Microbiological Analysis Of Food And Water
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FDA's Bacteriological Analytical Manual (BAM) presents the agency's preferred laboratory procedures for microbiological analyses of foods and cosmetics. AOAC International published previous editions of this manual in a loose-leaf notebook format, and, more recently, on CD-ROM. This online BAM is now available to the public. Some changes have been made to methods since the previous version. A listing of chapters updated since the last hard-copy version (Edition 8, Revision A /1998) can be found in About the Bacteriological Analytical Manual. The members of the BAM Council are listed below. In addition recent changes for most Chapters are documented in a brief Revision History at the beginning of the Method. There is also e-mail contact information for each Chapter. Chapter numbers have been retained from the previous version. However, for this Table of Contents, chapters have been grouped by category. Please send comments to Karen Jinneman.
Innovations in methods for the microbiological analysis of food continue to appear at a rapid pace. Edition 8 (1995) of the Bacteriological Analytical Manual (BAM-8) contained numerous refinements of procedures and updates of references from the 1992 edition. The list of commercially available test kits and the discussion of rapid methods in Appendix 1 were thoroughly revised. Three chapters were added: the use of reverse transcription (RT) and the polymerase chain reaction (PCR) to detect and quantify contamination of shellfish with hepatitis A virus (Chapter 26); new procedures for the alkaline phosphatase test to determine whether dairy foods were prepared with pasteurized milk (Chapter 27); and the use of PCR to detect toxigenic Vibrio cholerae in foods (Chapter 28). For this printing (BAM - 8A), the following has been revised or added: Campylobacter (Chapter 7), Yeast and Molds (Chapter 18), Cyclospora [Chapter 19 (Parasites)] and Staphylococcus enterotoxins (Chapter13). In addition, there are updated tables in Appendix 1 on Rapid Methods and revised and corrected tables in Appendix 2 on MPN. Appendix 3 reflects changes in media and corrects errors in the 8th Edition. A table summarizing changes from BAM-8 to BAM-8A is included.
The methods described in Chapters 1 to 28 are those preferred by FDA for the microbiological analysis of foods, drinks, and cosmetics as well as for their containers, contact materials, and the production environment. This is not necessarily the case for the rapid methods listed in Appendix 1: this appendix is a listing of different kits that are commercially available. These methods have not necessarily been evaluated by FDA, and listing of a method in this appendix does not constitute a recommendation.
The most effective way to check water supplies for faecal contamination is microbiological analysis, and a range of test methods designed for that purpose has been developed for the water industry. Instead of carrying out separate tests for each of the potential pathogens, viruses, or parasites that might be in the water, microbiologists test for indicator organisms that are always present when enteric pathogens and viruses are.
Membrane filtration: A typical MF method for water analysis is performed by passing a known volume of water through a sterile membrane filter with a pore size small enough to retain bacterial cells (typically 0.45µm). The filter is then transferred aseptically to the surface of an agar plate, or an absorbent pad saturated with a suitable selective medium and incubated. Colonies are allowed to develop on the surface of the filter and can be counted and examined directly. MF methods are quick and easy to perform, require little incubator space, and can handle large volumes of water if needed. Over the last 30 years, they have become the preferred methods for the microbiological examination of water for indicator organisms. There are several official published methods based on MF, notably a series of ISO methods, such as ISO 9308-1 for coliforms and E. coli and ISO 7899-2 for enterococci.
In addition to tests for indicator organisms and certain specific pathogens, non-selective colony counts are also routinely carried out to determine the population of heterotrophic bacteria present. Counts at two temperatures (22oC and 37oC) are typically performed to provide information on the general microbiological population of the water and detect sudden changes in water quality. Counts at 37oC have been used to indicate faecal contamination in the past, but this is not generally considered to be reliable.
Rapid methods: Although most official methods for microbiological water analysis still rely on traditional culture methods and MF methods, the time is taken to obtain results, typically 24-48 hours, has focused attention on alternative rapid methods. Combining MF with QPCR detection and enumeration is a particularly rapid and effective means of analysing water samples. The main disadvantage of this method is that it may detect non-viable cells and overestimate the population, but it seems likely that QPCR-based methods will become increasingly important in water microbiology, leading to the development of commercial products similar to those already used for food analysis.
In recent years, water microbiologists have become increasingly aware of the importance of biofilms for microbiological populations in water systems. Biofilms are now recognised as complex microbial communities, which form on surfaces. Biofilms typically consist of a variety of microbial cells, potentially including pathogens, within a matrix composed of exopolysaccharides (EPS) secreted by certain bacterial species. Biofilms develop over time, becoming more complex and extensive, and can protect individual bacterial cells from chlorine and other antimicrobial compounds in water.
Biofilms are also notoriously difficult to remove from surfaces and can act as a sporadic source of microbial contamination as bacterial cells are sloughed off from the matrix into the surrounding water. It is now recognised that most of the bacteria in drinking water distribution systems are present within biofilms rather than free-living in the water itself. Pathogens isolated from within biofilms include Salmonella Typhimurium, Campylobacter sp., Pseudomonas aeruginosa and Aeromonas hydrophila. Legionella pneumophila is now known to be a major pathogen in residential water systems, due to its proclivity to form biofilms on water distribution pipes and also within cells of microbial parasites. Biofilms may affect general microbiological water quality, cause objectionable tastes and odours and accelerate corrosion within distribution systems.
The presence of significant biofilm growth may make it difficult to obtain representative water samples and may influence the results of microbiological analysis. High heterotrophic plate counts may be indicative of biofilm formation in distribution systems. In some cases, it may be necessary to sample biofilms directly using swabs or by allowing a film to develop on the surface of removable metal coupons or within specially designed sections of pipework.
A typical MF method for water analysis is performed by passing a known volume of water through a sterile membrane filter with a pore size small enough to retain bacterial cells (typically 0.45µm). The filter is then transferred aseptically to the surface of an agar plate, or an absorbent pad saturated with a suitable selective medium and incubated. Colonies are allowed to develop on the surface of the filter and can be counted and examined directly. MF methods are quick and easy to perform, require little incubator space and can handle very large volumes of water if required.
Over the last 30 years, they have become the preferred methods for the microbiological examination of water for indicator organisms. There are several official published methods based on MF, notably a series of ISO methods, such as ISO 9308-1 for coliforms and E. coli and ISO 7899-2 for enterococci.
The US Environmental Protection Agency (EPA) has published official microbiological methods for water testing. Laboratories routinely testing drinking waters, recreational waters and environmental samples should use the appropriate official method recommended by their local enforcement agency. Laboratories testing water supplies for industrial use, such as food processing, are advised to use the same methods when the water supply is required to be of potable quality.
Disposable single-use sterile filter funnels are also available for convenience. Suppliers include Millipore, Sartorius and Membrane Solutions. The filter unit is connected to a suitable vacuum source to draw the samples through the filter. This may be a vacuum line or a stand-alone pump unit. Compact pump units specifically designed for use with MF methods are now available. An example is the Millipore EZ-Stream unit, which can run the filtered sample straight to drain, thus saving time spent emptying and cleaning the waste sample containers used with traditional laboratory vacuum pumps. A variety of membrane filters are available for different applications, but microbiological water analysis is typically carried out with 47mm diameter mixed cellulose ester-based filters of 0.45µm pore size. The filters are usually marked with a grid to aid colony counting.
Numerous outbreaks of foodborne illness have been linked to the consumption of raw sprouts. Sprout producers have been advised by the Food and Drug Administration to include microbiological testing of spent irrigation water during production as part of an overall strategy to enhance the safety of sprouts. Alfalfa sprouts and irrigation water were analyzed to show the feasibility of using irrigation water for monitoring the microbiological safety of sprouts. Sprouts and water were produced and harvested from both commercial-scale (rotary drum) and consumer-scale (glass jars) equipment. Rapid increases of aerobic mesophiles occurred during the first 24 h of sprouting, with maximum levels achieved after 48 to 72 h. The counts in irrigation water were on average within approximately 1 log of their respective counts in the sprouts. Similar results were obtained for analysis of Escherichia coli O157:H7 in irrigation water and sprouts grown from artificially inoculated seeds. Testing of spent irrigation water indicated the contamination status of alfalfa sprouts grown from seeds associated with outbreaks of Salmonella infection. 59ce067264
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