Sampling Methods for Microbiological Analysis of Red Meat and Poultry Carcasses

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Journal of Food Protection, Vol. 67, No. 6, 2004, Pages 1303–1308 Copyrightq, International Association for Food Protection

Review

Sampling Methods for Microbiological Analysis of Red Meat and Poultry Carcasses

ROSA CAPITA,*MIGUEL PRIETO,^ANDCARLOS ALONSO-CALLEJA

A´rea de Nutricioń y Bromatolog‡á, Escuela Superior y Tećnica de Ingenier‡á Agraria, Avenida de Astorga, s/n, 24400-Ponferrada, Leoń, Spain MS 03-212: Received 12 May 2003/Accepted 2 February 2004

ABSTRACT

Microbiological analysis of carcasses at slaughterhouses is required in the European Union for evaluating the hygienic performance of carcass production processes as required for effective hazard analysis critical control point implementation.

The European Union microbial performance standards refer exclusively to the excision method, even though swabbing using the wet/dry technique is also permitted when correlation between both destructive and nondestructive methods can be established. For practical and economic reasons, the swab technique is the most extensively used carcass surface-samplingmethod.

The main characteristics, advantages, and limitations of the common excision and swabbing methods are described here.

During processing, red meat and poultry carcasses may become contaminated with spoilage and pathogenic microorganisms from fecal material, stomach contents, and the hide. Additional sources of microbial contamination are the processing tools and equipment, structural components of the facility, human contact, and carcass-to-carcass contact (28, 30).

Traditional meat inspection procedures cannot always assure that consumers will not be exposed to infectious dos- es of meatborne pathogens(9). The hygienic condition of raw meats can be assured only by the development of hazard analysis critical control point (HACCP) systems for meat production processes. Bacteriological analysis of animal carcass surfaces has become an important source of information in developing and implementing HACCP systems for red meat and poultry slaughter and dressing op- erations. European Union (EU) Decision EC/471/2001(13) requires EU meat and poultry industries to implement HACCP systems and to meet proposed microbial criteria.

According to EU regulations, microbiological testing for the objective evaluation of the hygienic performance of meat production processes is based on the enumeration of indicator organisms (total aerobic counts andEnterobacte- riaceaecounts) on carcasses at the end of the process. The established microbial performance standards used to assess the hygienic condition of carcasses are based exclusively on the results of destructive methodologies. Swabbing is also permitted but only when a correlation between the excision and swabbing techniques has been previously found.

According to numerous authors, swabbing should be acceptable only when substantial fractions of the bacteria present on sampled areas are recovered (15, 44) and when

* Author for correspondence. Tel: int-34-987-442031; Fax: int-34-987- 442070; E-mail: [email protected].

precision is high(50).However, the swabbing technique is commonly used in practice without previous assessment of the relationships between results from both destructive and swab methods.

Knowledge of the relative numbers of bacteria recovered by the different sampling methods used is necessary for proper evaluation of the microbiological data obtained by the different techniques. Here, we describe characteristics of the main destructive and nondestructive sampling techniques and review the comparative results of previous studies on this topic.

ATTACHMENT OF BACTERIA TO MEAT SURFACES

The mechanism of bacterial attachment is generally accepted as a two-step process(49).The initial stage involves retention of bacteria in a liquid lm on the surface of the skin or meat. Reversible adhesion at this stage is associated with a complex interaction between charges and hydrophobicity on the surfaces of the support or cells. When the bacteria are$50 nm from the surface, only van der Waals forces are involved and speci c interactions are virtually ruled out, whereas at separations of 10 to 20 nm, electro- static interactions could play a role. Adhesion is also associated with the interactions of external appendages ( a- gella, mbriae, extracellular polysaccharides) of microbial cells with speci c surface receptors. The second stage is characterized by a time-dependent, irreversible exopolymer (glycocalyx) formation by bacteria. Extracellular polymers provide a favorable environment for growth and subsequent attachment of more bacteria, other microbes, and debris and favor the proliferation of bio lms under certain conditions.

Bacteria are also believed to be closely associated with and entrapped in inaccessible sites (crevices, devices) mainly by physical forces(5, 34, 37, 38, 49).Irreversible attachment

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TABLE 1. Advantages and limitations of the most commonly used sampling methods for enumerating bacteria on meat carcass surfaces (16, 22, 25, 35, 41, 44, 45, 50)

Method Advantages Limitations

Destructive Excision

Coring, scrapping, and abrading

Provides most reliable and least variable counts because of almost complete recovery of strongly attached bacteria

Destructive nature (devalues carcasses); limited sampling area; time and skill requirements (unsuitable for routine carcass sampling) Non-destructive

Swabbing Little or no damage of surfaces; usual-

ly covers a large area, suitable for detection of bacteria with low inci- dence and uneven distribution on carcass

Poor and variable results for removing bacteria because only loosely attached bacteria are recovered; percentage of bacteria removed from a surface depends on numerous factors

Contact methods (agar syringes, RODAC

plates,^aagar sausages, membrane lter blots,

self-adhesive tape, impression plates)

No damage to surfaces; possibility of direct microscopic examination or plating and incubating; provides mirror image of distribution of bacteria on surfaces; simple and quick, requiring few materials

Inapplicable when bacterial count is

.100 CFU/cm²because contact sur-

face becomes overcrowded; counts are not precise; counts are often ,1% of those obtained with excision or blending; unsuitable for ir- regular surfaces or crevices (excep- tion: adhesive tape methods);

because large areas cannot be sampled, a suf cient number of sites must be sampled to yield represen- tative data

Rising and shaking in diluents Little or no damage to surfaces; total

bacteria removed close to that for stomaching and about 10 times more than that for swabbing

Only suitable for poultry carcasses and small meat cuts (metal cylinders must be used for large meat carcasses)

aRODAC, replicate organism detection and counting.

of cells to a surface may occur between 30 min (10, 21, 34) and a few hours (11),depending on numerous factors such as bacterial type and temperature. Cells at the rst stage of attachment have been described as loosely attached or planktonic bacteria, whereas irreversibly attached bacteria are considered strongly attached or attached bacteria (14).

Various factors such as pH, contact time, temperature, medium, bacterial species or types, nature of the contact surface, cell density, and osmolarity could in uence the attachment of bacteria to meat surfaces(2, 5, 17, 20, 21, 42, 43, 49, 52).During storage of contaminated meat, the ratio of irreversibly attached bacteria to reversibly attached bacteria initially increases and then decreases. The initial increase is related to the secretion of bers attaching bacteria to the surface, and the decrease is due to the formation of microcolonies(46).

The main potential consequences of attachment are the increase of resistance to decontamination treatments and the increase of quantitative and qualitative differences of microbial counts depending on the sampling method used. The nondestructive methods (rinse, swab, or contact) will recover only those bacteria that are weakly attached; rmly attached bacteria may not be dislodged. Thus, sampling by methods that involve excision of tissue allows recovery of

larger numbers of bacteria from a surface than do nondestructive sampling techniques. The types of microorganisms recovered may differ also because some bacterial strains attach better than others(21).

METHODS FOR MICROBIOLOGICAL SAMPLING OF CARCASS SURFACES

Over a period of almost 100 years, many techniques have been developed for enumerating microorganisms on the surfaces of meat and poultry carcasses. Advantages and limitations of the main sampling methods are shown in Ta- ble 1. Excision and swabbing methods have found the wid- est acceptance because they are easy to use and require only small amounts of specialized material and because the data are generally more reproducible. Excision is considered the most accurate method, and swabbing is the most practical one(48).

The majority of relevant studies indicate that the excision and blending or stomaching of surface tissue (generally 2 mm thick in red carcasses or skin only in poultry carcasses) is the most effective carcass sampling method for bacteria because it provides more reliable and less variable bacterial counts than other sampling techniques and results in an almost complete recovery of rmly attached bacteria(15, 22, 26, 33, 36, 41, 44, 55).However, in meat

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processing facilities, excision is often neither practical nor acceptable because of the time and expertise needed and the invasive and destructive nature of the procedure. Be- cause the excision technique is economically undesirable and not very practical for HACCP monitoring (24, 44), nondestructive techniques are generally used in EU coun- tries for collecting data from carcass surfaces.

The nondestructive techniques do not recover all the microorganisms on carcass surfaces. The reliability of nondestructive methods is related to numerous factors. Recov- ery of bacteria from a carcass surface can vary greatly depending on the nondestructive method and material used.

For exemple, Nortje et al.(41)observed that results of the agar sausage technique was more closely correlated with those of the excision technique than with those of the swab technique. Werlein(55) did not nd any substantial differences between the rinsing and excision techniques for detection of bacteria on pig carcasses. The adhesive tape method was satisfactory when bacterial counts on carcass surfaces were low (23). With regard to poultry carcasses, Sharpe et al.(50) found that rinsing removed about 20%

of the ora and serial scrubbings removed 50%, and shaking resulted in higher total bacteria counts than those obtained with the other two methods. Izat et al.(31)reported that the whole poultry carcass rinse procedure resulted in signi cantly greater recovery of bacteria than did the surface swab or breast massage sampling methods. Other studies have demonstrated that the rinse and swab techniques are comparable, but that the results obtained with the rinse method are less variable (31, 39). A summary of counts obtained using different sampling methods is provided in Table 2.

Swabbing has been the most popular nondestructive surface sampling method used. Both the swabbing and excision techniques are included in the EU regulations for microbiological analysis of carcasses at slaughterhouses.

There is no consensus on the relative amounts of bacteria recovered by swabbing, which range from 0.01 to 89% of the bacteria recovered by excision(18, 26).Other percent- ages reported are 6 to 16%(1),10%(56),7.65 to 25%(3), 9%(7), 1 to 14% (36), 13%(6), 33 to 47% (19),and 16 to 45% (27). This high variability is related to numerous factors.

An important source of variation in the swabbing data is the material used for obtaining the samples. Swabs are usually made of alginate, cotton, or gauze. Cellulose or polyurethane sponges or polyester-bonded cloths are also used. More abrasive sampling material seems to improve bacterial recovery, especially at lower contamination levels (15, 26, 53).Thus, swabbing with cotton or calcium alginate, which are not abrasive, has been reported to recover ,10% of the numbers that were recovered by excision, whereas swabbing with more abrasive materials results in bacterial numbers similar to those obtained with excision (1, 4, 6, 15, 16, 26, 36, 48).Surface swabbing with alginate swabs has produced results that are more accurate, less variable, and easier to collect than has surface swabbing with cotton swabs (31, 35). The swabbing method also has a signi cant effect on bacterial recovery. In studies where a

single cotton swab was applied to a surface, the numbers of bacteria recovered are reported to be relatively few com- pared with the numbers recovered by excision(1, 6, 8, 10, 15, 16, 24, 36). However, when swabbing using the wet- dry technique (wet and dry cotton swabs are applied suc- cessively), the numbers recovered by swabbing can be about 50% of those recovered by excision (22, 35, 40).

With the wet-dry technique, Palumbo et al.(44)found higher counts of aerobic bacteria, total coliforms, and Esche- richia coli on swine carcass surfaces using the three-site swab method than when using the one-site swab method.

Pressure in the application of the swab, time of swabbing, and operator-related differences can also in uence the ef- fectiveness of bacterial removal by swabbing(6, 51).

High microbial loads imply considerable formation of easily removed colonies and higher recoveries of bacteria by swabbing(50).Thus, results from arti cially inoculated samples with a high inoculum concentration should be in- terpreted with caution.

Storage time of carcasses before sampling is also a signi cant factor in bacterial recovery by swabbing. Thus, as the storage period increases, the number of recorded bacteria decreases (4, 21). The period following inoculation may have allowed for rmer bacterial attachment, penetra- tion, or bio lm formation(11, 12, 21, 36, 53),thereby de- creasing cell recovery during sampling and subsequently reducing the ef cacy of the swab sampling method.

Carcass species also appears to in uence the percentage of bacteria removed (29). Ingram and Roberts (29) found counts from swabbing ranging from 1 to 24% (average 10%) for fresh beef carcasses, 27 to 52% (average 37%) for fresh mutton, 13 to 67% (average 44%) for fresh pork, and 25 to 89% (average 39%) for chilled pork com- pared with counts from excised and blended samples.

The percentage of the bacterial ora recovered by swabbing also depends on bacterial types present. Prieto et al. (45) reported that the mean percentage of bacteria recovered on lamb carcasses using the swab method ranged from 38.42 to 47.74% for mesophiles and from 43.26 to 53.48% for psychrotrophs. The substantial in uence of microbial type has also been reported by Izat et al.(31).

Other factors in uencing relative numbers of bacteria recovered by swabbing are type of tissue (fat or muscle), humidity, and differences in the texture of the sample surface(24, 26). Lower relative counts are found on adipose than on lean tissue using the swab method, especially when bacterial numbers are low(6). Investigations by Anderson et al. (1) and Firstenberg-Eden (21) indicated that when using the swab method moist carcass surfaces yield more accurate total aerobic bacterial counts than do dry carcasses.

The nondestructive methods of microbial sampling are considered less effective at recovering microorganisms from carcass surfaces than are the destructive methods, especially for red meat carcasses. However, because the excision method of sampling is time consuming, requires expertise, is destructive in nature, and involves sampling of only a limited area, nondestructive sampling is presently used in industry for collecting data from carcass surfaces

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TABLE2.Enumeration(mean6SDlogCFU/cm2)ofmicroorganismsoncarcasssurfacesfollowingdifferentsamplingmethods Countsbysamplingtechniquea CarcasstypenMicroorganismDestructive (excision)

Nondestructive SwabbingContactagarRinseCountryReference Redmeatcarcassesb,c7Pseudomonasuorescens3.5260.612.3161.34d2.8660.65eSouthAfrica41 Beefb,f5Totalaerobiccounts Pseudomonasfragi Escherichiacoli SalmonellaTyphimurium Listeriamonocytogenes

7.8660.37 7.0160.42 7.3960.25 7.2960.35 7.3060.43 7.5460.21 6.8960.49 7.3960.17 7.3160.25 7.2960.35 UnitedKingdom54 Beefg Beefh Beef

26 240 25

Totalaerobiccounts E.colibiotype1 Totalaerobiccounts Coliforms E.coli 4.20A 1.2260.61 1.65–2.05 1.08–1.60 0.60–1.51 3.20B 1.2160.69i 1.67–2.53i;1.58–2.37j; 1.47–2.09k 1.26–2.63i;2.61–4.54j; 1.52–2.29k 0.78–2.52i;1.40–3.20j; 0.60–2.17k

UnitedStates UnitedStates Canada

36 47 26 Beef Beef64 30Totalaerobiccounts Totalaerobiccounts Coliformsb E.colib

2.6A 1.6A 8h 4h

2.1Bi;1.3Cl 1.3Bi 2h,i 1h,i

UnitedStates UnitedStates15 16 Beef(adiposetissue)b Beef(adiposetissue)g10 15Aerobicplatecounts Totalaerobiccounts4.760.05A 3.153.460.38Bi 1.31UnitedStates UnitedStates4 6 Lamb30Totalaerobiccounts Totalcoliforms Fecalcoliforms E.coli

4.8 3.7 3.3 1.5 4.0m 3.0m 1.7m 0.1m

2.8n 2.2n 1.3n 0.1n

Tunisia22 Lamb(adiposetissue)g15Totalaerobiccounts3.172.90UnitedStates6 Pork120Totalaerobiccounts Coliforms E.coli

4.7060.72A 2.1260.99A 2.3560.99A

4.0260.70Bi,o 0.2660.87Bi,o 0.2760.95Bi,o

UnitedStates44 Pork(adiposetissue)g Pork15 25Totalaerobiccounts Totalaerobiccounts Coliforms E.coli

2.12 2.13–2.20A 1.78–1.89 1.28–1.72 1.78 1.83–1.87ABi;1.87– 1.90ABj;1.55–1.57Bk 2.73–4.01i;2.77– 4.19j;1.40–1.95k 2.45–2.73i;2.41–4.04j; 0.85–1.81k

UnitedStates Canada6 26

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TABLE2.Continued Countsbysamplingtechniquea CarcasstypenMicroorganismDestructive (excision)

Nondestructive SwabbingContactagarRinseCountryReference Poultry16Aerobicmesophiles Presumptivecoliforms Yeastandmoldsq

4.7360.01A 2.9860.07A 1.3560.03A

4.5260.05Bp 1.9860.07Bp 1.2960.03Ap

5.0960.03C 3.1160.03A 1.0560.02B

UnitedStates31 Chicken101Salmonella Campylobacterspp.26%positiver 84%positiver13%positives 86%positivesUnitedKingdom32 aMeansinthesamerowwithnolettersincommonaresignicantlydifferent(P,0.05).bPreviouslyinoculated.cAmountin6.42cm2.dDoubledryswabtechnique.eAgarsausage.f Amountin35cm2.gAmountin6.45cm2.hAmountin100cm2.iSpongeswabbing.jMedicalgauze.kCottonball.lCottonswabs.mDoublemoistswabtechnique.nSterilepetri disheswithnutrientagar.oThree-siteswab.pCalciumalginateswabs.qAmountin10cm2.rNeckskin.sWholecarcass.

for HACCP monitoring and to ensure that microbiological performance criteria are being met.

Swabbing is an accepted nondestructive sampling technique listed in EU regulations for microbiological analysis of carcass surfaces, but only if operators can demonstrate that it is possible in practice to obtain data correlated with those obtained with the excision method. The inability of the swabbing method to recover all bacteria on carcass surfaces is related to the attachment of microorganisms to meat surfaces. Because this attachment process is very complex and is affected by many factors, a universal quantitative conversion factor between excision results and swabbing results has not yet been established. Correlations between results of both destructive and nondestructive (swab) methods should be established for each meat and poultry plant and for various circumstances, i.e., sampling technique, carcass species, microbial group, stage and time of sampling, type of tissue, and location of sampled area.

ACKNOWLEDGMENT

The authors thank the Consejer‡´a de Sanidad y Bienestar Social de la Junta de Castilla y Leo´n (Spain) for nancial support (project LE 08/

02).

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