MICROBIOLOGICAL ANALYSIS | Total Bacterial Count

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MICROBIOLOGICAL ANALYSIS/Total Bacterial Count

Griffiths MW (2000) Nucleic acid-based assays: overview. In: Robinson RK, Batt CA and Patel PD (eds) Encyclopedia of Food Microbiology, pp. 1599–1608. London: Academic Press. Hill WE and Jinneman KC (2000) Principles and application of genetic techniques for detection, identification, and subtyping of food-associated pathogenic microorganisms. In: Lund BM, Baird-Parker TC and Gould GW (eds) The Microbiological Safety and Quality of Food, vol. II, pp. 1813–1851. Gaithersburg, MD: Aspen. McPherson MJ, Møller SG, Beynon R and Howe C (2000) PCR (Basics: From Background to Bench), 1–119, 241–265. New York: Springer-Verlag. O’Connor L and Maher M (2000) Molecular biology in microbiological analysis—DNA-based methods for the detection of food-borne pathogens. In: Robinson RK, Batt CA and Patel PD (eds) Encyclopedia of Food Microbiology, pp. 1475–1481. London: Academic Press. Olsen JE, Aabo S, Hill W et al. (1995) Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. International Journal of Food Microbiology 28: 1–78. Rudi K, Nogva HK, Moen B et al. (2002) Development and application of new nucleic acid-based technologies for microbial community analyses in foods. International Journal of Food Microbiology 78: 171–180. van der Vossen JMBM and Hofstra H (1996) DNA based typing, identification and detection systems for food spoilage microorganisms: development and implementation. International Journal of Food Microbiology 33: 35–49.

Total Bacterial Count J M Jay, University of Nevada, Las Vegas, NV, USA © 2004 Elsevier Ltd. All Rights Reserved.

Introduction Determining the number of bacteria in a meat or meat product is done routinely for a number of reasons, and the reason dictates the type of method that is best to use. Methods for determining total bacterial counts fall into three broad groups, summarized in Table 1. These methods may be used for all meat, poultry or seafood products. For determining bacterial numbers on the surface of meat, cotton or calcium alginate swabs may be used to remove bacteria from a measured surface area such as 5 cm or 10 cm2. After the organisms are removed from the swabs in a dilution blank, bacterial enumeration may be made in the same way as for other samples. Each of these methods is described below in some detail, but a standard reference should be consulted for actual bacterial count determinations.

Direct Microscopic Methods Direct Smear

The best known, and most widely used, of these methods is the Breed slide, which was developed for numbering of microorganisms in raw milk. This method is not useful if the number of bacteria per cm2 is < 10 000. It can be used for meat homogenates following filtration through Whatman no. 1 filter paper (to remove meat particles and lipids). It has been used for certain dried foods such as powdered milk and eggs, and it may be used to estimate numbers of bacteria on the surface of meat cuts and poultry parts, especially where high levels of bacterial contamination are suspected. Direct smear methods provide only estimates of the bacterial load of product, since both living and dead cells are enumerated, and the occurrence of single cells versus clumps is unpredictable. However, since results can be obtained in around 5 minutes, it can be of value as a rapid screening method. The classical Breed slide method employs specially made slides that have 1 cm2 areas marked, or ordinary microscope slides that are placed over templates with 1 cm2 marked areas. Special pipettes are used to deliver 0.01 ml of test sample to the 1 cm2 area, and the sample is spread over this area with the aid of a wire needle. After air drying and heat fixing, the smear is stained with a dye such as methylene blue for 2 minutes or more, followed by rinsing, drying and viewing in a microscope equipped with an oilimmersion objective. Before a direct smear slide is examined, the microscope factor (MF) of the microscope needs to be determined (eqn [1]). The MF represents the number of microscope fields in 1 cm2, 100 mm2. MF =

10 000 3.1416 × r

2

[1]

In eqn [1], r = half of the field diameter. With 10× ocular and an oil-immersion objective with a numerical aperture of at least 1.40, common MFs typically fall between 400 000 and 600 000 in round numbers. The number of microscope fields to be counted depends upon the overall numbers of cells or clumps on the slide. In general, only 10–15 fields may be counted if numbers per cm2 are several millions, while 30–40 fields need to be counted if numbers per cm2 are <500 000 (see standard procedure). Counting Chambers

These methods are quite similar to the direct smear methods except that the liquid samples are placed

MICROBIOLOGICAL ANALYSIS/Total Bacterial Count

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Table 1 Summary of total bacterial count methods (see text for details) Microscopic methods • Direct smear (breed type). The fastest of all count methods – results in around 5 minutes. Both viable and nonviable cells are enumerated. Numbers by these methods are higher than for all others. • Counting chambers. Similar to the above except that special devices are employed for samples. More quantitative than the above. • Direct epifluorescent filter technique (DEFT). Cell numbers are enumerated with a microscope as for the two techniques above, but a fluorescent stain is used. More flexible than other microscope methods in that the cells in liquids can be concentrated. Colony (plate) count methods • Pour plate. This agar plate method has been the most widely used in the past several decades. Only viable and culturable cells are enumerated. Along with the other colony methods, it continues to be the reference method. • Surface plate. Specimens are applied directly onto the surface of prepoured and dried agar plates. Especially valuable for strict aerobes and psychrotrophs, and when selective culture media are used. • Dry films. These methods, using dry, rehydratable culture media, are employed in the same way as the surface plating methods. They do not allow colonial features of colonies to be observed. • Microcolony-DEFT. Membranes containing viable cells are placed on plates of culture media to allow cells to form colonies after a short incubation. Microcolonies are enumerated with a microscope. Statistical culture methods • Most probable number (MPN). The 3-tube and 5-tube dilution methods are widely used to make estimates of numbers of viable organisms that can grow in a given culture medium. The MPN is determined by use of special MPN tables. Bacterial counts tend to be higher than those from colony count but lower than from direct microscopic methods. • SimPlate. Consists of a single s pecial plate containing wells and culture medium along with a fluorogenic substrate. Numbers of viable cells are determined by reference to special MPN tables. • Hydrophobic grid membrane filter (HGMF). Special filters that contain 1600 wax grid squares on a single membrane are used to collect cells from liquid suspensions. Membranes are then placed on agar plates containing suitable growth medium. The ISO-GRID method is the best known of the HGMF methods.

into special counting chambers such as the Petroff– Hausser, Helberg or Hawksley chambers. The Petroff–Hausser chamber, which is a modification of the haemocytometer, is described below. As in the direct smear method, both viable and nonviable cells are enumerated. The Petroff–Hausser chamber is ruled with squares. A liquid sample is added to the gridded chamber, which is designed to fill with a total volume of 0.02 mm3. The chamber has an etched grid that is depressed 0.02 mm from the surface of the cover slip; this has an area of 1 mm2 and is divided into a matrix of small squares. After a coverslip is added, the cells in a set number of grid squares are counted. Since the chamber factor is typically 4 × 106 to 2 × 107, this method is not suitable for meats that contain fewer than approx. 107 cells per g or per cm2. Membrane Filter Methods

Unlike the two general microscope methods above, which require large numbers of cells, membrane filter methods can be used for specimens that contain low numbers. These methods combine membrane filtration and microscopy, and are typified by the direct epifluorescent filter technique (DEFT). To carry out DEFT, all reagents must be filtered through 0.22 µm pore membranes. Meat extracts need first to be filtered through Whatman no. 1 filter paper to remove large particles. The filtered extract is added to a filter assembly equipped with a 0.6 µmpore black polycarbonate membrane with the shiny side up. After the added volume is subjected to a vacuum to collect cells on the membrane surface, the

filter is rinsed with about 5 ml of 0.1% Triton X-100. After rinsing, the membrane is overlaid with approx. 2 ml of acridine orange (3,6-bis[dimethylamino] acridinium chloride) and allowed to stain for at least 2 minutes followed by rinsing, first with approx. 2.5 ml of 0.l mol l-1 citrate–NaOH buffer (pH 3.0) and then with about 2.5 ml of ethanol. After drying, the filter mounted on a microscope slide, a coverslip and a drop of immersion oil are added. The number of cells is counted using an epifluorescence microscope equipped for acridine orange. The number of fields to count is determined as follows: 0–10 cells 15 fields 11–25 cells 10 fields 26–50 cells 6 fields 51–75 cells 3 fields 76–100 cells 2 fields The number of cells per ml is obtained by multiplying the average number of cells per field by the membrane filter (MF). Multiply this value by the dilution of the prefiltered sample. Since varying volumes of meat homogenates can be filtered through the membranes, this and related methods can be used on products that contain very low numbers of cells. Both viable and nonviable cells are enumerated. As colony viable count membrane, microcolony-DEFT is described below.

Colony (Plate) Count Methods These methods are the most widely used for determining numbers of bacteria in meats. They are often

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MICROBIOLOGICAL ANALYSIS/Total Bacterial Count

referred to as standard plate count (SPC) or aerobic plate count (APC) methods. Only viable cells are enumerated. The standard APC requires 48–72 h for results, while a psychrotroph count requires up to 10 days. The value of these cell count methods for meats is due to the fact that only viable cells are important for meat preservation and safety. With the appropriate choice of culture media and incubation conditions, colony count methods make it possible to determine the presence and relative numbers of bacteria of particular importance in fresh or processed meats. The Pour Plate Method

This method, and the surface method below, are the reference methods for the enumeration of viable bacteria. Although it has its detractors, it provides information that no other existing method can: it reveals the presence and numbers of bacteria that can reproduce under the conditions that are specified. To execute the APC, one needs sterile dilution pipettes, Petri dishes and dilution tubes or bottles containing sterile diluent consisting of Butterfield’s phosphate buffer (34 g KH2PO4 + 500 ml distilled water with pH adjusted to 7.2 with 1 mol l-1 NaOH; sterilize and store in refrigerator; for use, add 1.25 ml of above stock to 1 litre of distilled water (or use 0.1% peptone water). Other needs include a balance, a stomacher or sterile Waring blender flasks, and sterile plate count (standard methods) agar (PCA). A suitable test sample of minced meat is 25 g, which is homogenized with 225 ml (1:10 dilution) of sterile diluent for 2 min. If 50 g samples are used, add 450 ml of diluent. Although not highly recommended, 11 g samples of meats may be taken and added to 99 ml dilution blanks (1:10 dilution). In this case, the blanks are shaken briskly before plating. When meat, poultry or fish surfaces are sampled with swabs, a common method is to aseptically break off the cotton or calcium alginate swab into a 99 ml blank, followed by brisk shaking to dislodge the bacterial cells. Duplicate or triplicate plates are prepared for serial decimal dilutions of the original sample. Ideally, only 1.0 ml or 0.1 ml volumes are placed in plates. As soon as possible (but not later than 20 min) after placing diluted sample in Petri dishes, molten PCA cooled to approx. 45 °C is poured and mixed well while not allowing agar to get on the Petri dish lid. After the agar has solidified, the Petri dishes are inverted and incubated at 25 to 30 °C for 72 h or 48 h for APC. For a psychrotroph count (mesophiles that can grow at refrigerator temperatures), incubation is at 5–7 °C for 7–10 days. Colonies are enumerated with the aid of a Quebec colony counter with numbers on duplicate or triplicate plates averaged and rounded to two significant digits, and then multiplied by the dilution factor.

The Surface Plate Method

Surface plating can be done either manually or by use of an automated plating device. The manual method requires sterile bent glass rods (‘hockey sticks’) and prepoured PCA plates preferably poured 24–48 h earlier so that the agar surface is dry. The manual plating procedure is essentially the same as for the pour plate except that some 9 ml dilution blanks are needed, and only 0.1 ml of dilutions are plated onto the agar surface, followed by distribution of the inoculum by use of a bent glass rod. The automated surface plating procedure is carried out by use of a commercially available device that inoculates the surface of prepoured plates. The Spiral Plater (Spiral System Instruments, Bethesda, Maryland) dispenses a diluted sample onto a rotating agar plate in an Archimedes spiral, resulting in progressively fewer colonies per length of the spiral from the centre of the plate to the outer edge. Colonies are counted in a specified area of the plate according to instructions from the manufacturer. An automated counting device is available, and the spiral plate method has been determined to give results that are comparable to those obtained by the manual method. The spiral plating device is simple to use, and a large number of samples can be processed in a short time. Sample homogenates that are added must not contain particles that will plug the stylus tip. Among the advantages of surface plating over pour plating is the presence of all bacterial colonies on the surface, which makes for better assessments of colonial features and possible identifications of meatborne bacteria. Strictly aerobic bacteria such as Pseudomonas spp. grow better on the surface, and this method is preferred for psychrotroph counts since some of these organisms are quite heat-sensitive and their numbers may be reduced by the molten agar during pour plating. Surface plating is preferred for the enumeration of certain generic groups of bacteria for which selective and differential culture media are often used. The major disadvantage of surface plating onto a nonselective medium, such as PCA, is the tendency for some colonies to spread over the agar surface and thus obscure smaller colonies and make proper enumeration more difficult. Dry Film Methods

The best-known and most-used of the dry film methods is Petrifilm, a product of the 3M Company (St Paul, MN, USA). This is a rehydratable culture medium that is enclosed by a see-through plastic film and a thicker base that holds the culture medium. A number of culture media have been incorporated into this format, including PCA. The latter formulation

MICROBIOLOGICAL ANALYSIS/Total Bacterial Count

includes triphenyltetrazolium chloride (TTC), which turns the small developing colonies red to aid in their enumeration. For the inoculation of dry films, only 1.0 ml portions can be used. This portion is added to the centre of a well-defined circle under the film cover. With a special device provided by the manufacturer, the inoculum is spread throughout the dehydrated agar resulting in its rehydration. Petrifilm ‘plates’ are incubated in the same way as agar plates. Bacterial colony counts by Petrifilm are comparable to those by the agar pour or surface methods. Spreading growth is minimized and colony enumeration is made simpler by the presence of TTC in the medium. In spite of the smaller size of individual colonies, they can be recovered by use of a straight inoculating needle and transferred to other media for further study. Microcolony-DEFT

This is a variation of DEFT (described above as a direct microscopic method) that is a viable count method. After a meat homogenate has been filtered through a DEFT membrane, the latter is placed on the surface of a poured and dried PCA plate and incubated at 30 °C. Microcolonies will be evident within 4 to 10 h, with Gram-negative bacteria requiring less time than Gram-positives. Since these colonies are too small to be correctly enumerated with the unaided eye, this is best done using low-power magnification under a microscope. Although this method provides the fastest way to determine total viable bacterial numbers, it does not lend itself to routine use because of the high variability that exists among meat and meat products in terms of relative bacterial load and types of bacteria. It can be used as a one-day screening method on a limited number of samples. Perhaps the best use of this method is in the examination of meats for specific groups of bacteria that can be cultured on selective media. For example, as a one-day method to determine the presence and relative numbers of Gramnegative or enteric bacteria, the DEFT membranes can be placed on prepoured and dried plates of MacConkey agar and incubated at 30–35 °C for 4–5 h.

Statistical Culture Methods Each of the three methods described below may be used to determine bacterial numbers, but the numbers determined are not actual counts of cultivable cells: thus are the most probable number (MPN). When determining the presence and relative numbers of certain specific bacteria (such as pathogens) in meats, an

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MPN method may be quite suitable, and this is especially true for the first MPN method described below. Most Probable Number (MPN)

The classical MPN method is a 3-tube or 5-tube dilution method that has been in use for over 50 years to determine coliforms in foods and water. It can be used to make estimates of the number of total viable and cultivable organisms by use of nonselective broth media, or of specific groups for which selective culture media exist. An example of how an MPN test is carried out is illustrated below for determining the numbers of psychrotrophic bacteria in minced beef. For a 3-tube MPN, one needs nine tubes of culture medium for each sample. For psychrotrophic bacteria, a general growth medium is used. Trypticase soy broth (TSB) is one such medium, and it contains glucose. The nine tubes of TSB will each hold 9.0 ml. Sterile dilution blanks and pipettes are needed. For minced beef, the dilution blanks may contain distilled water rather than 0.1% peptone or Butterfield’s phosphate buffer. To homogenize samples, a Waring blender or stomacher is needed. The 3-tube method is carried out as follows under the assumption that the minced beef in question contains a low number of viable bacteria. A 25 g sample is homogenized for 2 min with 225 ml of sterile distilled water. From this 1:10 dilution, add 1.0 ml to each of three 9.0 ml TSB tubes (1:100). From the original homogenate, transfer 1.0 ml to a 9.0 ml blank (1:100), and from this blank, transfer 1.0 ml to a second set of three 9.0 ml TSB tubes (1:1000). To the remaining three 9.0 ml TSB tubes, transfer 1 ml from a 1:1000 blank (1:10 000). Incubate the tubes at 5–7 °C and observe after 5 days for evidence of bacterial growth (turbidity). Record the number of tubes in each dilution series that display growth. Re-incubate the tubes and re-examine after 10 days. The MPN is determined by reference to an MPN table available in Standard Methods (see Further reading). In the example used, if growth occurs within 5 days in any tubes, it represents the presence of stenopsychrotrophic bacteria, which because of their faster growth, are of greater importance in refrigerated meats than the eurypsychrotrophs that are reflected in the 10-day incubation. The 5-day results are of special importance for poultry and fish since these products undergo bacterial spoilage faster than red meats. The example of an MPN test outlined above is given simply to illustrate how this statistical method differs from viable count methods. A 5-tube MPN test (total of 15 tubes) is more accurate than a 3-tube. This method has proved its utility for detecting and enumerating specific groups of bacteria from meats

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MICROBIOLOGICAL ANALYSIS/Total Bacterial Count

and other food products. Although a lot of glassware is required, reading tubes as positive or negative is fairly definitive and this aspect recommends the procedure for use by different laboratories when testing the same products. SimPlate

SimPlate is a product of IDEXX Laboratories (Westbrook, ME, USA), that consists of a plastic plate that contains either 84 or 198 small wells. The plate contains a general growth medium formulation in addition to a fluorogenic substrate that is released by the enzymatic activities of growing bacteria. SimPlate is inoculated with a meat homogenate and, after swirling of the plate to allow inoculum to enter the wells, the excess is poured off and the plate is incubated at the desired temperature for at least 24 h. The plates are read by exposing then to long wave or shortwave UV light and counting the number of wells that fluoresce. The estimated or most probable number of bacteria in the original sample is determined by reference to special MPN tables for this method. Plates with 84 wells are used if relatively low numbers are suspected, while the 198-well plate is used when high numbers of bacteria are anticipated. This method obviates the need for multiple dilutions of the original homogenate. Because of the simplicity of its use, this method is of value in determining the overall load of bacteria that exists on fresh meats. It has an advantage over the classical tube MPN in its requirement for less glassware. Hydrophobic Grid Membrane Filter (HGMF)

This method employs a membrane filter that contains 1600 wax grid squares (simply ‘grids’ hereafter). When liquid homogenates are passed through the filter membrane, ideally a single grid is occupied by an individual cell because of the hydrophobic nature of the waxy line that surrounds each grid, though sometimes several cells will occupy a single grid. After collecting cells, the HGMFs are placed on the surface of a suitable growth medium – PCA in the case of total viable numbers in meats. Following incubation, the grids are inspected under low magnification for the presence of small colonies. After the colonies are counted, the number per g in the original sample is determined by reference to a special MPN table. As few as 10 cells per g or per ml can be detected by this method, and up to 9 × 104 can be enumerated by MPN from a single sample dilution. One of the best known applications of HGMF is the ISO-GRID method. This method consists of a special filtration apparatus that consists of a 5 µm

prefilter to remove particulates from test samples. Before a test sample is applied, 10–15 ml of sterile diluent is passed through the prefilter to wet the HGMF membrane. One ml of the homogenate is now added and drawn through the HGMF by vacuum. After the sample has passed, 10–15 ml of sterile diluent is added to rinse cells from walls of apparatus. With the aid of sterile forceps, the membrane is placed on the surface of agar plates as noted above. For meat samples with low numbers of bacteria, the target is a count of 20–200 per filter. If a membrane contains more than 1520 positive grids, the procedure needs to be repeated with the use of a more dilute homogenate. The counting of HGMF plates has been automated, and this method has been found to give results comparable to those of several other widely used methods.

Important Concerns for Colony Counts and their Meaning Although each of the steps and procedures that are involved in determining bacterial counts in meats can have measurable effects on the final results, the two most important are choice of culture medium and temperature and time of incubation. It is also important to keep in mind the objectives and reasons for determining numbers of bacteria in or on meats, poultry and seafood products. It is assumed that the method chosen is an acceptable one. Culture Medium

The choice of culture medium to be used when determining numbers of bacteria in fresh meats received much debate and discussion in the 1950s. One outcome was the recommendation by a committee of the American Public Health Association (APHA; Recommended Methods for the Microbiological Examination of Foods, 1958) to use ‘total plate count agar’ (also referred to as tryptone–glucose–yeast agar, standard methods agar and plate count agar). Unlike some of the media used earlier, this medium contains 1.0% glucose and does not obscure the counting of small colonies, since it is essentially colourless. It is imperative that culture media for determining viable bacteria in meat, poultry and seafood products contain a simple carbohydrate, and glucose appears to be ideal. Without glucose, the large group of lactic acid bacteria would not be represented in an APC result following 48 h incubation. Also, the simple sugar enables many of the other typical meat-borne bacteria to grow at a much faster rate. The use of a growth medium such as nutrient agar, which does not contain a simple carbohydrate, will lead to undercounts.

MICROBIOLOGICAL ANALYSIS/Indicator Organisms Temperature/Time of Incubation

In its 1958 recommendation for plating medium and temperature/time of incubation for fresh meats, the APHA recommended 21°C for 72 h. Twenty years later, the ICMSF (International Commission on Microbiological Specifications for Foods) recommended 29–31°C for 48 h for foods in general. In spite of these recommendations, a number of published studies indicate the use of 35 °C for 24 h for fresh meats. It is well established that higher numbers of bacteria are found when fresh meats are examined if PCA plates are incubated between 21 and 30 °C for 72–48 h than at higher temperatures for shorter times. A review of microbiological literature for the past 20 years or so reveals the use of incubation temperatures from 20 to 37 °C for fresh meats. The use of the temperature/time combination of 30 °C/48 h is recommended. Since the psychrotrophic bacteria are of paramount importance for the shelf-life of fresh meat, and since these bacteria are defined by their growth at 5–7 °C within 10 days, the use of incubation temperatures at or below 30 °C for 48–72 h is recommended. An incubation temperature of 35 °C for 24 h may be suitable for certain groups of bacteria found in meat, such as coliforms and some human pathogens, but this temperature/time combination is not suitable for determining the APC of fresh meat, poultry or seafood products. Significance and Meaning of Total Bacterial Numbers in Meats

It should be assumed that any or all fresh meat, poultry and seafood products contain bacteria at some level. Thus, the finding of numbers on the order of 103–105per g or per cm2 is of little or no significance in relation to product safety. It is generally accepted that fresh meats will display some offodours when bacterial numbers reach around 107 per g or per cm2 (assuming that proper attention is paid to choice of plating medium and time and temperature of incubation as noted above). For product shelflife, it is especially important that psychrotroph numbers are determined for any food product that is stored at chill or refrigerator temperatures. With regard to the microbial safety of meat products, total bacterial number determinations are of little value since even a low number, such as 103 per g or per cm2, could consist of all pathogens in the case of meat from diseased animals. Overall, the numbers of bacteria in meats may be taken to reflect the handling, processing and storage conditions of the products. Since the tissues of healthy animals are generally sterile, it is theoretically possible to produce meats

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(such as beef, pork and lamb) that are entirely free of bacteria. However, this is not practical when one considers that even under optimal conditions for slaughtering 300–500 steers per hour in a nonsterile environment, some bacteria from the air, the hands of workers and storage containers will enter finished products. See also: Microbiological analysis: Sampling and testing; Standard methods; Rapid methods; DNA methods; Indicator organisms. Microbiological safety of meat: Salmonella spp.; Escherichia coli O157:H7; Clostridium botulinum; Clostridium perfringens; Thermotolerant Campylobacter; Listeria monocytogenes; Yersinia enterocolitica; Staphylococcus aureus; Bacillus cereus; Aeromonas spp.; Yeasts and moulds; Prions and viruses.

Further Reading Downes FP and Ito K (2001) Compendium of Methods for the Microbiological Examination of Foods, 4th edn. Washington, DC: American Public Health Association. ICMSF (1978) Microorganisms in Foods. 1. Their Significance and Methods of Determination. Toronto: University of Toronto Press. Jay JM (2002) A review of aerobic and psychrotrophic plate count procedures for fresh meat and poultry products. Journal of Food Protection 65: 1200–1206. Kepner RL Jr and Pratt JR (1994) Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiological Reviews 58: 603–615. Marshall RT (ed) (1993). Standard Methods for the Examination of Dairy Products, 16th edn. Gaithersburg, MD: AOAC International.

Indicator Organisms D W Schaffner and S Smith, Rutgers University, New Brunswick, NJ, USA © 2004 Elsevier Ltd. All Rights Reserved.

Introduction Routine examination of meats for a multitude of potential pathogens is impractical, yet regular testing for selected pathogens may be necessary when evidence suggests that they may be present. Difficulties in pathogen detection due to their low concentration and uneven distribution in food samples have prompted the use of ‘indicator microorganisms’. Such an organism ‘indicates’ the possibility that a pathogen may be present below the limit of detection in a given food sample (or related sample). There is still controversy over the degree to which the presence of various