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Comparison of the ISO and IDF methods for detection of Listeria monocytogenes in blue veined cheese
International Dairy Journal 9 (1999) 149}155
Comparison of the ISO and IDF methods for detection of Listeria monocytogenes in blue veined cheese Elisabet Waak *, Wilhelm Tham, Marie-Louise Danielsson-Tham Arla FoU, S-105 46 Stockholm, Sweden Department of Food Hygiene, Swedish University of Agricultural Sciences, P.O. Box 7009, S-750 07 Uppsala, Sweden Received 17 April 1998; accepted 18 March 1999
Abstract The International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) is compared with the ISO Draft International Standard ISO/DIS 11290-1 (1995) for detection of Listeria monocytogenes in inoculated samples of the blue veined cheeses Gorgonzola and AG delost (Swedish blue mould cheese) and in non-inoculated market cheeses of mostly soft mould cheese. The ISO method detected L. monocytogenes signi"cantly more frequently than the IDF method when inoculated Gorgonzola samples were analysed. For inoculated AG delost, and non-inoculated market cheeses the methods were equally good. The selective isolation media PALCAM and Oxford Listeria selective agar, were compared for recovery of Listeria spp. Less growth of non-Listeria colonies was obtained on PALCAM agar compared to Oxford Listeria selective agar especially after the primary enrichment step of the ISO method. Consequently, it was easier to identify and isolate growth of Listeria species from the cheeses on the PALCAM agar. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Listeria; ISO; IDF; Method; Cheese; PALCAM; Oxford
1. Introduction It is essential for the dairy industry to avoid Listeria monocytogenes in their cheeses. These products, especially soft cheeses, often provide excellent conditions for growth (Terplan, Schoen, Springmeyer, Degle & Becker, 1986; Ryser & Marth, 1987), and have also been implicated in outbreaks of listeriosis (James et al., 1985; Bille & Glauser, 1988; Jensen, Fredericsen & GernerSmidt, 1994; Goulet et al., 1995). There are several cultural methods available for detection of L. monocytogenes in food, e.g. the standard methods from FDA, AOAC, ISO, IDF and NMKL. The IDF method is recommended for milk products. However, in a collaborative study by Twedt, Hitchins and Prentice (1994) the IDF method showed a low sensitivity for detection of L. monocytogenes in some red smear cheeses and blue cheeses.
* Corresponding author. E-mail address: [email protected] (E. Waak)
The aims of the present study were: (i) to compare the method of the International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) with the method stated in ISO Draft International Standard ISO/DIS 11290-1, 1995 (1995) for detection of L. monocytogenes in blue veined cheese, and (ii) to investigate the importance of two parallel selective plating media (PALCAM agar and Oxford agar), as prescribed by the ISO method.
2. Materials and methods 2.1. Cheese samples Four matrices of AG delost and 13 of Gorgonzola were used for arti"cial contamination. In addition to these cheeses another 61 cheeses (Market cheese samples); 16 made out of goats' milk, 1 of ewes' milk and 44 of cows' milk were analysed for the possible natural presence of L. monocytogenes with both methods (ISO and IDF). All of the 61 cheeses, except two hard cheeses, were soft or semi-hard with a smeared surface, white mould surface or
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blue veined internal mould. Fifty-"ve were made out of raw milk. Most cheeses (46) were of French origin, 5 were of Italian, 7 of British and 3 of Spanish origin. 2.2. Strains Two L. monocytogenes strains were used for inoculation; L. monocytogenes no. SLU 92, serovar 3b, isolated from a cheese-producing dairy plant (Unnerstad, Bannerman, Bille, Danielsson-Tham, Waak & Tham, 1996) and L. monocytogenes no. SLU 592, serovar 4b, from a cheese-associated outbreak (Bille & Glauser, 1988). Each strain was grown overnight in Trypticase2+ Soy Broth (BBL) at 373C. The cultures were diluted in peptone water and the "nal dilution was done in autoclaved (1153C, 20 min) 10% (w/v) reconstituted skim milk to achieve a population of 10}10 organisms per ml. Portions of these skim milk cultures were stored at !203C while awaiting further experiments. 2.3. Sample preparations 2.3.1. Market cheese samples Small pieces were taken from di!erent parts of the cheeses both of surface material and inner material. 2.3.2. Artixcial contamination One brand of Swedish blue veined cheese (AG delost) and three di!erent brands (A, B and C) of Gorgonzola were used for arti"cial contamination. At least 250 g of each cheese was used as a matrix and was mashed in a stomacher bag to evenly distribute the microbial #ora in the cheese sample. For the AG delost, four di!erent matrices were used (di!erent batches of production). One matrix was an old cheese stored at 43C after the sell-by date with a fat-in-dry-matter content of 50%, the other matrices were within sell-by date and with a fat-in-dry-matter of 60%. Pre-cut wedges of Gorgonzola were used. For
brand A, three matrices were used, for brand B four, and brand C six matrices. Portions of 25 g of the matrices were macerated with 225 ml Listeria enrichment broth (IDF method) or with 225 ml of the Half Fraser broth (ISO method). The skim milk cultures were thawed in water at about 203C and 0.1}2 ml were added to the macerated cheese portions in broths and blended just before adding the selective agents for each of the methods. A non-inoculated portion in each matrix was analysed for any possible natural presence of L. monocytogenes. To obtain inoculum counts 0.1 ml of the skim milk cultures were surface-plated onto Oxford agar and incubated at 373C for 48 h. 2.4. IDF method This method was performed according to the International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) (IDF method). Listeria enrichment broth base was prepared using Trypticase2+ soy broth (BBL) and Yeast extract (Oxoid L21) and the standard selective supplements. The IDF method was extended so that in addition to &Oxford agar' (Oxford Listeria Selective agar, Oxford Formulation, agar base CM856 and supplement SR140, Oxoid) also &PALCAM agar' (PALCAM Listeria Selective Agar, PALCAM agar base CM877 and supplement SR150E, Oxoid) were used. Plates were streaked after 48 h as in the method and in addition also after 24 h. The streak after 24 h was done to eliminate the di!erence between the two methods with reference to the number of times the broths were streaked out and the number of plates used. 2.4.1. Modixed IDF method In a test with two of the Gorgonzola cheese brands the IDF method was altered. The Listeria enrichment broth base in the IDF method was modi"ed (FDA, 1998) by replacing the dipotassium phosphate with the bu!er used
Table 1 Diagram of procedures Day
IDF method
ISO method
Monday
Test portion of 25 g#selective Enrichment medium 225 ml. 48 h at 303C Streak onto Oxford and PALCAM medium. 48 h at 373C
Test portion of 25 g#Half Fraser 225 ml (Primary enrichment). 30 h at 303C. Streak onto Oxford and PALCAM agar. 48 h at 373C. Secondary enrichment 48 h at 373C.
Tuesday Wednesday Thursday
Streak onto Oxford and PALCAM medium. 48 h at 373C Select 5 characteristic colonies for conxrmation from each agar medium
Friday
Select 5 characteristic colonies for con"rmation from each agar medium.
Monday
Modi"cations are marked with italics.
Streak onto Oxford and PALCAM media. (48) 96 h at 373C. Select 5 characteristic colonies for con"rmation from each agar medium.
Select 5 characteristic colonies for con"rmation from each agar medium
E. Waak et al. / International Dairy Journal 9 (1999) 149}155
in the ISO procedure: Na HPO ) 2H O (12 g l\) and KH PO (1.35 g l\) and pH adjusted to 7.2$0.2 (the pH of the ordinary broth is 7.3$0.1) (Table 1).
Table 2 Recovery of L. monocytogenes in inoculated AG delost samples Strain
2.5. ISO method This method was performed according to the ISO Draft International Standard ISO/DIS 11290-1 (ISO method). Half Fraser broth was prepared from Oxoid Fraser broth base CM895 and supplements were added to 225 ml broth. Fraser broth was prepared from Oxoid Fraser broth base CM895 and the supplements were added to 10 ml. After a preliminary check after 24 h, the incubation time of the second isolation step on Oxford and PALCAM agar, was lengthened to 96 h instead of 48 h to avoid work at weekends (Table 1). 2.6. Conxrmation Market cheeses: Five presumptive L. monocytogenes colonies, or all when fewer were present, were isolated from both PALCAM and Oxford agar. The ISO procedure was used for con"rmation with a few modi"cations. The motility test and CAMP test were excluded and when haemolytic activity was weak, the commercial probe AccuProbe (GENE-PROBE威) was used to determine whether the isolates were L. monocytogenes or not. Two extra carbohydrates were used, mannitol as a negative control for Listeria and glucose as a positive control of fermentation. The control strain L. monocytogenes NCTC 7973 was used to test all media in each test. L. monocytogenes isolates were in addition serotyped with Listeria O Antiserum 1 and 4 (Difco laboratories, Detroit, Michigan, US). From the inoculated cheeses the con"rmation of Listeria was simpli"ed. All typical isolates were tested for haemolytic activity on horse blood agar and production of catalase. Cell shape and tumbling motility (203C) were studied on one out of the "ve isolates. Non-haemolytic Listeria spp naturally occurring were in addition tested (one isolate per occasion) for fermentation of rhamnose, xylose, mannitol and glucose. 2.7. Statistical analyses The McNemar Chi-square test was used to detect di!erences between proportions (McNemar, 1947). A difference at the 5% level was considered to be statistically signi"cant (Wardlaw, 1985).
3. Results and discussion 3.1. Detection of L. monocytogenes in A$ delost (Table 2) The recovery of L. monocytogenes from the inoculated AG delost samples was nearly 100% and the methods were
151
No. of inoculated Inoculation samples level/25 g
SLU 92
16 8 6 SLU 592 10 10 Total 50
1}9 10}99 100}200 1}9 10}99
ISO
IDF
PE
SE 24 h
48 h
14 8 6 10 10 48
15 8 6 10 10 49
15 8 6 10 10 49
15 8 6 10 10 49
Primary enrichment. Secondary enrichment.
Table 3 Recovery of L. monocytogenes strain SLU 92 in Gorgonzola samples Cheese brand
No. of inoculated Inoculation samples level/25 g
No. samples positive for L. monocytogenes ISO
A
B C
Total
2 2 1 9 1 12 1 28
1}9 10}99 100}200 10}99 1}9 10}99 100}200
IDF
PE
SE 24 h
48 h
2 0 1 2 0 3 1 9
2 1 1 5 0 3 1 13
2 0 0 0 0 2 1 5
2 0 0 0 0 2 1 5
Primary enrichment. Secondary enrichment.
equally good. Of 16 cheeses inoculated with 1}9 cfu L. monocytogenes (strain SLU 92) per 25 g, 15 were con"rmed as L. monocytogenes positive with both methods. Both methods failed to detect the L. monocytogenes strain when inoculation was performed in a very old cheese of a lower fat content that had been stored far beyond the best-before date. This cheese had a #ora of Bacillus spp, which grew well with blackened colonies on both Oxford and PALCAM agar and disturbed the interpretation of the plates. All inoculations made with 10 or more L. monocytogenes per 25 g with strain SLU 92 were detected with both methods. With strain SLU 592 all inoculations were found positive with both methods. No other species of Listeria were found in the AG delost. 3.2. Detection of L. monocytogenes in Gorgonzola cheese (Tables 3 and 4) The recovery of the inoculated L. monocytogenes in the Gorgonzola cheeses was much lower than in the AG delost with both methods. L. monocytogenes was recovered in
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only about 50% of the samples (13/28; Table 3, 15/28; Table 4) with the ISO method, and in 18% (5/28; Tables 3 and 4) with the IDF method. Even inoculation rates of more than 10 cfu per gram could not be recovered by the IDF method when the Gorgonzola brands A and B were used. The di!erence in recovery rate between the two methods is signi"cant (P"0.005, Table 3 and P"0.002, Table 4). The two-stage enrichment procedure and another bu!ering system may explain the higher performance of the ISO method. The di$culty in recovering L. monocytogenes in the Gorgonzola cheeses can be explained by several factors. A contamination #ora of L. innocua was frequently found in these cheeses, especially in brand C (see Table 7). This #ora grows faster than the inoculated L. monocytogenes strains (Petran & Swanson, 1993). The outgrowth of L. monocytogenes may also be a!ected by other competitive micro-organisms (In't Veld, Notermans & van de Berg, 1995; Vlaemynck & Moermans, 1996). Bacteriocin-producing enterococci (Martin, Friedrich, Beyer & Terplan, 1994) and lactobacilli (Asperger, Url & Brandl, 1989; Farias, De Ruiz Hogado & Sesma, 1993; Gira!a, Picchioni, Neviani & Carminati, 1995; Ali, Lacroix, Thuault,
Table 4 Recovery of L. monocytogenes strain SLU 592 in Gorgonzola samples Cheese brand
No. of inoculated Inoculation samples level/25 g
No. samples positive for L. monocytogenes ISO
A
B C Total
3 1 1 9 1 13 28
1}9 10}99 100}200 10}99 1}9 10}99
Bourgeois & Simard, 1995; Stecchini, Aquili & Sarais, 1995) may also inhibit the growth. Presence of free medium-chain fatty acids in blue mould cheese has also been suggested to inhibit the growth of Listeria. Free medium-chain fatty acids dissolved in butteroil have been shown to inhibit growth of a test strain of L. monocytogenes (Kinderlerer, Matthias & Finner, 1996). 3.3. Comparison of 24 h and 48 h selective enrichment Previous studies (McClain & Lee, 1988; Slade & Collins-Thompson, 1987) have shown that two-stage enrichment procedure, e.g. the ISO method, improves the detection of Listeria when a competitive #ora is present. However, six days are needed to perform the ISO analyses. Thus, work must be done also at weekends. It is also a disadvantage for the food industry to wait an extra day (compared with the IDF method) for results. With the ISO method, 77 of 106 (72.6%) inoculated samples were found positive for the presence of L. monocytogenes after the secondary enrichment. Of these, 69 were found to be positive already after the primary enrichment (Tables 2}4). The IDF method di!ers from the ISO method in that the total number of samples positive for the presence of L. monocytogenes was lower. The IDF method detected the same number of positive samples after 24 and 48 h 59/106 (55.5%) (Tables 2}4). 3.4. Modixed IDF method (Table 5)
IDF
PE
SE 24 h
48 h
2 0 1 4 1 4 12
2 0 1 7 1 4 15
2 0 0 0 0 3 5
2 0 0 0 0 3 5
Primary enrichment. Secondary enrichment.
A small test with 16 samples of Gorgonzola was performed to see if the IDF method was improved by bu!ering the enrichment medium. The results indicate that the recovery of L. monocytogenes was improved, as 5/16 (31%) became positive with the modi"cation compared with 3/16 (18%) with the non-modi"ed method (Table 5). When pH was measured in the non-inoculated portion on the second day of incubation the ordinary enrichment medium had a pH of 6.1 and the modi"ed enrichment medium a pH of 6.8. Media maintaining their pH at neutral values have been reported to perform better than
Table 5 Results of a modi"ed IDF method (mIDF) compared to the methods of ISO and IDF Cheese brand
B B C C Total
Strain
SLU SLU SLU SLU
92 592 92 592
Inoculation level/25 g
10}99 10}99 10}99 10}99
These samples are also included in Tables 3 and 4.
No. of inoculations
4 4 4 4 16
No. of samples positive for L. monocytogenes ISO method
IDF method
mIDF
2 3 1 0 6
0 2 1 0 3
3 2 0 0 5
E. Waak et al. / International Dairy Journal 9 (1999) 149}155
153
Table 6 Market cheeses where L. monocytogenes was detected Cheese no.
Type of cheese
Type of milk
Methods of detection ISO
2 16 8 18 19 13 20
Semi-hard, Bv Semi-hard, Bv Soft, Sm Soft, Sm Soft, Sm Soft, Sm Hard, smear
HT, cows' HT, cows' R, cows' R, cows' R, cows' R, ewes' *
Sero group IDF
PE
SE
24 h
48 h
Pos Pos Pos Pos Neg Neg Pos
Pos Pos Pos Pos Pos Neg Pos
Pos Pos Neg Pos Pos Pos Pos
Pos Pos Neg Pos Pos Pos Pos
1/2 1/2 1/2 ND ND 1/2 1/2
Classi"cation according to Fox (1993). Bv"Blue veined internal mould, Sm"white surface mould, HT"heat treated, R"raw milk, ND"not determined. Primary enrichment. Secondary enrichment.
Table 7 Market cheeses and Gorgonzola types A, B and C where L. innocua was detected Cheese number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Gorgonzola Gorgonzola Gorgonzola Total no. of
Type of cheese
Soft, smear Semi-hard, Bv Soft, smear Soft, Sm Soft, Sm Soft, Sm and smear Soft, Sm Soft, Sm Soft, Sm Soft, Sm Soft, Bv Soft, smear Soft, Sm Soft, smear type A, 3 samples type B, 4 samples type C, 6 samples positive samples
Method of detection/ L. innocua"#
(Table 7). L. innocua was detected by use of the ISO method in 20 of the samples (matrices included) and by use of the IDF method in 14 of the samples. The di!erence is close to signi"cance (P"0.058), but did not quite reach the 5% level.
ISO
IDF
3.6. PALCAM and Oxford agar (Table 8)
# # # # # # # # # # # # ! ! 1#, 2! 1#, 3! 6# 20
! ! # ! ! # ! # # # ! ! # # 1#, 2! 4! 6# 14
On PALCAM agar Listeria spp. form colonies which are approximately 2 mm in diameter, grey}green in colour with a black sunken centre and a black halo against a cherry-red medium background (van Netten, Perales, van de Moosdijk, Curtis & Mossel, 1989). On Oxford agar the colonies are black with a sunken centre and a black halo (Curtis, Mitchell, King & Gri$n, 1989). The sizes are the same as on PALCAM medium. On some occasions in the present study, a black halo was observed on Oxford agar, but the actual colonies were more or less overgrown. When making a streak from such a plate onto PALCAM agar, typical Listeria colonies appeared. The high selectivity of the PALCAM medium (van Netten et al., 1989; Cantoni, Valenti & d'Aubert, 1990) made it easier to detect the Listeria colonies, especially after primary enrichment (ISO method) in the present study. Improvement in the detection of Listeria spp. by use of PALCAM agar when a high background #ora is present has also been reported by other authors (Hammer, Hahn, Kirchho! & Heeschen, 1990; Gunasinghe, Henderson & Rutter, 1994). The number of plates with typical Listeria spp. was calculated for each of the two agar types for all analyses (Table 8). The use of two parallel agar media after the secondary enrichment (ISO method) seems excessive. No presumptive Listeria spp. would have been missed if only PALCAM agar had been used and only one out of 130 positive "ndings (0.8%) if only Oxford agar had been used (Table 8).
media with a low bu!ering capacity (Warburton et al., 1992; Beumer, Gi!el, Anthonie & Cox, 1996). 3.5. L. monocytogenes in market cheese samples (Tables 6 and 7) L. monocytogenes was found in 7 of the market cheeses (9%). Both methods detected L. monocytogenes in the same number of samples but not always in the same cheeses (Table 6). Cheese nos. 2, 8 and 13 not only harboured L. monocytogenes but also Listeria innocua
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E. Waak et al. / International Dairy Journal 9 (1999) 149}155
Table 8 Total number of Listeria spp. positive analyses Method
ISO IDF
Number of positive analyses
130 110
Number of plates with typical Listeria colonies Primary enrichment (ISO) 24 h (IDF)
Secondary enrichment (ISO) 48 h (IDF)
PALCAM
Oxford
PALCAM
Oxford
116 106
102 101
130 110
129 106
4. Conclusions The ISO and the IDF methods detected L. monocytogenes in the same number of inoculated AG delost samples and market cheese samples. However, the recovery of L. monocytogenes in inoculated samples of Gorgonzola was signifcantly higher with the ISO method. The PALCAM agar more frequently revealed Listeria colonies in the present study as less growth of nonListeria colonies occurred on this substrate than on the Oxford agar.
5. Addendum Since completion of this study, the Revised Provisional IDF standard 143:1990 (1990) has become International IDF standard 143A: 1995 (1995). The only di!erence from the provisional standard is that the references for guidance of sampling and preparation have been updated and given new names. The recommended pH of the enrichment broth and blood agar base has also been changed from 7.3$0.1 to 7.3$0.2. The ISO draft international standard ISO 11290-1 has become International standard ISO 11290-1. The changes from the draft standard are; a margin to the incubation time of the "rst incubation step is included (24$2 h), colony appearance on Oxford and PALCAM agar are somewhat di!erently described, and a choice between three incubation temperatures can be made (30, 35 or 373C). The incubation time and temperature of the carbohydrate utilisation tests has been changed from 373C for up to 7 d to 35 or 373C for up to 5 d. In the draft standard, a choice between horse and sheep de"brinated blood can be made, in the standard only sheep de"brinated blood is mentioned. The composition of phosphate-bu!ered saline is also added in the standard.
Acknowledgements The authors wish to express thanks to Arla R & D for funding the study, Janet Ha kansson for excellent tech-
nical assistance, Prof. J. Bille for providing L. monocytogenes strain SLU 592, and Dr. Anders Christiansson for statistical support.
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Comparison of the ISO and IDF methods for detection of Listeria monocytogenes in blue veined cheese Elisabet Waak *, Wilhelm Tham, Marie-Louise Danielsson-Tham Arla FoU, S-105 46 Stockholm, Sweden Department of Food Hygiene, Swedish University of Agricultural Sciences, P.O. Box 7009, S-750 07 Uppsala, Sweden Received 17 April 1998; accepted 18 March 1999
Abstract The International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) is compared with the ISO Draft International Standard ISO/DIS 11290-1 (1995) for detection of Listeria monocytogenes in inoculated samples of the blue veined cheeses Gorgonzola and AG delost (Swedish blue mould cheese) and in non-inoculated market cheeses of mostly soft mould cheese. The ISO method detected L. monocytogenes signi"cantly more frequently than the IDF method when inoculated Gorgonzola samples were analysed. For inoculated AG delost, and non-inoculated market cheeses the methods were equally good. The selective isolation media PALCAM and Oxford Listeria selective agar, were compared for recovery of Listeria spp. Less growth of non-Listeria colonies was obtained on PALCAM agar compared to Oxford Listeria selective agar especially after the primary enrichment step of the ISO method. Consequently, it was easier to identify and isolate growth of Listeria species from the cheeses on the PALCAM agar. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Listeria; ISO; IDF; Method; Cheese; PALCAM; Oxford
1. Introduction It is essential for the dairy industry to avoid Listeria monocytogenes in their cheeses. These products, especially soft cheeses, often provide excellent conditions for growth (Terplan, Schoen, Springmeyer, Degle & Becker, 1986; Ryser & Marth, 1987), and have also been implicated in outbreaks of listeriosis (James et al., 1985; Bille & Glauser, 1988; Jensen, Fredericsen & GernerSmidt, 1994; Goulet et al., 1995). There are several cultural methods available for detection of L. monocytogenes in food, e.g. the standard methods from FDA, AOAC, ISO, IDF and NMKL. The IDF method is recommended for milk products. However, in a collaborative study by Twedt, Hitchins and Prentice (1994) the IDF method showed a low sensitivity for detection of L. monocytogenes in some red smear cheeses and blue cheeses.
* Corresponding author. E-mail address: [email protected] (E. Waak)
The aims of the present study were: (i) to compare the method of the International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) with the method stated in ISO Draft International Standard ISO/DIS 11290-1, 1995 (1995) for detection of L. monocytogenes in blue veined cheese, and (ii) to investigate the importance of two parallel selective plating media (PALCAM agar and Oxford agar), as prescribed by the ISO method.
2. Materials and methods 2.1. Cheese samples Four matrices of AG delost and 13 of Gorgonzola were used for arti"cial contamination. In addition to these cheeses another 61 cheeses (Market cheese samples); 16 made out of goats' milk, 1 of ewes' milk and 44 of cows' milk were analysed for the possible natural presence of L. monocytogenes with both methods (ISO and IDF). All of the 61 cheeses, except two hard cheeses, were soft or semi-hard with a smeared surface, white mould surface or
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blue veined internal mould. Fifty-"ve were made out of raw milk. Most cheeses (46) were of French origin, 5 were of Italian, 7 of British and 3 of Spanish origin. 2.2. Strains Two L. monocytogenes strains were used for inoculation; L. monocytogenes no. SLU 92, serovar 3b, isolated from a cheese-producing dairy plant (Unnerstad, Bannerman, Bille, Danielsson-Tham, Waak & Tham, 1996) and L. monocytogenes no. SLU 592, serovar 4b, from a cheese-associated outbreak (Bille & Glauser, 1988). Each strain was grown overnight in Trypticase2+ Soy Broth (BBL) at 373C. The cultures were diluted in peptone water and the "nal dilution was done in autoclaved (1153C, 20 min) 10% (w/v) reconstituted skim milk to achieve a population of 10}10 organisms per ml. Portions of these skim milk cultures were stored at !203C while awaiting further experiments. 2.3. Sample preparations 2.3.1. Market cheese samples Small pieces were taken from di!erent parts of the cheeses both of surface material and inner material. 2.3.2. Artixcial contamination One brand of Swedish blue veined cheese (AG delost) and three di!erent brands (A, B and C) of Gorgonzola were used for arti"cial contamination. At least 250 g of each cheese was used as a matrix and was mashed in a stomacher bag to evenly distribute the microbial #ora in the cheese sample. For the AG delost, four di!erent matrices were used (di!erent batches of production). One matrix was an old cheese stored at 43C after the sell-by date with a fat-in-dry-matter content of 50%, the other matrices were within sell-by date and with a fat-in-dry-matter of 60%. Pre-cut wedges of Gorgonzola were used. For
brand A, three matrices were used, for brand B four, and brand C six matrices. Portions of 25 g of the matrices were macerated with 225 ml Listeria enrichment broth (IDF method) or with 225 ml of the Half Fraser broth (ISO method). The skim milk cultures were thawed in water at about 203C and 0.1}2 ml were added to the macerated cheese portions in broths and blended just before adding the selective agents for each of the methods. A non-inoculated portion in each matrix was analysed for any possible natural presence of L. monocytogenes. To obtain inoculum counts 0.1 ml of the skim milk cultures were surface-plated onto Oxford agar and incubated at 373C for 48 h. 2.4. IDF method This method was performed according to the International Dairy Federation Revised Provisional IDF Standard 143:1990 (1990) (IDF method). Listeria enrichment broth base was prepared using Trypticase2+ soy broth (BBL) and Yeast extract (Oxoid L21) and the standard selective supplements. The IDF method was extended so that in addition to &Oxford agar' (Oxford Listeria Selective agar, Oxford Formulation, agar base CM856 and supplement SR140, Oxoid) also &PALCAM agar' (PALCAM Listeria Selective Agar, PALCAM agar base CM877 and supplement SR150E, Oxoid) were used. Plates were streaked after 48 h as in the method and in addition also after 24 h. The streak after 24 h was done to eliminate the di!erence between the two methods with reference to the number of times the broths were streaked out and the number of plates used. 2.4.1. Modixed IDF method In a test with two of the Gorgonzola cheese brands the IDF method was altered. The Listeria enrichment broth base in the IDF method was modi"ed (FDA, 1998) by replacing the dipotassium phosphate with the bu!er used
Table 1 Diagram of procedures Day
IDF method
ISO method
Monday
Test portion of 25 g#selective Enrichment medium 225 ml. 48 h at 303C Streak onto Oxford and PALCAM medium. 48 h at 373C
Test portion of 25 g#Half Fraser 225 ml (Primary enrichment). 30 h at 303C. Streak onto Oxford and PALCAM agar. 48 h at 373C. Secondary enrichment 48 h at 373C.
Tuesday Wednesday Thursday
Streak onto Oxford and PALCAM medium. 48 h at 373C Select 5 characteristic colonies for conxrmation from each agar medium
Friday
Select 5 characteristic colonies for con"rmation from each agar medium.
Monday
Modi"cations are marked with italics.
Streak onto Oxford and PALCAM media. (48) 96 h at 373C. Select 5 characteristic colonies for con"rmation from each agar medium.
Select 5 characteristic colonies for con"rmation from each agar medium
E. Waak et al. / International Dairy Journal 9 (1999) 149}155
in the ISO procedure: Na HPO ) 2H O (12 g l\) and KH PO (1.35 g l\) and pH adjusted to 7.2$0.2 (the pH of the ordinary broth is 7.3$0.1) (Table 1).
Table 2 Recovery of L. monocytogenes in inoculated AG delost samples Strain
2.5. ISO method This method was performed according to the ISO Draft International Standard ISO/DIS 11290-1 (ISO method). Half Fraser broth was prepared from Oxoid Fraser broth base CM895 and supplements were added to 225 ml broth. Fraser broth was prepared from Oxoid Fraser broth base CM895 and the supplements were added to 10 ml. After a preliminary check after 24 h, the incubation time of the second isolation step on Oxford and PALCAM agar, was lengthened to 96 h instead of 48 h to avoid work at weekends (Table 1). 2.6. Conxrmation Market cheeses: Five presumptive L. monocytogenes colonies, or all when fewer were present, were isolated from both PALCAM and Oxford agar. The ISO procedure was used for con"rmation with a few modi"cations. The motility test and CAMP test were excluded and when haemolytic activity was weak, the commercial probe AccuProbe (GENE-PROBE威) was used to determine whether the isolates were L. monocytogenes or not. Two extra carbohydrates were used, mannitol as a negative control for Listeria and glucose as a positive control of fermentation. The control strain L. monocytogenes NCTC 7973 was used to test all media in each test. L. monocytogenes isolates were in addition serotyped with Listeria O Antiserum 1 and 4 (Difco laboratories, Detroit, Michigan, US). From the inoculated cheeses the con"rmation of Listeria was simpli"ed. All typical isolates were tested for haemolytic activity on horse blood agar and production of catalase. Cell shape and tumbling motility (203C) were studied on one out of the "ve isolates. Non-haemolytic Listeria spp naturally occurring were in addition tested (one isolate per occasion) for fermentation of rhamnose, xylose, mannitol and glucose. 2.7. Statistical analyses The McNemar Chi-square test was used to detect di!erences between proportions (McNemar, 1947). A difference at the 5% level was considered to be statistically signi"cant (Wardlaw, 1985).
3. Results and discussion 3.1. Detection of L. monocytogenes in A$ delost (Table 2) The recovery of L. monocytogenes from the inoculated AG delost samples was nearly 100% and the methods were
151
No. of inoculated Inoculation samples level/25 g
SLU 92
16 8 6 SLU 592 10 10 Total 50
1}9 10}99 100}200 1}9 10}99
ISO
IDF
PE
SE 24 h
48 h
14 8 6 10 10 48
15 8 6 10 10 49
15 8 6 10 10 49
15 8 6 10 10 49
Primary enrichment. Secondary enrichment.
Table 3 Recovery of L. monocytogenes strain SLU 92 in Gorgonzola samples Cheese brand
No. of inoculated Inoculation samples level/25 g
No. samples positive for L. monocytogenes ISO
A
B C
Total
2 2 1 9 1 12 1 28
1}9 10}99 100}200 10}99 1}9 10}99 100}200
IDF
PE
SE 24 h
48 h
2 0 1 2 0 3 1 9
2 1 1 5 0 3 1 13
2 0 0 0 0 2 1 5
2 0 0 0 0 2 1 5
Primary enrichment. Secondary enrichment.
equally good. Of 16 cheeses inoculated with 1}9 cfu L. monocytogenes (strain SLU 92) per 25 g, 15 were con"rmed as L. monocytogenes positive with both methods. Both methods failed to detect the L. monocytogenes strain when inoculation was performed in a very old cheese of a lower fat content that had been stored far beyond the best-before date. This cheese had a #ora of Bacillus spp, which grew well with blackened colonies on both Oxford and PALCAM agar and disturbed the interpretation of the plates. All inoculations made with 10 or more L. monocytogenes per 25 g with strain SLU 92 were detected with both methods. With strain SLU 592 all inoculations were found positive with both methods. No other species of Listeria were found in the AG delost. 3.2. Detection of L. monocytogenes in Gorgonzola cheese (Tables 3 and 4) The recovery of the inoculated L. monocytogenes in the Gorgonzola cheeses was much lower than in the AG delost with both methods. L. monocytogenes was recovered in
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only about 50% of the samples (13/28; Table 3, 15/28; Table 4) with the ISO method, and in 18% (5/28; Tables 3 and 4) with the IDF method. Even inoculation rates of more than 10 cfu per gram could not be recovered by the IDF method when the Gorgonzola brands A and B were used. The di!erence in recovery rate between the two methods is signi"cant (P"0.005, Table 3 and P"0.002, Table 4). The two-stage enrichment procedure and another bu!ering system may explain the higher performance of the ISO method. The di$culty in recovering L. monocytogenes in the Gorgonzola cheeses can be explained by several factors. A contamination #ora of L. innocua was frequently found in these cheeses, especially in brand C (see Table 7). This #ora grows faster than the inoculated L. monocytogenes strains (Petran & Swanson, 1993). The outgrowth of L. monocytogenes may also be a!ected by other competitive micro-organisms (In't Veld, Notermans & van de Berg, 1995; Vlaemynck & Moermans, 1996). Bacteriocin-producing enterococci (Martin, Friedrich, Beyer & Terplan, 1994) and lactobacilli (Asperger, Url & Brandl, 1989; Farias, De Ruiz Hogado & Sesma, 1993; Gira!a, Picchioni, Neviani & Carminati, 1995; Ali, Lacroix, Thuault,
Table 4 Recovery of L. monocytogenes strain SLU 592 in Gorgonzola samples Cheese brand
No. of inoculated Inoculation samples level/25 g
No. samples positive for L. monocytogenes ISO
A
B C Total
3 1 1 9 1 13 28
1}9 10}99 100}200 10}99 1}9 10}99
Bourgeois & Simard, 1995; Stecchini, Aquili & Sarais, 1995) may also inhibit the growth. Presence of free medium-chain fatty acids in blue mould cheese has also been suggested to inhibit the growth of Listeria. Free medium-chain fatty acids dissolved in butteroil have been shown to inhibit growth of a test strain of L. monocytogenes (Kinderlerer, Matthias & Finner, 1996). 3.3. Comparison of 24 h and 48 h selective enrichment Previous studies (McClain & Lee, 1988; Slade & Collins-Thompson, 1987) have shown that two-stage enrichment procedure, e.g. the ISO method, improves the detection of Listeria when a competitive #ora is present. However, six days are needed to perform the ISO analyses. Thus, work must be done also at weekends. It is also a disadvantage for the food industry to wait an extra day (compared with the IDF method) for results. With the ISO method, 77 of 106 (72.6%) inoculated samples were found positive for the presence of L. monocytogenes after the secondary enrichment. Of these, 69 were found to be positive already after the primary enrichment (Tables 2}4). The IDF method di!ers from the ISO method in that the total number of samples positive for the presence of L. monocytogenes was lower. The IDF method detected the same number of positive samples after 24 and 48 h 59/106 (55.5%) (Tables 2}4). 3.4. Modixed IDF method (Table 5)
IDF
PE
SE 24 h
48 h
2 0 1 4 1 4 12
2 0 1 7 1 4 15
2 0 0 0 0 3 5
2 0 0 0 0 3 5
Primary enrichment. Secondary enrichment.
A small test with 16 samples of Gorgonzola was performed to see if the IDF method was improved by bu!ering the enrichment medium. The results indicate that the recovery of L. monocytogenes was improved, as 5/16 (31%) became positive with the modi"cation compared with 3/16 (18%) with the non-modi"ed method (Table 5). When pH was measured in the non-inoculated portion on the second day of incubation the ordinary enrichment medium had a pH of 6.1 and the modi"ed enrichment medium a pH of 6.8. Media maintaining their pH at neutral values have been reported to perform better than
Table 5 Results of a modi"ed IDF method (mIDF) compared to the methods of ISO and IDF Cheese brand
B B C C Total
Strain
SLU SLU SLU SLU
92 592 92 592
Inoculation level/25 g
10}99 10}99 10}99 10}99
These samples are also included in Tables 3 and 4.
No. of inoculations
4 4 4 4 16
No. of samples positive for L. monocytogenes ISO method
IDF method
mIDF
2 3 1 0 6
0 2 1 0 3
3 2 0 0 5
E. Waak et al. / International Dairy Journal 9 (1999) 149}155
153
Table 6 Market cheeses where L. monocytogenes was detected Cheese no.
Type of cheese
Type of milk
Methods of detection ISO
2 16 8 18 19 13 20
Semi-hard, Bv Semi-hard, Bv Soft, Sm Soft, Sm Soft, Sm Soft, Sm Hard, smear
HT, cows' HT, cows' R, cows' R, cows' R, cows' R, ewes' *
Sero group IDF
PE
SE
24 h
48 h
Pos Pos Pos Pos Neg Neg Pos
Pos Pos Pos Pos Pos Neg Pos
Pos Pos Neg Pos Pos Pos Pos
Pos Pos Neg Pos Pos Pos Pos
1/2 1/2 1/2 ND ND 1/2 1/2
Classi"cation according to Fox (1993). Bv"Blue veined internal mould, Sm"white surface mould, HT"heat treated, R"raw milk, ND"not determined. Primary enrichment. Secondary enrichment.
Table 7 Market cheeses and Gorgonzola types A, B and C where L. innocua was detected Cheese number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Gorgonzola Gorgonzola Gorgonzola Total no. of
Type of cheese
Soft, smear Semi-hard, Bv Soft, smear Soft, Sm Soft, Sm Soft, Sm and smear Soft, Sm Soft, Sm Soft, Sm Soft, Sm Soft, Bv Soft, smear Soft, Sm Soft, smear type A, 3 samples type B, 4 samples type C, 6 samples positive samples
Method of detection/ L. innocua"#
(Table 7). L. innocua was detected by use of the ISO method in 20 of the samples (matrices included) and by use of the IDF method in 14 of the samples. The di!erence is close to signi"cance (P"0.058), but did not quite reach the 5% level.
ISO
IDF
3.6. PALCAM and Oxford agar (Table 8)
# # # # # # # # # # # # ! ! 1#, 2! 1#, 3! 6# 20
! ! # ! ! # ! # # # ! ! # # 1#, 2! 4! 6# 14
On PALCAM agar Listeria spp. form colonies which are approximately 2 mm in diameter, grey}green in colour with a black sunken centre and a black halo against a cherry-red medium background (van Netten, Perales, van de Moosdijk, Curtis & Mossel, 1989). On Oxford agar the colonies are black with a sunken centre and a black halo (Curtis, Mitchell, King & Gri$n, 1989). The sizes are the same as on PALCAM medium. On some occasions in the present study, a black halo was observed on Oxford agar, but the actual colonies were more or less overgrown. When making a streak from such a plate onto PALCAM agar, typical Listeria colonies appeared. The high selectivity of the PALCAM medium (van Netten et al., 1989; Cantoni, Valenti & d'Aubert, 1990) made it easier to detect the Listeria colonies, especially after primary enrichment (ISO method) in the present study. Improvement in the detection of Listeria spp. by use of PALCAM agar when a high background #ora is present has also been reported by other authors (Hammer, Hahn, Kirchho! & Heeschen, 1990; Gunasinghe, Henderson & Rutter, 1994). The number of plates with typical Listeria spp. was calculated for each of the two agar types for all analyses (Table 8). The use of two parallel agar media after the secondary enrichment (ISO method) seems excessive. No presumptive Listeria spp. would have been missed if only PALCAM agar had been used and only one out of 130 positive "ndings (0.8%) if only Oxford agar had been used (Table 8).
media with a low bu!ering capacity (Warburton et al., 1992; Beumer, Gi!el, Anthonie & Cox, 1996). 3.5. L. monocytogenes in market cheese samples (Tables 6 and 7) L. monocytogenes was found in 7 of the market cheeses (9%). Both methods detected L. monocytogenes in the same number of samples but not always in the same cheeses (Table 6). Cheese nos. 2, 8 and 13 not only harboured L. monocytogenes but also Listeria innocua
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E. Waak et al. / International Dairy Journal 9 (1999) 149}155
Table 8 Total number of Listeria spp. positive analyses Method
ISO IDF
Number of positive analyses
130 110
Number of plates with typical Listeria colonies Primary enrichment (ISO) 24 h (IDF)
Secondary enrichment (ISO) 48 h (IDF)
PALCAM
Oxford
PALCAM
Oxford
116 106
102 101
130 110
129 106
4. Conclusions The ISO and the IDF methods detected L. monocytogenes in the same number of inoculated AG delost samples and market cheese samples. However, the recovery of L. monocytogenes in inoculated samples of Gorgonzola was signifcantly higher with the ISO method. The PALCAM agar more frequently revealed Listeria colonies in the present study as less growth of nonListeria colonies occurred on this substrate than on the Oxford agar.
5. Addendum Since completion of this study, the Revised Provisional IDF standard 143:1990 (1990) has become International IDF standard 143A: 1995 (1995). The only di!erence from the provisional standard is that the references for guidance of sampling and preparation have been updated and given new names. The recommended pH of the enrichment broth and blood agar base has also been changed from 7.3$0.1 to 7.3$0.2. The ISO draft international standard ISO 11290-1 has become International standard ISO 11290-1. The changes from the draft standard are; a margin to the incubation time of the "rst incubation step is included (24$2 h), colony appearance on Oxford and PALCAM agar are somewhat di!erently described, and a choice between three incubation temperatures can be made (30, 35 or 373C). The incubation time and temperature of the carbohydrate utilisation tests has been changed from 373C for up to 7 d to 35 or 373C for up to 5 d. In the draft standard, a choice between horse and sheep de"brinated blood can be made, in the standard only sheep de"brinated blood is mentioned. The composition of phosphate-bu!ered saline is also added in the standard.
Acknowledgements The authors wish to express thanks to Arla R & D for funding the study, Janet Ha kansson for excellent tech-
nical assistance, Prof. J. Bille for providing L. monocytogenes strain SLU 592, and Dr. Anders Christiansson for statistical support.
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