Chilled and frozen foods

Traditional and accelerated methods of microbiological analysis

Р. П. Бете, Campden and Chorleywood Food Research Association


The identification and determination of the number of microorganisms in a food product or on surfaces that come into contact with it is an integral part of any control and quality assessment system. Microbiological tests of food products can be divided into two types: a) quantitative, in which groups of microorganisms in the sample are counted, and the result is expressed as the number of organisms present per unit mass of the sample, and b) qualitative (“presence / absence”), which are reduced to detecting the presence or absence of a particular microorganism in a known sample mass.

The basis of the methods used to detect microorganisms in food is well known and relies on the introduction of food into a nutrient medium in which microorganisms can be reproduced, which leads to their apparent growth. Such methods are simple, flexible, convenient and usually cheap, but they have two drawbacks: first, the tests are based on the growth of microorganisms in the environment, which can take a long time and leads to a long test duration; secondly, the methods are focused on their implementation manually and therefore time consuming.

In recent years, a large amount of research has been done in the field of accelerated automated microbiological methods (express analysis). The goal of this work was to reduce the duration of the tests (by applying methods other than growing microorganisms to detect and / or counting microorganisms) and to reduce the labor intensity (by maximizing automation). Developed automated rapid methods gained some recognition in the food industry and could form an important tool for controlling the quality of chilled products. The results of express analysis can increase the shelf life of refrigerated products by one to two days compared with the traditional microbiological method. In addition, the presence of accelerated methods of microbiological rapid analysis indicates the presence of real-time control capabilities and their application in the quality assessment system according to the method of AS CP.

 Sample selection

Although this chapter discusses the methods used for food testing, the issue of sampling is particularly important for a microbiologist. No matter how good a particular method is, if the sample is taken incorrectly and is not representative of the batch of the product from which it is taken, the test results do not make sense. It is useful to develop a sampling plan, in which the results are determined on the basis of a series of analyzes, rather than one by one. Currently, microbiologists usually use sampling plans with two or three sets of samples, which indicate the number of individual tested samples from the same batch (along with the current microbiological limits). Such sampling plans are described in detail in [2].

After the sampling plan has been developed, a representative portion of the product should be taken for analysis. To do this, the microbiologist should be sufficiently versed in the product and its microbiology. Many cooled products are not homogeneous mixtures, but consist of layers or parts (a ready-made sandwich is a good example). It must be decided whether a microbiological result is necessary for the whole sandwich (i.e. bread and filling), only for bread or only for filling. Indeed, in some cases it may be necessary to check one part of the mixed filling. After the decision has been taken, a sample can be taken for analysis using an appropriate aseptic method and sterile means of sampling [76]. After the development of the sampling procedure, the microbiologist can be confident that the samples taken are representative samples of the product being tested, and that verification methods can be trusted.

 Traditional microbiological methods

As shown in the introduction to this chapter, traditional microbiological techniques are based on the generally accepted method: food samples are placed in a nutrient medium and maintained there for some time for the growth of microorganisms. The determination of microorganisms or their counting is then carried out by the usual visual assessment of culture growth. These methods are technically simple and relatively cheap because they do not require sophisticated equipment. At the same time, they can be easily adapted to determine the number of microorganisms of various groups.

Before testing, food samples must be converted to liquid form so that they can be mixed with growing medium. This is usually done as follows: the sample is precisely weighed into a sterile vessel and a known volume of sterile solvent is added (the ratio of sample to solvent is usually 1: 10); then the mixture is homogenized using a homogenizer, which crushes the sample, and all the microorganisms get into the solution. The right choice of solvent is important here - if microorganisms in a sample are adversely affected by improperly chosen pH or low osmotic pressure, they may be damaged or die, which will affect the final result of the microbiological test. The solvent must be well buffered at a pH corresponding to the product being tested and be osmotically balanced. When testing certain products (for example, dried ones), which may contain microorganisms that have undergone severe adverse effects before the test, it may take a certain period to restore them so that the cells do not die in its initial phase [39].

Traditional quantitative methods

Counting the number of microorganisms in the samples is usually made by the method of counting on a plate or by the method of the most probable number (MPN). The first of these methods is most widely used, while the second is usually used only for certain microorganisms (for example, Escherichia coli) or their groups (for example, coliforms).

Calculation method on the plate

The method of counting on a plate is based on placing the sample in an agar layer in or on a petri dish. Individual microorganisms or small groups of them occupy a separate place in agar, and after incubation they grow to form separate colonies, the number of which is counted visually. To count the number of different types of microorganisms, you can use different types of agar medium. Using a non-selective nutrient medium that is maintained at 30 ° C under aerobic conditions will give a total score of viable microorganisms, or a mesophilic aerobic score. Changing the incubation conditions to anaerobic, get a total anaerobic score. Changes in temperature will lead to changes in the types of microorganisms capable of growth, demonstrating some flexibility in the traditional approach using agar. If there is a requirement to determine in the sample the number of microorganisms of a certain type, then to make possible the growth of only the desired type of microorganisms, in most cases it is necessary to change the composition of the medium. There are three approaches to creating an environment that allow you to create special environments: elective, selective, and differential procedures.

Elective procedures are those in which reagents are included in the medium or growth conditions are used that promote the development of the identified microorganisms, but not inhibit the growth of others. Such reagents can serve as sugars, amino acids or other growth factors. Selective procedures include procedures for the inclusion of reagents or the use of growth conditions that suppress the development of microorganisms other than those determined. It should be noted that in many cases, selective factors will also adversely affect the growth of the microorganisms that are detected, but this effect will be less than their effect on other cells. Examples of selective procedures are the introduction of antibiotics into the medium or the use of anaerobic growth conditions. Finally, differential procedures allow to distinguish microorganisms by the reactions that their colonies cause in the environment. An example is the introduction of a pH indicator into the medium to distinguish the microorganisms that form the acid. In most cases, the environment will use a system with an integrated approach, containing elective, selective and differential components, so that the user can identify and count the identified microorganism.

The number of the currently available types of agar is too high, even to just listing them. Their characteristics are to be found in reference manuals firms producing these environments (eg, Oxoid, LabM, Difco, Merck).

MPN method

The second of the counting procedures mentioned above is the MPN method, which allows you to estimate the number of viable microorganisms in a sample based on a statistical method. Evaluation is obtained by preparing tenfold dilutions of the sample and transferring the obtained samples of each dilution into three (usually) tubes of the nutrient medium. These tubes are incubated, and those in which there is any growth (turbidity) are recorded and compared with the standard results table [2], which indicates the level of infection of the product.

This method is used only for certain types of tests, since it is more laborious and requires more materials than the method of counting on the plate. In addition, the boundaries of the confidence interval are large, and thus, this method is usually less accurate than the plate counting method.

Traditional qualitative methods

Qualitative procedures are used when it is not required to know the number of microorganisms in the sample, but only to find out if they are present or absent. Typically, such methods are used to identify potentially pathogenic microorganisms, such as Salmonella, Listeria, Yersinia and Campylobacter. To perform the analysis, it is necessary to homogenize an accurately weighed sample (usually 25 g) in the primary nutrient broth and maintain for a specified time at a given temperature. In some cases, the sample after the primary broth may require transfer to the secondary nutrient broth and additional exposure (incubation). The resulting product is usually applied to a selective agar plate, on which growth of the detected microorganism is possible. A long growing procedure is used because the sample may contain very few detectable microorganisms and a large number of “background”. In addition, in the processed products, the detected microorganisms can be in a damaged state, and therefore the methods of cultivation make it possible to repair damaged cells and their subsequent selective cultivation in the presence of a large number of competing microorganisms.

The identified microorganism in the broth culture is usually invisible, so the broth must be applied on a selective / differential agar plate. Then microorganisms can be detected by the appearance of their colonies. Formation of colonies typical for certain microorganisms on agar is described as putative colonies. To confirm that these colonies are composed of microorganisms to be determined, additional biochemical and serological determinations are usually carried out on pure cultures of the microorganism. This usually requires that, to ensure the purity of the colony from the plates of the primary selection were re-transferred. Purified colonies are then tested biochemically, growing them in an environment that indicates whether the microorganism produces certain enzymes or uses certain sugars.

Currently, a number of firms are selling miniature systems for biochemical testing, allowing microbiologists to quickly and easily perform rapid biochemical tests or automated tests. Serological tests are performed on pure cultures of some isolated microorganisms (for example, Salmonella) using commercially available antisera.

Express methods and automated methods

The general interest in new microbiological methods is partly determined by the increased volume of food production. This leads to:

  •  increase the volume of stored samples to produce a positive outcome (reduced analysis time would reduce storage costs);
  •  an increase in the number of samples analyzed in the laboratory (the only possibility of their processing is an increase in the size of the laboratories and their staff, as well as the use of more accelerated automated methods);
  •  longer shelf life chilled products (reduction of analysis time could accelerate output and thereby increase shelf life);
  •  increased application of HACCP procedures (express methods can be applied in quality control procedures according to the HACCP method).

There are several methods called “express methods”, and most of them are very different from each other and from the traditional procedures that they replace. These methods can be divided into quantitative and qualitative tests, and the first give the value of the number of microorganisms in the sample, and the second indicate only their presence or absence. In laboratories where the use of rapid methods for standard (routine) tests is being considered, one should carefully determine one’s own requirements before purchasing an accelerated analysis system. Each new method is unique and gives a slightly different result, it has certain time characteristics, level of automation and performance. In addition, some methods do not work well with certain types of products or cannot identify certain microorganisms or groups that need to be identified. All these points must be considered before making a decision on the inclusion of one or another method in the laboratory's arsenal. It is also important that personnel using new methods understand the principles that underlie them, and therefore, with clearly incorrect results, could discover the reason for their receipt.

Electrical methods

Quantification of microorganisms in a solution with two electric methods could be achieved, one of which determines the number and size of the particles and the other controls their metabolic activity.

particle counting

Counting and determining particle size can be performed on the basis of the “Coulter” principle using the Coulter Counter (from Coulter Electrics, Luton, UK). The method is based on the transmission of electric current between two electrodes placed on the sides of the partition with a small hole. When particles or cells suspended in an electrolyte pass through a hole, they displace an electrolyte volume equal to them in volume, causing a drop in electrical conductivity with a constant current that depends on the size of the cell. These changes in conductivity are determined by the device and can be converted into a sequence of voltage pulses, with the amplitude of each pulse proportional to the volume of the particle, and the number of pulses corresponds to the number of particles.

This method is widely used in research laboratories for experiments that require determining the size of cells or their distribution, as well as application in clinical microbiology, which requires the determination of bacteria [1]. In food microbiology, this method, however, has so far been little applied. There are reports on the determination of the number of cells in milk [49] and the definition of yeast in beer [83], but information about other studies is clearly not enough. Any use of particle counting in food microbiology would probably be limited to inviscid liquid samples or liquids free from suspended particles, since a very small amount of the sample can cause significant distortion of the results and clogging of the hole.

Metabolic Activity

[123] was the first to report the use of electrical measurements to control the growth of microorganisms. The author used conductivity measurements to control blood decomposition, and concluded that electrical changes caused ions produced by bacterial decomposition of blood components. After this first report, the use of electrical measurements to control the growth of microorganisms was studied by a number of researchers, and their work was largely successful. However, this method is not widespread until reliable instruments capable of controlling electrical changes in cultures of microorganisms are available.

Four instruments are currently available for detecting microorganisms using electrical measurements. The Malthus System (IDG, Bury, UK), based on [105], monitors conductivity changes in the growing medium, similar to the Rabit System {Don Whitley Scientific, Yorkshire). Devices Bactometer (bioMerieux, Basingstoke, UK) and Batrac (SyLab, Purkersdorf, Austria) [6] can monitor both active conductivity and capacitance. All of these devices have the same basic components:

A) an incubator system for storing samples at a constant temperature during the test;

b) a control unit that measures the active conductivity and / or capacitance of each cell at regular intervals (usually every 6 minutes);

c) a computerized data processing system that presents the results in an easy-to-use format.

The determination of the growth of microorganisms using electrical systems is based on the measurement of ionic changes occurring in the environment due to the metabolism of microorganisms. The changes caused by the metabolism of microorganisms and the electrochemical processes used in these systems are described in some detail in the literature [23, 45, 46]. The principle is that with the growth and metabolism of bacteria, the conductivity of the medium increases. Electrical changes caused by small numbers of bacteria cannot be detected using current instruments. In order for changes to be registered, approximately 1 microorganisms must be present in 106 ml. This value is known as the detection limit, and the time it takes to reach this point is called the detection time.

When using electrical systems to determine the number of microorganisms in food, the sample must first be homogenized. The homogenized sample is placed in the cell or tube with the growth medium located in the device and the control unit of the incubation chamber or thermostat is connected to it. The electrical properties of the growth medium are recorded during the incubation period. A sample container is usually a glass or plastic test tube or cell that holds two electrodes. The tube is filled with a suitable microbial growth medium and a homogenized food sample is added to the tube. Electrical changes occurring in the growth medium during the metabolism of microorganisms are monitored using electrodes and recorded by the device.

During the growth and metabolism of microorganisms, new substances are formed in the environment. Usually, uncharged or weakly charged substrates are converted into highly charged final products [44], increasing the active conductivity of the medium. The growth of some microorganisms (for example, yeast) does not lead to a significant increase in conductivity, which is probably due to the fact that these microorganisms do not produce ionized metabolites, and this can lead to a decrease in conductivity during growth.

When using an impedance meter, the electrical resistance of the growth medium is recorded automatically during the incubation period at regular intervals (eg 6 min). When a change in the monitored electrical parameter is detected, the elapsed time since the start of the test is calculated by the computer and is usually displayed as the detection time. The complete curve of changes in the electrical parameter over time (see Fig. 8.1) is similar to the curve of bacterial growth, which has a sigmoidal shape with three sections:

a) inactive area, where all electrical changes lie below the detection threshold of the device;

B) active region where rapid electrical changes occur and

C) a stationary region or a region of decay located immediately after the active region and indicating a deceleration of electrical changes.

The electrical response curve should not be regarded as similar to the growth curve of microorganisms. It is believed [45] that the lag phase and the logarithmic phase of the growth of microorganisms fall on the inactive and active regions of the electrical response curve, up to and beyond the detection threshold of the device. The logarithmic and stationary phases of bacterial growth correspond to the active region and the decay region of the electrical response curves.

When using time-of-detection data from electrical instruments, a calibration must be performed to assess the microbiological quality of a product sample. Calibration consists of testing samples with a traditional plate test and an electrical test. Results are presented graphically, with the traditional result on the y-axis and the detection time on the x-axis (see Figure 8.2). The result is a negative curve withThe active conductivity curve obtained with the growth of bacteria in a favorable environment

Fig. 8.1. The active conductivity curve obtained with the growth of bacteria in a favorable environmentCalibration curve showing the change in the detection time for conductivity and the total number of viable microorganisms

Fig. 8.2. Calibration curve showing the change in the detection time for conductivity and the total number of viable microorganisms

data covering 4-5 logarithmic cycles of microorganisms with a correlation coefficient of more than 0,85 [45]. Calibrations must be performed for each type of sample tested using electrical methods; different samples will contain different types of microflora with different growth rates. If the calibration is incorrect, it can significantly affect the detection time and lead to incorrect results.

So far, we have considered the use of electrical devices to estimate the total abundance of microorganisms. These systems, however, are based on the use of growing media, and therefore allow, using specially selected media, to develop methods for assessing the abundance or detection of certain microorganisms or their groups. There is enough work devoted to electrical measurements to detect / quantify specific microorganisms. Thus, Enterobacteriaceae were studied in [34,95, 7], Pseudomonas - [132], Yersinia enterocolitica - [31], yeast - [42], E. coli - [24], and Campylobacter - [XNUMX].

In the future, the number of identified microorganism species will undoubtedly increase. A large amount of research is underway on media for the detection of Listeria, after which media for other microorganisms will begin to emerge.

Most of the eclectic methods described above are based on the use of contact measurement, that is, electrical changes are monitored using electrodes immersed in the cultivation medium. Some authors [93] pointed out the potential of contactless conductometry for the determination of microorganisms. In this case, the growth medium is located in one compartment of the cell, and the electrode in another. The liquid surrounding the electrode absorbs the gas (for example, for carbon dioxide, this is potassium hydroxide). Placed on the growing medium

sample, and gas is released as the microorganisms grow. This gas is absorbed by the liquid surrounding the electrode, causing a detectable change in conductivity.

This method solves the problem of microorganisms causing only a small change in conductivity in traditional contact conductometry cells. Such microorganisms (for example, many types of yeast) are very difficult to detect using traditional methods of direct conductometry, but when using non-contact conductometric control, the determination becomes quite simple [10]. The development of indirect methods in the future could significantly improve the ability of electrical systems to detect microorganisms that cause small electrical changes in contact systems, facilitating the application of this method in the food industry.

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