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Bioljuminesцenцija adenozintrifosfata (tackling)

Non-biological synthesis of ATP in the extracellular environment is known [104], but it is generally accepted that such sources of ATP are very rare [62].

ATP is a high-energy substance found in all living cells [104], and a necessary component in the initial biochemical stages of using the substrate, as well as in the synthesis of cellular material.

[84] was the first to show that the emission of light in the bioluminescence reaction of RkoyptrugIe [firefly (flying)] is stimulated by ATP. The method for determining ATP concentrations using untreated extracts of fireflies is described in [84], and since then it has been used in many areas as a sensitive and accurate method for measuring ATP. The catalyst of the reaction, accompanied by the emission of light, is the enzyme luciferase, which is found in glowing fireflies. Luciferase is involved in the following reaction:1.1

The reaction is accompanied by the emission of light, is effective; lyutseferina oxidation of each molecule is accompanied by the emission of a single photon, and thus uses every molecule of ATP [112].

In [78], the use of quantitative determination of ATP based on the bioluminescence of fireflies to detect the presence of viable microorganisms was first described. After the appearance of this first report, a large amount of work was carried out to determine viable microorganisms in environmental samples [118] using the bioluminescence method. Since all viable microorganisms contain ATP, it can be considered that the bioluminescence method can be simply used to quickly determine the number of microorganisms. Studies, however, showed that the amount of ATP in different microbial cells is different depending on the type, nutrition, adverse effects and growth stage [118,119]. Therefore, when using bioluminescence, it is important to consider the following:

  •  defined type of microorganism (usually vegetative bacteria contain 1 femtogrammu (fg) of ATP in the cell [72], yeast contain it ten times more [118], and spores do not contain ATP [113]);
  •  if negative cells were exposed to (e.g., nutrient depletion, cooling or changing pH); in such cases may require short recovery prior to testing;
  •  Whether the cells are in an environment in which relatively little ATP (such as a growth medium), or in a complex matrix (such as food) with a very high content of background ATP.

When testing food samples, one of the biggest problems is the last of the above points. All products contain ATP, and its content is usually significantly higher than its content in microorganisms found in these products. The [113] data shows that the ratio of ATP products to ATP bacteria varies from 40 LLC: 1 (in ice cream) to 15: 1 (in milk). Therefore, in order to use the definition of ATP as a rapid test for the determination of microorganisms in food products, methods for the separation of microbial ATP have been developed. Two types of methods were studied: the physical separation of microorganisms from other ATP sources and the use of specific extracting substances for the removal and destruction of non-microbial ATP. Filtration methods have been successfully applied to isolate microorganisms from beverages [77,79] and brewing samples [65]. These methods, however, are difficult to apply to solutions containing particulates, as the filters quickly become clogged. A possible way to solve this problem is based on the application of the double filtration [80] scheme, in which the first filter removes food residues, but passes microorganisms, and the second filter retains microorganisms before lysis and bioluminescent analysis. Other researchers [8,120] used ion-exchange resins to selectively separate product residues or microorganisms prior to bioluminescent tests.

As a result of careful studies of selective chemical extraction, it was shown that it can be successfully used to separate microbial and non-microbial ATP for milk [25] and meat [18]. Usually, this method uses the lysis of somatic (food) cells with the subsequent destruction of the isolated ATP enzyme (apirase). Then you can apply a stronger extractant for the decomposition of microbial cells, and then - testing using luciferase. This method allows to determine only microbial ATP.

A number of devices are available that are specifically designed to determine microbial ATP. Firms Lumac (Netherlands), Foss Electric (Denmark), Bio Orbit (Finland) and Biotrace (United Kingdom) produce systems using separation techniques specifically designed to identify microorganisms in food products. Typically, these systems work well and have similar characteristics, including the minimum threshold for determining 104 bacteria (yeast 103 cells) and the analysis time less than 1 h.

In addition to determining the total number of viable microorganisms in food samples, there are a number of reports regarding the possible applications of ATP-bioluminescence in the food industry. The use of ATP analysis for rapid determination in sanitation is considered in [59] as a method for rapid assessment of microbiological contamination and as a method for measuring the overall cleanliness of surfaces. It is in the second case that the ATP measurement can give a unique result. As noted above, almost all food products contain ATP in large quantities, so using the bioluminescent method, which allows you to quickly check the health status, food residues on the production line can be detected within a few minutes. ATP-bioluminescence to control the hygienic state of surfaces is now widely used in industry. The availability of relatively inexpensive, portable and easy-to-use luminometers allows many food manufacturers to introduce rapid hygiene testing methods, ideal for monitoring within the HACCP system, where the hygienic surface condition is a critical control point. The [51] data indicate that in all surveyed enterprises that regularly apply the methods of hygienic ATP control, after the introduction of these methods, an improvement in the sanitary and hygienic state was noted. Such control systems based on the definition of ATP can be used on most food processing installations, in catering organizations and in retail trade, as well as for assessing the sanitary and hygienic state of vehicles (for example, tank trucks).

One of the tasks that ATP bioluminescence cannot cope with so far is the determination of specific microorganisms. You can use the selective growing medium for certain microorganisms when they are selectively grown before ATP analysis. Such an approach can, however, significantly increase the duration of the analysis, in connection with which erroneously high results can be expected. The ability to successfully use selective lysogenic factors that release ATP only from the cells to be determined is shown in the study described in [119]. The set of such specific substances is quite small, and therefore the method may have limited use. Perhaps the most promising method developed for the determination of individual microorganisms is the use of bacteriophages obtained by genetic engineering [111,126,127].

Bacteriophages are viruses that infect bacteria. A study of bacteriophages has shown that some of them are highly selective and infect only certain types of bacteria. Thus, it is possible to enter into the bacteriophages genetic information that causes the production of bacterial luciferase. Thus, when a bacteriophage infects a certain “host” bacterium, it produces luciferase and begins to luminesce. This method requires careful selection of a bacteriophage to eliminate erroneous positive or negative results, but research shows that in the future, methods based on luminescence can be used for the rapid determination of specific microorganisms [124].

In conclusion, it can be noted that the use of ATP-bioluminescence in the food industry has already been developed to a state in which it can be reliably used for the rapid determination of viable microorganisms (if an effective separation method for microbial ATP is used). The potential use of this method in rapid hygienic state analyzes is now generally recognized. Studies have also shown that luminescence makes it possible to expressly identify specific microorganisms, but such a system should be well developed before it is widely used in the food industry.

Methods microscopy

Microscopy is a recognized and uncomplicated method for determining the number of microorganisms. One of the first descriptions of its use relates to the rapid counting of bacteria in milk films stained with methylene blue [26]. One of the main advantages of microscopic methods is the speed with which individual analyzes can be performed; however, a large amount of manual work and the possibility of operator fatigue caused by continuous counting should be considered.

The use of fluorescent dyes instead of traditional colored compounds allows to simplify the counting, and therefore these dyes have become the subject of many studies. Environmental microbiologists first used such compounds to make microorganisms visible in natural water and count them [50, 67]. The use of polycarbonate membrane filters from the nuclepore (nuclepore) for the separation of microorganisms before fluorescent staining was first described in [58]. The calculation was described in detail in [100], and the method developed by the authors is known as the direct epifluorescent filter technique (DEFT).

The DEFT method is a time-consuming manual method, which gave impetus to research in the field of automated microscopic fluorescence methods (with automatic sample preparation and high productivity). The first fully automated instrument based on fluorescence microscopy was Bactoscan (from Foss Electric, Denmark), which was designed to count bacteria in milk and urine. Milk samples placed in the device undergo chemical treatment to decompose somatic cells and dissolve casein micelles. The bacteria are then separated by continuous centrifugation to a dextran / sucrose gradient. Then the microorganisms were grown with a protease to remove residual protein, after which they were stained with acridine orange and applied in a thin film on a disk rotating under a microscope. Luminescent radiation from an image in a microscope is converted into electrical pulses and recorded. Bactoscan is widely used to test raw milk in continental Europe and gives a good correlation of results with data obtained by traditional methods [71]. The method, however, has a low sensitivity (approximately 5 x 104 cells per ml), which prevents its use for samples with a small number of bacteria.

For the food industry, an instrumental fluorescence counting method was developed in which samples were applied to a thin plastic tape. The Autotrak instrument applied samples to a tape that was passed through a staining and wash solution and then passed in front of a fluorescent microscope. Light pulses from the stained microorganisms were then counted by a photomultiplier. Analysis of food samples using this instrument showed [13] that food residue samples interfered with staining and counting, and the results were significantly higher than those obtained by counting the total number of viable microorganisms.

Probably the newest method of fluorescence microscopy used in the food industry for express counting is flow cytometry. The colored sample is passed under a fluorescent microscope as a liquid in a flow cell. Light pulses caused by light on the colored particles enter the photomultiplier and are counted. This automated rapid method is potentially very flexible. Of the microscopy methods discussed above, DEFT is probably the most widely used, and flow cytometry is very promising. Below we consider them in more detail.

Метод DEFT

The DEFT method was developed to expressly count the number of bacteria in raw milk [99,100] samples. The method is based on the pretreatment of a milk sample in the presence of a proteolytic enzyme and surfactant at 50 ° C, followed by membrane filtration that separates microorganisms. Pretreatment is used to decompose somatic cells and soluble fats that could block the membrane filter. After filtration, the membrane is stained with a fluorescent dye that binds nucleic acid, orange acridine, then washed and placed on a glass slide. After that, the membrane is examined through an epifluorescence microscope, which illuminates the membrane with ultraviolet light (the dye emits visible light, which can be observed through the microscope). Because the dye binds to nucleic acids, it concentrates in the cells of microorganisms, binding to DNA and RNA molecules; thus, any microorganism on the membrane can be easily seen and counted. Full preprocessing and counting can be done in just 30 minutes.

Although the DEFT method made the calculation very fast, it was very laborious, since all the preliminary processing and calculation was done manually, and therefore the performance of this method was very small. The development of semi-automatic counting methods based on image analysis [99] made it possible to overcome some of the problems of manual counting and thus made the method more user-friendly.

The work of using DEFT to count cells in raw milk was followed by an analysis of other types of products. It was soon found that a good correlation between the results of DEFT and the traditionally obtained total number of viable microorganisms in raw milk samples is not available when analyzing milk that has undergone heat treatment [98]. Initially it was believed that this was due to changes in gram-positive cocci during the heat-indicating staining [98], but later it was shown [3] that similar changes occur in both gram-positive and gram-negative microorganisms. Similar dyeing phenomena were also observed in the case of heat treatment of yeast [106] and in irradiated food products [14], and therefore the use of DEFT as a method to quickly determine the total number of viable microorganisms is mainly limited to raw products.

The list of types of products for which the DEFT method can be used has now been expanded. The literature describes the application of this method for frozen meat and vegetables [108], raw meat [114], alcoholic beverages [33, 115], tomato paste [101], confectionery [96], dehydrated food [92] and sanitary hygiene control [60]. In addition, some researchers [107] wrote about a possible modification of the method for identifying and counting certain groups of microorganisms.

In conclusion, DEFT is a very fast method of counting the total number of viable microorganisms in raw foods, which has been successfully used in industry. Its disadvantages include the lack of selectivity and the inability to give an accurate estimate of the number of viable microorganisms in processed foods. The first problem can be solved by applying short stages of selective growth or antibodies with fluorescent labeling, but such solutions require time and money. The problem of treated products can only be solved by examining other staining systems that mark viable cells; [15] showed the fruitfulness of this approach and the promise of the method for the production and introduction of fluorescent dyes for viable microorganisms. Nevertheless, at present, the high labor intensity and low productivity of the DEFT method limits its use in the food industry.

Flow Cytometry

Flow cytometry is a method based on the rapid measurement of cells as they pass through the fluid flow past the reading point [28]. The studied cells are injected into the center of the fluid flow (carrier fluid). This causes them to pass one by one on the sensor and makes it possible to register each particle, rather than to obtain average values ​​for the entire population. At the point of reading there is a beam of light (ultraviolet or laser), aimed at a controlled flow, and one or more detectors measuring light scattering or fluorescence when particles pass under a beam of light. The expansion of flow cytometry in research laboratories is mainly due to the development of robust instruments and multiple staining systems. Dyes that can be used with flow cytometry instruments allow for a wide variety of measurements. Fluorescence sensors based on enzyme activity, nucleic acid content, membrane potential and pH were studied; the use of fluorescent dyes combined with antibodies gives the system selectivity.

Flow cytometers were used to study a number of eukaryotic and prokaryotic organisms. Working with eukaryotes included the study of pathogenic amoeba [89] and yeast culture [64], and bacterial studies included the study of Escherichia coli [122] growth, the counting of bacterial cultures [103] and the determination of Legionella species in water [125] cooling towers.

Flow cytometry methods for the food industry are covered in [130]. [41] investigated the use of antibodies labeled with fluorescent substances to detect Listeria monocytogenes in milk, and encouraging results were obtained. The method used in this work is based on the selective growth of organisms during the day and subsequent staining with polyvalent Listeria antibodies labeled with fluorescein isothiocyanate. Then, the stained cells were passed through a flow cytometer and L. monocytogens was determined. It has been suggested that this system can be used with other types of products. A similar approach was also used in [82] to determine Salmonella typhimurium in dairy products.

[94] investigated the use of an Argus flow cytometer to calculate bacteria in pure cultures and foods. The results obtained with pure cultures showed a good correlation of the counts obtained on the flow cytometer with the counts on the plates to the concentrations of 103 cells per gram, but inconsistent results were obtained for food products. The use of this method for meat samples gave a good match with the counts on the plates and made it possible to count up to 105 cells per gram. The results for milk and pate were worse, and the sensitivity of the system for pate was 106 cells per 1 ml, and cells introduced into the milk were not detected at more than 107 per 1 ml. The low sensitivity of this flow cytometer when measured on products was attributed to the influence of the counting system caused by food residues; the authors suggested that this problem could be solved by applying the methods of separating microorganism cells and food residues.

The most successful flow cytometry methods are applied to food using the Chemunex Chemflow Rjin system for detecting contaminating yeast in dairy and fruit products [6]. The technique used when working with this system requires incubation of the product for 16-20 h followed by centrifugation to separate and concentrate the cells. A dye is then added and the sample is passed through a flow cytometer for analysis. Evaluation of the system [97] on the example of non-alcoholic beverages, fermented with yeast, showed that it is reliable and easy to use. The data of the cytometer well coincided with the data of the DEFT method, but the author did not give the results of comparing the data of the system with the results of calculations on the plates.

In the Chemflow system studies described in [6], various dairy and fruit products, fermented with yeast, were used. The results showed that the yeast could be detected in dairy products in a day if there were only 1 cells per 25 gram. A similar sensitivity was also obtained in fruit juices, but to achieve it, 48 h was required. The system showed its reliability and convenience in operation and is currently adapted to detect both yeast and bacterial cells; There are also options for its use for biomass fermenter and counting the total microflora in vegetables. The Chemflow system has been fully tested under production conditions when analyzing fermented milk products [43]. The authors of this work point to a very good agreement between the counting results on the cytometer and on the plate (r = 0,98), and the results are obtained in 24 hours, which means a saving of three days compared with the classical methods.

In conclusion, flow cytometry can provide a fast and sensitive method for the rapid counting of microorganisms. Successful operation of the system depends on the design and application

a) the relevant systems and staining

b) procedures for the separation of microorganisms from food residues, which otherwise interfere with the operation of the detection system. In the future, a flow cytometer equipped with a number of photosensor systems could allow for the analysis of samples simultaneously for many parameters, greatly simplifying the test modes.

Solid-Phase Cytometry

A relatively new version of cytometry was developed by Chemunex (r. Mezon-Alfort, France) based on solid-phase cytometry. In this embodiment, the samples pass through a membrane filter that retains the infectious microorganisms. Then, in order to mark the metabolically active cells of the microorganisms, a fluorescent dye is applied to the filter. After staining, the membrane is transferred to a Chemscan RDI instrument, which scans the entire membrane with a laser, counting fluorescent cells. The procedure takes about 90 min and allows you to identify individual cells in the sample on the filter. The Chemscan RDI solid phase cytometry system is a powerful tool for quickly counting small quantities of microorganisms. It is ideally suited for analyzing water or other clean filtrating liquids, and the use of specific labeling methods can be used to selectively identify certain microorganisms. Foods with large particles may, however, interfere with the analysis, since microorganisms must be separated from the food material before filtering and analysis.

Immunological methods. Antibodies and antigens

Immunological methods are based on the specific binding reaction that occurs between the antibody and the corresponding antigen. Antibodies are protein molecules that are produced by white blood cells in response to contact with a substance that causes an immune response. The region to which an antibody attaches to a molecule is known as an antigen. The antigens used in immunochemical methods are of two types: the first occurs when the substance being detected has a low molecular weight and therefore does not stimulate the immune response — such substances are called haptens (defective antigens), and they must cause be bound to a larger carrier molecule. The second type of antigen is immunogenic and can itself trigger an immune response.

Two types of antibodies can be used in immunological tests: monoclonal and polyclonal antibodies. Polyclonal antibodies are formed by using large molecules (such as proteins or whole bacterial cells) to stimulate the immune response. The presence of a large number of antigenic sites leads to the formation of many different antibodies in response to a molecule or cell. Monoclonal antibodies are formed using cell cultures from a single leukocyte; this means that they target one antigenic site. Antigen binding is very specific, and therefore immunological methods can be used to detect certain microorganisms or proteins (for example, toxins). In many cases, when using these methods, a label is attached to an antibody so that it will be easily visible upon binding.

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Many types of labels can be used with antibodies, including radioactive labels, fluorescent substances, luminous substances, and enzymes; in addition, agglutination reactions can be used to detect the binding of an antibody to an antigen.

Radioisotopes are widely used as labels mainly because of the high sensitivity that can be achieved with their help. Nevertheless, they have some disadvantages, the main of which is the potential danger of reagents. This prevents their use anywhere, except in special laboratories, and naturally, the possibility of their use in the food industry raises great doubts.

Fluorescent labels are widely used to study microorganisms. The most commonly used reagent is fluorescein, but others are also used, such as rhodamine and umbelliferone. The simplest case of using fluorescent antibodies is microscopy. Recent advances in this area are the use of flow cytometry for multiparameter flow analysis of stained preparations and the development of an immunofluorescence analysis method using an immobilized enzyme (ELIFA), some of which have been automated.

Luminous (fluorescent) labels were investigated as an alternative to potentially dangerous radioactive [75]. Labels can be both chemiluminescent and bioluminescent, and their advantage compared to radioactive is the ease of use and measurements that can be performed on simple equipment with similar sensitivity [109]. A number of research papers have successfully used immunoluminometric analysis [81], but so far there is no evidence of its introduction into production.

Antibodies are used to detect antigens in precipitation and agglutination reactions. Such analyzes are usually more difficult to quantify than other types of immunological analysis, and therefore they are used for qualitative assessment. Analyzes are carried out simply and quickly, requiring minimal hardware.

A number of precipitation reactions have already been successfully introduced into the food industry. These methods were used mainly to confirm the identity of microorganisms, and not to identify the specified microorganisms. The analysis time using these methods is relatively small, they are simple to use and usually they do not require special equipment, which makes them ideal for laboratories performing standard tests.

Several Latex Agglutination Kits are available for the determination of Salmonella in food. These include the Oxoid Salmonella Latex Kit (Oxoid), designed for use with the Oxoid Rapid Salmonella Test Kit [61]; Micro Screen Salmonella Latex Slide Agglutination Test Kit (Mercia Diagnostics Ltd.); Wellcolex Color Salmonella Test kit (Wellcome Diagnostics) [53,54] and Spectate Salmonella test (Rhone Poulenc Diagnostics Ltd.) [29]. The last two kits use mixtures of colored latex particles, which allow not only to define, but also to reveal the serological groups of Salmonella. Latex agglutination test kits are also available for Campylobacter (Microscreen, Mercia Diagnostics), Staphyloccocus aureus (Staphaurex, Wellcome Diagnostics), Shigella (Wellcolex Color Shigella Test, Wellcome Diagnostics) and Escherichia coli 0157: # 7 {Oxoid). Agglutination tests have also been developed for the determination of microbial toxins, for example, the Oxoid Staphylococcal Enterotoxin Reverse Passive Latex Agglutination Test [5,110].

Enzyme immunoassay has been extensively researched as a method for rapid detection of foodborne microorganisms. The advantage of such an analysis is its specificity, achieved through the use of specific antibodies in combination with colored or fluorescent endpoints, which are easy to detect visually or with a spectrophotometer. Most commercially available immunoassay kits use the antibody sandwich technique to first capture and then identify specific microbial cells or toxins. The kits are supplied with two types of antibodies: immobilized and conjugated. The immobilized antibody is attached to a solid support such as a microtiter plate. An fortified food sample can be added to the well and antigens from any detectable cell present will bind to the antibodies. The well is washed to remove food debris and unbound microorganisms. Enzyme-conjugated antibodies can then be added to the well. They bind to the target cells, forming a "sandwich" of antibodies. Unbound antibodies can then be washed out of the well and an enzyme substrate added. Any enzyme present converts the colorless substrate into a colored product. A typical enzymatic microplate immunoassay takes 2-3 hours and shows the presence of putative bacterial cells. Sample analysis results showing a positive result should always be biochemically or serologically confirmed.

A number of enzyme immunoassay kits are available to detect Listeria, Salmonella, Escherichia coli 0157, Staphylococcal enterotoxins and Bacillus diarrhoeal toxin in food samples. The sensitivity of these systems is approximately 106 cells / ml, therefore an appropriate sample enrichment procedure must be followed prior to analysis. Thus, the results can be obtained after 2-3 days, and not after 3-5, as with traditional tests.

In recent years, a number of automated immunoassay options have been developed, making this accelerated method even less time consuming. Enzyme immunoassay automation is done in different ways - a number of manufacturers sell instruments that simply automate standard ELIS A (solid phase immunosorbent assay) methods on a microplate. These instruments contain reagent containers and use a robotic automatic pipette that dispenses the various reagents required in the correct sequence. Automation of analysis is completed by automatic rinsing and reading, reducing labor intensity. There are at least two manufacturers that produce original systems with kits for automated immunoassay.

The Vidas System (bioMerieux, Basingstoke, UK) uses a test strip containing all the reagents required for ELIS A. The first well of the strip is inoculated with an enriched food sample and placed into the Vidas instrument along with an immobilized antibody coated pipette tip. The instrument then uses the pipette tip to transfer the sample to be analyzed to other wells on the strip containing the various reagents required for the assay. All movements are fully automated, as well as obtaining the final test results. Vidas ELISA assays can be performed on a variety of microorganisms including Salmonella, Listeria, Listeria monocytogenes, E. coli 0157, Campylobacter, and staphylococcal enterotoxin. Based on the comparison [20,22], it was noted that the results of this automated analysis are at least equivalent to traditional test methods.

The EIAFOSS system (Foss Electiic, Denmark) is a fully automated system for solid-phase immunosorbent analysis. In this system, all reagents are automatically transferred into sample tubes in which all reactions take place. The EIAFOSS instrument implements an original procedure using magnetic beads coated with antibodies as a solid phase. During the analysis, these beads are immobilized using a magnet placed under the sample tube. EIAFOSS assay kits are available for Salmonella, Listeria, E. coli 0157 and Campylobacter, and studies have supported their good performance [68].

The latest commercially available version of the immunoassay is perhaps the simplest. Immunochromatography works with a dipstick consisting of an absorbent filler that contains colored particles coated with antibodies for a specific microorganism. The particles are on the bottom of the probe, and when it is dipped into broth for microbiological enrichment, they move up the filler (the liquid moves under the action of capillary forces). A number of immobilized specific antibodies are located at a certain point on the filler. In the presence of a detected microorganism, it binds to colored particles. This conjugate of cells and particles moves up the probe under the action of capillary forces until it encounters immobilized antibodies to which it "sticks". The accumulated colored particles form a clearly visible colored line indicating a positive test result.

A number of commercial kits are based on this procedure, including the Oxoid Listeria Rapid Test [69] and the Celsis Lumac Pathstik [70], which have shown good results. Immunochromatographic methods, like other methods of immunological analysis, require enrichment, but they do not require special instruments or equipment, and after contact of the probe with the sample, it takes only a few minutes to see a positive or negative result.

Brief conclusions

Immunological methods have already been comprehensively studied and implemented. Currently, there are a number of systems that make it possible to quickly identify the specific microorganisms for which they are intended. Numerous tests of industrially developed devices for immunological analysis have shown that their results generally correspond well to those obtained by traditional microbiological methods. In particular, enzyme immunoassay appears to be a simple way to reduce the assay time by 1–2 days. Automation or miniaturization of kits for such analysis has reduced the time it takes for the operator to complete the test and greatly simplified manual procedures.

The main problem with immunological assay systems is their low sensitivity. The minimum number of microorganisms required for an enzyme immunoassay system to obtain a positive result is approximately 105 / ml. If a microbiologist needs to determine the presence or absence of any microorganism in 25 g of a food product, an enrichment stage is always required, the use of which increases the total duration of the analysis by 1-2 days.

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