Microbiological and aseptic testing plays a significant role in quality assurance. It is not a guarantee of product safety; however, it is one component of an overall food safety system. Before microbiological testing is initiated, prerequisite programs must be in place.
These should include programs appropriate to the specific operation, such as Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP), Sanitation Practices, Hazard Analysis Critical Control Point (HACCP), Traceability, and Recall Management.
It helps to ensure the biological stability of the products. This is imperative because only a few microbes are all it takes to spoil large quantities of foods.
Quality control must be adopted in all food processes to ensure food is free from any trace of contamination. The microbiological assessment of food helps in the classification of microbiological quality into one of the following three classes:
1. Satisfactory: Test results indicating good microbiological quality.
2. Borderline: Test results that are not unsatisfactory but are also not satisfactory, are on the upper limit of acceptability, and which indicate the potential for development of public health problems and of unacceptable risk.
3. Unsatisfactory: Test results that indicate investigating reasons for high count may be considered. For hygiene indicator organisms, test results that require remedial action. For pathogens, test results at levels that indicate a product is potentially injurious to health and/or unfit for human consumption and require immediate remedial action.
Foodborne diseases are an important cause of morbidity and mortality because millions of people fall ill and many die after ingesting food unfit for consumption. They are caused by several microorganisms, including Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Salmonella spp, and pathogenic Escherichia coli, which cause various consumer disorders.
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Laboratory Methods for Assessing Microbiological Status of Beverages
For quality, safety of consumers, and longer shelf life of foods and beverages, they must meet certain microbiological criteria deemed safe. Because safety is very important, quality assurance cannot be limited to inspection of the final product alone; instead, continuous inspection of incoming raw materials and in-process quality control tests must be performed throughout production.
Although there is clearly a place for the direct examination of beverages for microorganisms, a full microbiological examination usually requires that individual viable cells are encouraged to multiply in broth or culture media. Contaminating indicator organisms grow in the media.
By ensuring optimal growth in the elective medium for one organism, it is desirable that conditions are sub-optimal, or even inhibitory, to others. The methods of assessment include:
Enumeration Methods of Counting Microbial Populations
Enumeration methods of microbiological assessment involve different direct cell counts using direct or cell counter. These techniques are particularly useful for determining the total numbers of microbes, including those that cannot be grown in culture. Unfortunately, they generally do not distinguish between living and dead cells in the specimen.
1. Microscopic Count
The microscopic count is a direct enumeration method. It is one of the most rapid methods of determining the cell concentration in a suspension. Here, a liquid specimen is added to a special glass slide designed specifically for counting cells.
The slide has a thin chamber that holds a known volume of liquid atop a microscopic grid. The contents of the chamber can be viewed under the light microscope, so the number of cells in a given volume can be counted precisely. At least 10 million bacteria (10^7) per milliliter are usually required for enough cells to be seen in the microscope field.
2. Use of Cell-Counting Instruments
A Coulter counter is an electronic instrument that counts cells in a suspension as they pass single file through a narrow channel. The suspending liquid must be an electrically conducting fluid, because the machine counts the brief changes in resistance that occur when non-conducting particles such as bacteria pass.
A flow cytometer is another cell counting instrument that is very useful. It is similar in principle to a Coulter counter except that it measures light scattered by cells as they pass a laser. The instrument can be used to count either total cells or a specific subset that has been stained with a fluorescent dye or tag.
3. Viable Cell Counts
Viable cell counts determine the number of cells capable of multiplying. They are particularly valuable when working with samples such as food and water that contain too few microbes for a direct microscopic count. In addition, by using appropriate selective and differential media, these methods can be used to count the cells of a particular microbial species.
4. Plate Counts
Plate counts measure the number of viable cells in a sample by taking advantage of the fact that an isolated microbial cell on a nutrient agar plate will give rise to one colony. A simple count of the colonies determines how many cells were in the initial sample. Plate counts are generally only done if a sample contains more than 100 organisms/ml.
Otherwise, few if any cells will be transferred to the plates. In these situations, alternative methods give more reliable results. When counting colonies, the ideal number on a plate is between 30 and 300. Numbers outside of that range are more likely to be inaccurate.
Samples usually contain many more cells than that, so they generally must be diluted by a stepwise process called serial dilution. This is done using a sterile liquid called the diluent, often physiological saline (0.85% NaCl in water). Dilutions are normally done in 10-fold increments, making the resulting math relatively simple.
Two techniques can be used to plate samples spread-plate and pour-plate. In the spread-plate method, 0.1 to 0.2 ml of the diluted sample is transferred onto a plate of a solidified agar medium. It is then spread over the surface of the agar with a sterilized bent glass rod that resembles a miniature hockey stick.
In the pour-plate method, 0.1 to 1.0 ml of the diluted sample is transferred to a sterile Petri dish and then overlaid with a melted agar medium cooled to 50°C. At this temperature, agar is still liquid. The dish is then gently swirled to mix the microbial cells with the liquid agar.
When the agar hardens, the individual cells become fixed in place; they form colonies when incubated. Colonies that form on the surface will be larger than those embedded in the medium.
In both methods, the plates are incubated and then the number of colonies is counted. From that number, the concentration of colony-forming units (CFUs) in the sample can be determined.
This measure of viable cells accounts for the fact that microbial cells often attach to one another and then grow to form a single colony. When calculating CFUs, three things must be considered: the number of colonies, the amount the sample was diluted before being plated, and the volume plated.
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Membrane Filtration: Concentrating Microbes for Accurate Counting
The membrane filtration technique is used for liquid samples that contain relatively few cells, as might occur in dilute environments such as natural waters. This method concentrates the microbes by filtration before they are plated.
A known volume of liquid is passed through a sterile membrane filter that has a pore size small enough to prevent microorganisms from passing through. The filter is then placed on an appropriate agar medium and incubated. The number of colonies that form on the filter indicates the number of cells in the volume filtered.
Most Probable Number (MPN): Statistical Estimation of Microbial Concentration
The most probable number (MPN) is a method for estimating the concentration of cells in a specimen. The procedure uses a series of dilutions to determine the point at which subsequent dilutions receive no cells. To determine the MPN, three sets of three or five tubes containing a growth medium are prepared.
Each set receives a measured amount of a sample such as water, soil, or food. The amount added is determined, in part, by the expected microbial concentration in that sample. What is important is that the second set receives 10-fold less than the first, and the third set 100-fold less. In other words, each set is inoculated with an amount 10-fold less than the previous set.
After incubation, the presence or absence of turbidity or other indication of growth is noted; the results are then compared against an MPN table, which gives a statistical estimate of the cell concentration.
Measuring Biomass: Assessing Microbial Mass in Beverages
In this method of microbiological assessment, instead of measuring the number of cells, the cell mass can be determined.
1. Turbidity: Measuring Microbial Density through Cloudiness
The cloudiness or turbidity of a microbial suspension is proportional to the concentration of cells and is measured with a spectrophotometer. This instrument shines light through a specimen and measures the percentage that reaches a light detector.
That percentage is inversely proportional to the optical density. To use turbidity to estimate cell numbers, a one-time test must be done to determine the correlation between optical density and cell concentration for the specific organism and conditions under study.
Once this correlation has been determined generally using a direct microscopic count or plate count to determine cell concentration the turbidity measurement becomes a rapid and relatively accurate assay. One limitation of using turbidity to measure biomass is that the medium must contain a relatively high concentration of cells to be cloudy.
A solution containing 1 million bacteria (10^6) per ml is still perfectly clear, and if it contains 10 times that amount, it is barely turbid. It is important to remember that although a turbid culture indicates that microbes are present, a clear solution does not guarantee their absence.
2. Total Weight: Measuring Wet and Dry Cell Mass
The total weight of a culture can be used to measure growth, but the method is tedious and time-consuming. Because of this, total weight is usually used to study only filamentous organisms that do not readily separate into the individual cells necessary for a valid plate count.
To measure the wet weight, cells in liquid culture are centrifuged and the liquid supernate removed. The weight of the resulting packed cell mass is proportional to the number of cells in the culture. The dry weight can be determined by heating the centrifuged cells in an oven before weighing them.
Frequently Asked Questions (FAQs)
- Why is microbiological testing important for beverage safety?
Microbiological testing ensures the biological stability of beverages, preventing spoilage and foodborne illnesses by detecting harmful microorganisms like Staphylococcus aureus and Escherichia coli. - What are the three classes of microbiological quality in beverages?
The three classes are Satisfactory (good quality), Borderline (upper limit of acceptability with potential health risks), and Unsatisfactory (requires immediate remedial action due to health hazards). - What is the microscopic count method, and how does it work?
The microscopic count is a direct enumeration method where a liquid specimen is placed on a special glass slide with a grid, viewed under a microscope, and cells in a known volume are counted. - How does the plate count method measure viable cells in beverages?
Plate counts measure viable cells by diluting a sample, plating it on nutrient agar using spread-plate or pour-plate techniques, incubating, and counting colonies to determine colony-forming units (CFUs). - What is the purpose of the membrane filtration technique in beverage testing?
Membrane filtration concentrates microbes from dilute liquid samples by passing them through a filter, which is then incubated on agar to count colonies, suitable for low-microbe environments like water. - How does the Most Probable Number (MPN) method estimate microbial concentration?
The MPN method uses serial dilutions in multiple tubes, incubates them, and compares growth patterns to an MPN table to statistically estimate the concentration of viable cells in the sample. - What are the limitations of using turbidity to measure microbial biomass?
Turbidity requires high cell concentrations to be detectable, and a clear solution does not guarantee the absence of microbes, necessitating prior correlation with cell counts for accuracy. - When is the total weight method used for microbiological assessment?
The total weight method, measuring wet or dry cell mass, is typically used for filamentous organisms that do not separate into individual cells, as it is tedious and less common for other microbes.
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