Vol. 54, No. 11

Methods for Improved Recovery of Listeria monocytogenesfrom Cheese

AHMED E. YOUSEF, ELLIOT T. RYSER, AND ELMER H. MARTH*

Department ofFood Science and The Food Research Institute, University of Wisconsin-Madison,Madison, Wisconsin 53706

Received 6 May 1988/Accepted 1 August 1988

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus

aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40°C) were compared for recovery ofListeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBridelisteria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i)homogenized for 2 min with the blender rather than the stomacher (P < 0.01), (ii) diluted in trisodium citraterather than TB (P < 0.01), and (iii) diluted in diluents at 40 rather than 20°C (P < 0.05). Based on these results,a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despitesuperior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheesesamples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated thatrecovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brickcheese increased when samples were diluted 1:10 in TBC at 45°C and stomached for 3 min, as compared withsimilarly treated samples diluted in TB at 25°C. A similar comparison of both diluents for recovery of L.monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different.Recovery of L. monocytogenes from cold-enriched (at 4°C for up to 8 weeks) samples of Colby cheese andcold-pack cheese food was generally similar for samples homogenized in TBC or TB.

Interest in detecting Listeria monocytogenes in dairyproducts has escalated over the past few years because atleast 160 cases of listeriosis, including 56 deaths, were linkedto consumption of pasteurized milk (2), Mexican stylecheese (4), Vacherin Mont d'Or soft-ripened cheese (Anon-ymous, Food Chem. News, 7 Dec. 1987), and feta cheese(M. P. Doyle, personal communication) contaminated withthe pathogen. In response to these outbreaks, increasedsurveillance of the dairy industry has led to isolation of L.monocytogenes from cheeses such as Mexican style (Anon-ymous, Food Chem. News, 10 Mar. 1986), Liederkranz(Anonymous, FDA Enforcement Rep., 4 Sept. 1985), ri-cotta, Cheddar, and Parmesan (J. G. Nichols, Abstr. J. FoodProt., vol. 50, p. 889, 1987), Brie (Anonymous, FDA En-forcement Rep., 9 Apr. 1986; Anonymous, Food Chem.News, 3 Mar. 1986; Anonymous, Food Chem. News, 17Feb. 1986; Anonymous, FDA Enforcement Rep., 23 Apr.1986), and semisoft and soft-ripened types (Anonymous,Food Chem. News, 18 Aug. 1986) imported from France.Recent efforts have focused on developing selective media

and shortened enrichment procedures to enumerate L.monocytogenes in food samples containing a mixed micro-flora; however, little attention has been given to the effectsof sample preparation on recovery of L. monocytogenesfrom cheese by using direct plating. According to standardmethods (13), preparation of cheese for microbiologicalanalysis involves diluting a sample of ground cheese inaqueous 2% trisodium citrate at 40 to 45°C, followed byhomogenization with a blender or stomacher, and thenplating the cheese homogenate on an appropriate medium.Although this method is reliable for quantitating a variety ofbacteria, the method lacks the sensitivity to detect the smallnumbers of L. monocytogenes occasionally found in cheese.Therefore, enrichment of the homogenized cheese sample is

* Corresponding author.

usually required before the organism can be isolated. SinceL. monocytogenes fails to grow in aqueous trisodium citrate,the present standard method is of no value for detecting lowlevels of the pathogen in cheese.

In 1948, Gray et al. (3) observed that L. monocytogenescould be isolated from infected bovine brains that werediluted in tryptose broth (TB) and held at 4°C for severalmonths. This method has since been used in medical labo-ratories to isolate the pathogen from clinical specimens.Although the extended incubation period makes this proce-dure of little value to the food industry, cold enrichment isstill of academic importance and remains among the mostreliable methods for detection of L. monocytogenes. Inrecent studies concerning the fate of L. monocytogenesduring manufacture of cottage (16), Cheddar (14), and Cam-embert (15) cheese, samples were diluted in TB at ca. 25°Cand homogenized in a stomacher for 30 s. If L. monocyto-genes was not found initially by direct plating on McBridelisteria agar, this procedure allowed possible detection of thepathogen in cheese homogenates during cold enrichment.However, using this method, Ryser and Marth (14, 15, 16)failed to observe expected increases in numbers of L.monocytogenes which occur during cheese manufacture as a

result of concentration of cells during curd formation. Theapparent insensitivity of this method led to the currentstudy, which examines multiple effects of various factors ofsample preparation on recovery of L. monocytogenes fromcheese. Results from the first part of this study led todevelopment of a new medium which increased recovery ofL. monocytogenes by direct plating and, at the same time,was suitable for cold enrichment.

MATERIALS AND METHODS

Media and diluents. The following bacteriological media(Difco Laboratories, Detroit, Mich.) and diluents were pre-pared, using deionized water: TB, TB containing 2% triso-

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1988, p. 2643-26490099-2240/88/112643-07$02.00/0Copyright C 1988, American Society for Microbiology

APPL. ENVIRON. MICROBIOL.

dium citrate (TBC), aqueous 2% trisodium citrate solution(CS), aqueous 0.5% peptone solution, and tryptose agar.McBride listeria agar was prepared from phenylethanol agarplus 1% glycine anhydride plus 0.05% (for Colby cheese) (7)or 0.5% (for cold-pack cheese food and brick cheese) (6)lithium chloride plus 5.0% defibrinated sheep blood (GIBCODiagnostics, Madison, Wis.).

Cultures. Four strains of L. monocytogenes were used inthis study: Scott A (serotype 4b, clinical isolate), V7 (sero-type 1, milk isolate), California (CA) (serotype 4b, isolatedfrom Mexican style cheese implicated in the 1985 outbreakof listeriosis in California), and Ohio (OH) (serotype 4b,isolated from recalled Liederkranz cheese manufactured inOhio). All cultures were maintained at 4°C on tryptose agarslants and were transferred bimonthly. Cultures of L. mono-cytogenes used to inoculate cold-pack cheese food and milkfor manufacture of brick cheese were prepared as describedpreviously (14).

Manufacture of cheeses: culture and rennet. A commercialStreptococcus cremoris starter culture (CC6; Marschall Di-vision, Miles Laboratories, Inc., Madison, Wis.) was used tomanufacture Colby and brick cheese. The starter culture wasincubated for 16 to 18 h at 21°C in previously steamed (45min, 100°C) reconstituted nonfat dry milk (10% total solids).A sufficient volume of the resulting starter culture wasprepared to provide an inoculum of 1% (wt/wt) (for Colbycheese) or 0.25% (wt/wt) (for brick cheese). Microbial rennetfrom the supplier that provided cultures was used in manu-facturing Colby and brick cheese.

Colby cheese. Colby cheese was made according to theprocedure described by Olson (11) or Kosikowski (5), asindicated in a previous study (22). Pasteurized (75°C for 16 s)whole milk (113 kg) was placed in a pilot-plant-size cheesevat, warmed to 31°C, and inoculated to contain ca. 103 L.monocytogenes (strain CA or V7) CFU/ml, after which lacticstarter culture (1%) was added. Each batch of Colby cheeseresulted in two 10-lb (ca. 4.6-kg) blocks which were vacuumpackaged and ripened at 4°C.

Brick cheese. Four duplicate batches of brick cheese weremanufactured according to the washed-curd method (10)from 190.5 kg of pasteurized (75°C for 16 s) whole milk. Milkwas inoculated to contain ca. S x 102 L. monocytogenes(strain Scott A, V7, CA, or OH) CFU/ml. Starter culture andmicrobial rennet were added to pasteurized whole milktempered to 31°C in the cheese vat. After the coagulum wascut, the temperature of the curd-whey mixture was graduallyincreased from 31 to 39°C. After 30 min of cooking at 39°C,half of the whey was drained and replaced with tap water at39°C. The whey-water mixture was drained after 30 min, andan approximately equal amount of curd was placed in each ofnine hoops. All hoops of cheese were periodically turned for6 h, after which time the finished cheese was placed in a 22%salt brine at 10°C. After 24 h, blocks of brick cheese wereremoved from brine, smeared with a culture of Brevibacte-rium linens (obtained from the surface smear of a commer-cially produced Limburger cheese), and ripened at 15°C and95% relative humidity for 2, 3, and 4 weeks to obtain mild,medium, and Limburger-like brick cheese, respectively.Cheese was ripened at 10°C and analyzed for numbers of L.monocytogenes and pH during a 6-month period.Cold pack cheese food. Cold-pack cheese food of three

different compositions was manufactured in duplicate 10.4-kg (22.9-lb) batches according to the following formula:8,000 g of Cheddar cheese (aged for 6 to 9 months), 471 g ofnonfat dry milk, 471 g of dried whey, 360 g of butter, and1,100 ml of water (1Sa). Duplicate batches of cheese food

were manufactured to contain either 0.30% sorbic acid or0.30% sodium propionate. Two additional batches wereprepared without preservative. All six batches of cheesefood had a pH of ca. 5.4. After Cheddar cheese was ground,all ingredients were combined and mixed for 3 min at highspeed, using a pilot-plant-sized sausage grinder. Each batchof cheese food was divided into four 2-kg portions whichwere inoculated to contain ca. S x 102 L. monocytogenes(strain Scott A, V7, CA, or OH) CFU/g. Inoculated portionsof cheese food were thoroughly mixed for 10 min to ensureuniform distribution of the organism. Cheese food was thenpacked into sterile 8-ounce (226.8-g) glass jars and stored at4°C until time of analysis for L. monocytogenes, pH, mois-ture, fat, and salt content. Results concerning the fate of L.monocytogenes in cold-pack cheese food during extendedstorage were reported elsewhere (1Sa).

Sampling cheese to test for L. monocytogenes: Colby cheese.Duplicate sets of samples were taken for enumeration of L.monocytogenes at the following points of cheese manufac-ture: (i) inoculated milk, (ii) curd after cooking, (iii) curdafter salting, (iv) curd after pressing (day 0), and (v) cheeseafter 42 and 84 days of ripening at 4°C. During cheesemanufacture, one set of duplicate curd (10-g) samples wasplaced in sterile (ca. 500-ml capacity) stomacher bags, di-luted in 90 ml of TB at 20°C, and homogenized in astomacher 400 (Tekmar Co., Inc., Cincinnati, Ohio) for 2min. The remaining set of 10-g samples was placed in anautoclaved stainless-steel blender jar (250-ml capacity), di-luted with 90 ml of CS at 40°C, and homogenized with aWaring blender (model 1042; Waring Products Co., Winsted,Conn.) at high speed for 2 min.The influence of the following variables in sample prepa-

ration on recovery of L. monocytogenes from 0-, 42-, and84-day-old samples of Colby cheese was examined: (i)method of homogenization, (ii) type of diluent in which thecheese was homogenized, and (iii) temperature of diluentadded to cheese before homogenization. In this study, asection of the cheese block was cut, exterior surfaces (ca.1-cm thick) were sliced and removed, and then the cheesesection was ground and mixed thoroughly. Duplicate 10-gsamples of ground cheese were diluted in 90 ml of TB or CSheld at either 20 or 40°C and were homogenized as previ-ously described, using a blender or stomacher.Two additional sets of duplicate 10-g samples were taken

from 5- to 8-month-old blocks of Colby cheese. One set ofsamples was diluted in 90 ml of TB at ca. 25°C, whereas theremaining set was diluted in TBC at 45°C. Both sets ofsamples were homogenized with the stomacher for 3 min.

Brick cheese. Two sets of duplicate 10-g samples weretaken for enumeration of L. monocytogenes during manu-facture of brick cheese according to the following scheme: (i)inoculated milk, (ii) curd after cutting, (iii) curd beforewashing, (iv) curd at dipping, (v) curd at fourth turn, and (vi)curd at brining. Both sets of samples were diluted in TB orTBC and homogenized with the stomacher as describedpreviously for Colby cheese.

Cold-pack cheese food. Six batches of cold-pack cheesefood were made as described previously. Each batch wasdivided into four equal portions and inoculated with one offour strains of L. monocytogenes. Two sets of duplicate 10-gsamples were taken from each portion of cheese food after 0,7, and 14 days of storage at 4°C. Both sets of samples werediluted in TB or TBC and homogenized with the stomacheras described previously for Colby cheese.

Detection of L. monocytogenes in cheese. Duplicate 10-gsamples taken during manufacture, ripening, or storage of

2644 YOUSEF ET AL.

IMPROVED RECOVERY OF L. MONOCYTOGENES FROM CHEESE 2645

cheese were homogenized according to methods indicatedearlier. Initial or subsequent dilutions of homogenizedcheese samples were surface plated in duplicate on McBridelisteria agar. All plates were incubated for 48 h at 35°C in anatmosphere of5% 02-10% C02-85% N2. Colonies typical ofthose formed by L. monocytogenes (smooth, bluish gray,slightly raised, translucent, watery consistency, 0.5 to 1.5mm in diameter, and weakly beta-hemolytic) were counted.Selected colonies were confirmed by the procedure of Ryserand Marth (14), which is based on a positive catalasereaction, tumbling motility in TB, appearance of bluishcolonies on tryptose agar, and results of biochemical testsfound on API 20S test strips (Analytab Products, Plainview,N.Y.).

In addition to direct plating, selected cheese samples werealso analyzed for L. monocytogenes by the cold-enrichmentmethod as described previously (14). In this procedure, theoriginal sample diluted 1:10 in TB or TBC was stored at 4°Cand surface plated on McBride listeria agar at 2-weekintervals for up to 8 weeks. Following 48 h of incubation at35°C under microaerobic conditions, all plates were exam-ined for colonies resembling L. monocytogenes. If present,the organism was confirmed as described previously (14).

Statistical analysis. In our work with Colby cheese, westudied the effect of three factors of cheese sample prepara-tion (two levels each, a 23 factorial design) on recovery of L.monocytogenes (log10) by direct plating. These factors were(i) method of homogenization (stomaching versus blending),(ii) type of diluent in which cheese was homogenized (TBversus CS), and (iii) temperature of diluent (20 verus 40°C).Data were analyzed statistically, using the analysis of vari-ance procedure of SAS statistical programs. The followingsplit-block linear model (19) was used to describe the data:

Y,kl = + pi + F, + Tk + PF + ,Tik + FTik + FTijk + bijklwhere Y,kl is the dependent variable (log1o CFU of L.monocytogenes per gram of cheese), P3i is batch of cheese(considered blocking factor, since two batches of cheesewere made at different times, using different initial popula-tions of L. monocytogenes in milk), F. is the treatmentfactors (eight combinations of the three factors studied), andTk is age of cheese (0, 42, and 84 days). The SAS generallinear models procedure was used to subdivide further thesum of squares for the F. term so that the factors studied(method of homogenization and diluent type and tempera-ture) and their interactions could be individually tested. Theerror term used to test each factor of interest was the meansum of squares of the interaction of this factor with theblocking factor (19).Data on brick cheese and cold-pack cheese food were

analyzed statistically by one- or two-way analysis of vari-ance, using MINITAB statistical programs.

RESULTS AND DISCUSSION

Colby cheese. During manufacture of Colby cheese, sam-ples of curd were prepared for enumeration of L. monocy-togenes by two methods. The first method is that describedin Standard Methods for the Examination ofDairy Products(13) and involves diluting curd (or cheese) in warm CS andthen homogenizing the mixture with a blender. The secondmethod, which is more suitable for handling pathogenicmicroorganisms, involves homogenizing a diluted curd orcheese sample with a stomacher. Cool TB was previouslyused in our laboratory as a diluent in the second method (14,15, 16). The major advantage of this method is that samples

C-.b\

lo$I *

10'-_ r 'X'

0 1 2 3 4 0 20 40 60 80 1 00

Manufacture(h) Ripening (d)

FIG. 1. Recovery of L. monocytogenes from samples takenduring the manufacture and storage of Colby cheese made frompasteurized milk inoculated with strain CA. Symbols: (0) sampleshomogenized with the blender, using CS at 40°C; (U) sampleshomogenized with the stomacher, using TB at 20°C. d, Days.

homogenized in TB also can be used for cold enrichment,which is usually needed to detect very small numbers of L.monocytogenes in cheese. Data in Fig. 1 indicate thatvariable numbers of the pathogen were detected in curdsamples analyzed by both methods. Differences in recoveryof L. monocytogenes from curd by the two methods in-creased as cheese manufacture progressed. Apparently, asthe curd became firmer, differences between the number ofL. monocytogenes recovered by the two methods becamelarger.Colby cheese was sampled during ripening to study the

effect of different parameters in preparing samples on thenumber of L. monocytogenes recovered from the cheese.The log1o values of the L. monocytogenes count per gram insamples obtained from 0-, 42-, or 84-day-old cheese andprepared by each of eight methods (three factors werevaried; each had two levels) are shown in Fig. 2. Theseresults suggest that fewer listeriae were recovered fromcheese that was homogenized with the stomacher than fromcheese homogenized with the blender. Statistical analysis ofthe data confirmed that assessment (Table 1). Sampleshomogenized in CS yielded significantly (P < 0.01) highernumbers of L. monocytogenes than did those prepared inTB. The effect of diluent temperature on recovery of L.monocytogenes from cheese cannot be ascertained directlyfrom data in Fig. 2. Statistical analysis of data, however,shows that significantly (P < 0.05) higher numbers of L.monocytogenes were recovered from samples homogenizedin warm (40°C) rather than cool (20°C) diluent.Some of the experimental factors had significant interac-

tions with age of cheese, implying that the effect of thesefactors was different at different points in the cheese-rip-ening process. The method of homogenization and the typeof diluent each had a significant (P < 0.01) interaction withage of cheese across the three sampling intervals duringcheese ripening (0, 42, and 84 days). For example, considercheese samples diluted in TB at 20°C and homogenized withthe stomacher (Al and Bi, Fig. 2) and samples diluted in CSat 40°C and homogenized in the blender (A8 and B8).Differences in recovery of L. monocytogenes from cheesesamples prepared by these two methods were quite large atthe early stages of ripening (0 and 42 days), but were minimalafter 84 days of ripening. This interaction can also be seen inFig. 1, which may be attributed to increased proteolysis ofcasein in older rather than younger cheese.

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2646 YOUSEF ET AL.

C_Dc,. 4 9D 111111 1CDCD 3.75

3.50

3.25

3.00

2.75

R1R2R3R4 R5R6R7 R8 B IB2B3B4B5 B6B7B8METHO

FIG. 2. Average numbers of L. monocytogenes (CFU per gramof cheese) in samples taken during the storage of Colby cheese andhomogenized by different methods. The letters A and B designatethe first and second batches, respectively. The digits designate themethods of preparing the samples as follows: 1 and 2, stomaching inTB at 20 or 40°C; 3 and 4, stomaching in CS at 20 or 40°C; 5 and 6,blending in TB at 20 or 40°C; 7 and 8, blending in CS at 20 or 40°C,respectively.

Although the split-block design allowed a formal factorialanalysis of the treatments, it is useful to order and comparethe eight procedures by using Fisher's least-significant-difference method at P = 0.05. Results of this analysisindicate that the numbers of L. monocytogenes recoveredfrom samples prepared with the blender are not significantlydifferent (Table 2). Numbers of L. monocytogenes, recov-ered from samples diluted in warm CS and homogenizedwith the stomacher are not significantly different from thoserecovered with the blender and warm TB or CS. Therefore,if the stomacher is to be used to prepare cheese samples, thesamples should be homogenized in warm CS.Thrasher and Richardson (20) reported no significant

difference in numbers of organisms recovered from stom-ached rather than blended Cheddar cheese samples thatwere diluted 1:10 in CS. von Asperger and Brandl (21)

TABLE 1. Results of analysis of variance for recovery ofL. monocytogenes from Colby cheese samples prepared by eight

different methods, using the split-block model

Source df Sum of psquares

Batch (l) 1 4.810 0.0001

Treatment factors (F) 7 2.770 0.0001Method of homogenization (Me) 1 1.840 0.0001Type of diluent (Dil) 1 0.353 0.0003Temp of diluent (Temp) 1 0.061 0.0300Me x Dil 1 0.330 0.0004Me x Temp 1 0.158 0.0032Dil x Temp 1 0.011 0.2956Me x Dil x Temp 1 0.017 0.1899

Error 1 (3 x F) 7 0.057 0.1240

Age of cheese (7) 2 17.50 0.0001Error 2 (j x 7) 2 0.179 0.0001

Treatment factors-cheese age interaction 14 0.963 0.0001(F x 7)

Error 3 (3 x F x 7) 14 0.135 0.0352

TABLE 2. Ranking and comparison of the eight methods ofhomogenizing Colby cheese samples by Fisher'sleast-significant-difference method at P = 0.05

Variables studiedMean

Method' Diluent T(eOC)p log1 CFUgb Grouping

Bl CS 20 4.05 ABl TB 20 4.04 ABJ TB 40 4.01 A BBl CS 40 4.01 A BSt CS 40 3.96 BSt CS 20 3.78 CSt TB 40 3.67 DSt TB 20 3.59 E

aMethod of homogenization was blending (BI) or stomaching (St).b Average log1o of L. monocytogenes count in 12 samples.Means with the same letters are not significantly different.

obtained lower bacterial counts from stomached rather thanblended samples or quarg, Gervais, and cottage cheese thatwere previously diluted in 2% trisodium citrate at 37°C. In astudy by Nanni (8), cheese samples of various ages, ripe-ness, structure, and consistency were immersed in physio-logical saline solution 15 min before homogenization with aStomacher for 10 to 25 min or with an Ultra-Turrax blenderfor 2 to 4 min. Populations of microorganisms recovered bythese two methods were not significantly different.Our data generally indicate that the blender was more

effective than the stomacher in preparing curd and cheesesamples for enumeratiQn of L. monocytogenes. The type andtemperature of the diluent, as well as the age of cheese, hada greater impact on recovery of listeriae from cheese thatwas homogenized with the stomacher than when the blenderwas used (Fig. 2).Development of a procedure for homogenizing and cold

enriching cheese samples. For years, TB has been the me-dium of choice for cold enrichment (at 3°C) of clinical andoccasionally food specimens so that small numbers of L.monocytogenes could be detected (17). Although cold en-richment in TB is not useful in the food industry (becauseseveral weeks are required to complete the test), this methodremains among the most reliable for detection of L. mono-cytogenes and is particularly valuable for detecting injuredcells (16). Therefore, TB was chosen as the medium tomodify.Based on our results from comparing methods of homog-

enization and type and temperature of diluent for recovery ofL. monocytogenes by direct plating, the procedure of Ryserand Marth (14) for homogenizing and cold enriching cheesesamples was modified as follows: (i) trisodium citrate (2%)was added to TB to improve the emulsification of cheesesamples, (ii) temperature of the diluent was increased from40 to 45°C as described in Standard Methods (13), and (iii)the time of homogenization in the stomacher was increasedfrom 0.5 to 3 min. Although larger numbers of listeriae wereroutinely detected in samples processed with the blenderrather than the stomacher, aerosols containing L. monocy-togenes can be produced during blending. Since cases ofhuman listeriosis attributable to the airborne pathogen havebeen reported (9), preparing contaminated samples with theblender in a poorly equipped laboratory may pose a healthhazard to the analyst and other laboratory workers. Otheradvantages of the stomacher include use of disposable,presterilized, Whirl-pak bags for diluting and cold enriching

APPL. ENVIRON. MICROBIOL.

IMPROVED RECOVERY OF L. MONOCYTOGENES FROM CHEESE 2647

10-

10 '-

0 2 4 6TIME (h)

FIG. 3. Average numbers of L. monocytogenes (four strains)recovered during manufacture of brick cheese. Symbols: (0) sam-

ples diluted in TBC at 45°C and stomached for 3 min; (U) samplesdiluted in TB at 25°C. and stomached for 3 min.

samples and the relative ease of processing large numbers ofsamples.Homogenization with the stomacher rather than the

blender also was shown to be less injurious to a variety ofgram-positive bacteria (18). Deibel and Banwart (1) foundthat stomaching was markedly less efficient than blending inbreaking up clumps and chains of some foodborne patho-gens. Yersinia enterocolitica, which is similar to L. mono-

cytogenes in that neither organism forms clumps or chains,was used as a control. After stomaching and blending, 97 and100% of yersiniae were present as single cells, respectively.Based on our observations and this discussion, we recom-

mend using the stomacher for homogenizing cheese samplesthought to contain listeriae. This procedure was evaluatedwith a variety of cheeses as indicated in the followingdiscussion.

Brick cheese. Results for recovery of L. monocytogenesduring manufacture of brick cheese are shown in Fig. 3. Twosets of samples taken at each point during cheese makingwere compared statistically, using the analysis of variance.Following inoculation of milk, significantly (P < 0.01) higher(2.2- to 3.0-fold) populations of L. monocytogenes were

consistently observed in curd samples diluted in TBC at 45°Crather than in TB at 25°C. Approximately 8.5 h after the startof brick cheese manufacture, populations of L. monocyto-genes were 22.4- and 7.6-fold greater in curd samples dilutedin TBC and TB, respectively, than in the orginal populationin the inoculated milk. Data from a previous study (22)indicate that the population of L. monocytogenes in Colbycheese (with a yield of ca. 10%) was ca. 10-fold higher thanthat in milk from which the cheese was made. This 10-foldincrease can be attributed to entrapment of L. monocyto-genes in curd during cheese manufacture. Similarly, num-

bers of L. monocytogenes in brick cheese are expected to beca. 10-fold higher than in milk from which this cheese ismanufactured. Our results indicate that growth of L. mono-

cytogenes during manufacture of brick cheese (22.4-foldincrease rather than the expected 10-fold increase resultingfrom curd formation) was only observed when curd sampleswere diluted in TBC. Using TB at 25°C, Ryser and Marthfailed to detect a 10-fold increase in numbers of L. monocy-togenes during manufacture of Cheddar (14) and Camembert(15) cheeses. In all probability, an increase of at least 10-foldwould have been observed if the samples had been diluted inTBC at 45°C and stomached for 3 min.

Cold-pack cheese food. Average populations of L. mono-cytogenes observed in 0-, 7-, and 14-day-old cheese food

TABLE 3. Comparison of TB and TBC for detection ofL. monocytogenes in cold-pack cheese food, using direct plating

(DP) and cold enrichment (CE)

No. of samples % False-

TB TBC negativeProduct

DP CE DP CE-_____ TB TBC

+- + - + _ + _

Colby cheese 3 27 20 10 6 24 18 12 33.3 33.3Cheese food 137 0 127 10 141 0 139 2 7.3 1.4

diluted in TBC and TB were 2.43 and 2.41 log1o CFU/g,respectively, and were not significantly different (results notshown). These results for direct plating were not totallyunexpected since cold-pack cheese food was readily spread-able and easily emulsified in both TB and TBC.

Since substantially larger populations of L. monocyto-genes were recovered during manufacture of brick cheesewith TBC rather than TB, the next consideration was todetermine the feasibility of using TBC as a cold-enrichmentmedium. Using 0-, 7-, and 14-day-old samples of cold-packcheese food in which L. monocytogenes was previouslyfound by direct plating, we failed to detect the organism in 2of 141 (1.4%) and 10 of 137 (7.3%) samples diluted in TBCand TB, respectively, after 8 weeks of cold enrichment(Table 3). Although cold enrichment was less successful thandirect plating for recovery of L. monocytogenes from cheesefood, TBC was an acceptable cold-enrichment medium forL. monocytogenes as evidenced by increased recovery ofthe organism with TBC rather than TB. According to Ralo-vich (12), trisodium citrate enhances growth of L. monocy-togenes in minimal media. Thus, presence of trisodiumcitrate may be partly responsible for increased recovery ofL. monocytogenes from cheese samples diluted in TBCrather than TB.

Colby cheese. The experiment with Colby cheese was doneto assess the ability of TBC when used as a cold-enrichmentmedium to recover low levels of L. monocytogenes from ahard cheese. In this study, TBC at 45°C and TB at 25°C werecompared for recovery of L. monocytogenes from 5- to8-month-old samples of Colby cheese, using both directplating and cold enrichment. Eight batches of Colby cheese,each consisting of two blocks (A and B), were studied, andtwo samples were taken from each cheese block. Data fordirect plating (Fig. 4) indicate that L. monocytogenes wasdetected in 6 of 30 (20%) and 3 of 30 (10%) samples dilutedin TBC and TB, respectively. Five of nine samples positivefor L. monocytogenes by direct plating came from theyoungest cheese (5 months old, batch 1). In contrast, theorganism was not detected by direct plating in the oldestcheese (8 months old, batch 8). With TBC as the diluent, acomparison between cold enrichment and direct platingindicates that L. monocytogenes was not detected by coldenrichment in two of six (33%) samples which were previ-ously positive by direct plating. Similarly, with TB as thediluent, the organism was not detected in one of three (33%)samples which were previously positive by direct plating.Thus, cold enrichment in either medium resulted in 33% ofthe samples being falsely reported as negative.

Cold-enrichment results (Fig. 4) indicate that 18 (60%) and20 (67%) of 30 samples were positive for L. monocytogeneswith TBC and TB, respectively. Two samples in which L.monocytogenes was detected after cold enrichment in TBCwere negative after cold enrichment in TB. Similarly, four

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2648 YOUSEF ET AL.

I

U

4

3

2

140

60 10

DP CE DP CE DP CE DP CE

TBC TB TBC TB

FIG. 4. Recovery of L. monocytogenes from eight batches of 5-to 8-month-old Colby cheese, each consisting of two blocks (A andB), by direct plating (DP) of samples prepared in TBC at 45°C or TBat 25°C and homogenized with the stomacher for 3 min. All sampleswere held at 4°C for cold enrichment (CE). Each square representsone cheese sample. Two adjacent vertical squares within the samebatch number represent samples from the same batch and block ofcheese. Open squares indicate that the organism was not detectedby direct plating or cold enrichment. Numbers within open squaresindicate the Listeria count (CFU per gram) in cheese as determinedby direct plating. Solid squares indicate that the organism was foundduring 8 weeks of cold enrichment, using either TBC or TB. (Note:Only one block of cheese from batch 5 [block A] was available forListeria analysis.)

samples which were positive for the pathogen after coldenrichment in TB were negative following cold enrichmentin TBC.

In contrast to results with cold-pack cheese food (Table 3),it appears that recovery of L. monocytogenes from Colbycheese was slightly enhanced after cold enrichment in TBrather than TBC. However, since populations of L. mono-

cytogenes were decreasing rapidly in our 5- to 8-month-oldColby cheese, it is possible that the organism could bepresent in one sample and absent from the other sampletaken at the same time from the same block of cheese. Thus,considering that both media resulted in 33% false-negativesfor Colby cheese and that TBC was superior to TB forrecovery of L. monocytogenes by cold enrichment fromcold-pack cheese food, it appears that TB and TBC are

equally acceptable as cold-enrichment media. Furthermore,since equal or greater populations of L. monocytogenes weredetected by direct plating of cheese samples diluted in TBCat 45°C rather than in TB at 25°C, we recommend dilutingground cheese 1:10 in TBC at 45°C and stomaching thesample for 3 min for detection of L. monocytogenes by directplating followed by use of the same sample for cold-enrich-ment procedures.

In conclusion, our results show that recovery of L. mono-

cytogenes from cheese curd or ripened cheese can beenhanced by diluting the sample in CS rather than TB.Recovery was further increased by warming the diluent to 40to 45°C. Although higher populations of L. monocytogeneswere detected in blended rather than stomached cheesesamples, it appears safer to homogenize Listeria-contam-

inated cheese samples with the stomacher. In addition toallowing greater recovery of listeriae from cheese by directplating, use of our enrichment medium (TBC) comparedfavorably with use of TB for recovery of the organism duringcold enrichment.

ACKNOWLEDGMENTS

Our research was supported by the College of Agricultural andLife Sciences; the National Dairy Promotion and Research Boardthrough a grant administered by the Dairy Research Foundation,Rosemont, Ill.; the Wisconsin Milk Marketing Board, Madison; andKraft, Inc., Glenview, Ill.We thank E. Nordheim for reviewing the statistical analysis of the

data. We also thank R. M. Twedt, Food and Drug Administration,Cincinnati, Ohio, for providing strains Scott A and V7; J. H.Silliker, Silliker Laboratories, Carson, Calif., for providing strainCA; and M. P. Doyle, The Food Research Institute, University ofWisconsin-Madison, for providing strain OH.

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VOL. 54, 1988