quality of meat, its freshness and hygiene has to be determined by rapid methods which reduces the time of testing by considerable amounts(Bülte and Reuter, 1984).
The bioluminescence technique used for the determination of adenosine tri-phosphate is a very reliable method to estimate living cell, for instance meat and meat products. This method is based on the luciferin-luciferase system(Bülte and Reuter, 1984). Luciferin is found in luminescent organasims like fireflies. Any technique or method utilizing the luciferin-luciferase system is based on the measurement of light produced during the oxidation of luciferin in the presence of adenosine tri-phosphate and magnesium(John, 1970). This method is typically used for testing meat and meat products which involves the use of samples from the surface of the meat product. In case of large meat particles and any form of debris centrifugation method is employed to separate them from the sample. The luciferin-luciferase system is then used to measure the microbial ATP(Bülte and Reuter, 1984).
DIRECT EPIFLUORESCENT FILTER TECHNIQUE (DEFT)
The direct epifluorescent filter technique more commonly known as DEFT is used to determine the microbial quality of milk and other kinds of food products. The most positive aspect about this method which acts in its favour is that the structural characteristics of micro-organisms can be determined. The principle of this method is based on a clump count in the milk sample which gives a closer result to plate counts which is used to measure the amount of bacteria present(Ubaldina and Kroll, 1985). This technique makes use of membrane fluorescence and epifluorescence microscopy and takes less than twenty five minutes to complete and is very accurate with milk containing 5 x 103 to 5 x 108 bacteria per ml. Bacteria present on the DEFT slides can be counted by use of television image analysis(Pettipher and Rodrigues, 1982).
The Coulter principle was developed in 1950's by Wallace H. Coulter who was later helped by his brother Joseph R. Coulter, Jr. during his research. It was a very straight forward method of passing cells through a sensing aperture. This technique laid a very strong base for the use of automated cell counting techniques and the instruments used for it in industries(Graham, 2003). Initially during the research on this method Wallace H. Coulter was working to attain a light path of very small dimensions for better particle dimensions, however this required an internal aperture but the laser had not yet been invented so instead Coulter made use of pulses which terminated in an electrical path of small dimensions. This led to the sensing of particle volume. This method can be employed for particle counting of all finely divided particulate matter for instance blood cells. The basic principle of this method is based on the regulation of the impedance of electrical current path of small dimensions by the use of D.C and an alternating field. The biological cells have very thin membranes hence, when a high electrical frequency is applied and if the applied aperture current is also of the same frequency the current flows through the cells and the simultaneous D.C produces a response with respect to the volume of the particle. The electrical sensing zone more commonly known as aperture impedance technique can be combined with other sensing means such as light scatter, polarization and fluorescence to accurately classify the particles or cells(Morgan, 1996).
Reflectometry is a method which measures the interference patterns of reflected waves in comparison with reference reflections to identify the reflection characteristics of the sample. This method is mostly employed to test meat samples. The first stage in this process is to set up the instruments to measure reflectivity of beef samples. The next stage involves the cooking the meat based on the parameters used in the shear force testing of meat, this is used as the reference sample. The last step is to compare the measurements obtained by the reflectometry system with that of the reference sample(Schlutz, 2008). It can also be used to test soil samples and other solute concentrations(Persson and Berndtsson, 1998). For measuring moisture content in porous materials time domain reflectometry method is employed(Cerný, 2009).
Impedance microbiology is an old study; it was first presented by Stewart in July 1898 in The Journal of Experimental medicine. In this Stewart measured the electrical response curves following the putrefaction of blood and serum which was similar to the curves obtained from the impedance systems in use presently. The only major difference was that Stewart was measuring impedance changes in excess of thirty days whereas impedance now is a rapid microbiological method having varied applications(Silley and Forsythe, 1996). Impedance measurements are based on the electrical changes in small amounts of solid or liquid media due to the growth and physiological activity of micro-organisms. During the process of measurement the main parameter provided is impedance detection time, this value is produced on sufficient production of microbial metabolites. This factor depends on initial concentration, the lag phase and the generation time of the growing organisms(Kleiss et al., 1995).
There are two main techniques for measuring in impedance microbiology, a direct method and an indirect method. In the direct method the electrodes are immersed in the same cell the inoculum is present in and the changes in impedance result from changes taking place in the bulk electrolyte which are produced by the metabolic activity of micro-organisms. The indirect method is employed when the salt concentration in the culture broth is very high. In this method the microbial metabolism is measured by keeping track of the carbon di-oxide produced in a chamber with electrodes and KOH(Felice et al., 1999).
The technique of impedance measurement can be utilized to a great extent in the food industry. The technique of total viable counts can be used to test frozen vegetables, grain products, UHT low acid foods, confectionery, fish and meat products(Silley and Forsythe, 1996).
In the milk industry impedance is used to monitor the growth of lactic acid bacteria and other food poisoning causing organisms. The lactic acid bacteria content is monitored within the starter culture by measuring the impedance changes caused due to the metabolism of the bacteria within the sample. Any instability caused in this is normally attributed to bacteriophages which can be detected by using a sensitive assay(Silley and Forsythe, 1996).
The technique of impedance measurement is used to a great extent to test for salmonella within food samples. This has resulted in impedance microbiology being a recognized method for screening of animal feeds. For the use of impedance in the detection of salmonella species within the sample, it is subjected to anaerobic conditions by providing a large volume with small surface area. The use of this condition reduces the time for detection. The impedance method for detection of salmonella species was accepted by the association of analytical communities after seventeen collaborative lab trials were performed(Silley and Forsythe, 1996).
In the year 1989, Phillips and Griffith reported that the Listeria species could be detected by impedance by making use of a slightly changed medium containing acriflavine, ceftazidime, nalidixic acid and aesculin. The impedance curve generated can be used to identify between Listeria and non-listeria species(Silley and Forsythe, 1996).
The indirect impedance method is utilized to detect coliforms and e. coli within samples. The principle was based on the fact that coliforms could ferment lactose which changed the broth colour from red to yellow and in case of e coli it was its property to cleave the substrate methylumbelliferyl-β-D-glucuronide to yield fluorescent methylumbelliferone(Silley and Forsythe, 1996).
The very first calorimetric measurement with respect to food was done by Dubrunfaut, but he studied the energy and heat balance of a fermentation vat as he did not have the modern calorimeters used today(Wadsö and Gómez Galindo, 2009).
The process of microcalorimetry is based on measuring a small amout of heat generated during biological processes. The main function of a microcalorimeter involves the measurement of heat formed during a thermodynamic process and can determine exact values of enthalpy changes or change in internal energy. It has great applications in the food industry due to its accuracy, non-destructivity and high amount of sensitivity. It can be utilized to explain the forces stabilizing the structures of biological molecules(Coimbra, 2009).
Microcalorimetry is of the isothermal titration and differential scanning type(Coimbra, 2009).Isothermal titration calorimetry is used to identify interaction between molecules like proteins, polysaccharides, DNA, lipids. It can also be utilized to measure the changes in heat within colloidal systems(Chanamai and McClements, 2001). Differential scanning microcalorimetry is used to measure heat changes in a sample under conditions in which the rise or fall in temperature is controlled(Coimbra, 2009). Differential scanning microcalorimetry can also be used to study changes in proteins as a function of temperature. When proteins are denatured by the use of thermal energy it leads to the disruption of various chemical forces and the changes which result from this are reflected on a differential scanning microcalorimetry thermogram(Li-Chan and Ma, 2002).
Differential scanning microcalorimetry is used more widely in the food industry as it is most adaptable in a varied range of applications and gives a better understanding of the food constituents like oil and fats. Microcalorimetry is mostly used to test vegetable oil and deep fried or microwaved samples by comparing the differential scanning microcalorimetry characteristics with that of the total polar compounds, viscosity and color changes(Li-Chan and Ma, 2002).
Rapid microbiological methods have become an integral part of the industry especially the food industry as these methods can give accurate results in a very short period of time as compared to conventional microbiological techniques. This saves the industry a lot of time, money and it gives the consumers a satisfaction of the product being safe as it has been tested by a reliable technique. The results obtained from these methods can be employed to maintain better standards of production and hygiene when manufacturing a food product.