International Journal of Food Microbiology, 13 (1991) 295-300 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
295
F O O D 00423
Recovery of Listeria monocytogenes on selective agar media in a collaborative study using reference samples P . H . in 't V e l d 1 a n d E. de Boer 2 1 Laboratory for Water and Food Microbiology, National Institute of Public Health and Enc,ironmental Protection, Bilthot~en, and 2 Inspectorate for Health Protection, Zutphen, The Netherlands" (Received 10 D e c e m b e r 1990; revision received 2 April 1991; accepted 22 May 1991)
Sixteen laboratories compared counts of Listeria monocytogenes in reference samples using Blood agar, Palcam(y) agar and Oxford agar. Significant differences were found between laboratories. T h e m e a n counts on Blood agar were significantly higher than on Palcam(y) or Oxford agar. The m e a n counts on Palcamy agar were somewhat higher than on Oxford agar (only after 48 h incubation), but no significant difference was found. Addition of egg yolk to Palcam agar seems to be benificial for the recovery of sublethally injured cells. Recovery of L. monocytogenes was higher after 48 h incubation for all media tested. Key words: L&teria monocytogenes; Recovery; Comparative study; Reference sample
Introduction
Recent outbreaks of listeriosis emphasized the need for effective methods for the isolation, enumeration and identification of Listeria monocytogenes. Though many Listeria isolation media are described, only a few of these are suitable for the selective isolation or enumeration of Listeria species, including L. monocytogenes from foods. Van Netten et al. (1989) described Palcam agar, a selective and differential agar for the isolation of Listeria. The selectivity of this medium is based on the inhibiting substances polymyxin B sulphate, acriflavin, lithium chloride and ceftazidime. Differential diagnosis is possible using the components aesculin, ferric ammonium citrate, mannitol and phenol red, resulting in gray-green coloured Listeria colonies with a black-brown halo. Recovery of sublethally injured ceils on Palcam agar can be improved by the addition of egg yolk.
Correspondence address." E. de Boer, Inspectorate for Health Protection, P.O. Box 9012, 7200 G N Zutphen, The Netherlands.
296
Oxford agar according to Curtis et al. (1989) contains colistin, cefotetan, fosfomycin, acriflavin, cycloheximide and lithium chloride as selective substances. The aesculin-ferric ammonium citrate indicator system results in Listeria colonies surrounded by black haloes. A collaborative study was conducted in 16 laboratories to evaluate the productivity of the Palcamy and Oxford media in relation to blood agar as enumeration media for L. monocytogenes. Also, five laboratories tested Palcam agar. For this purpose reference samples were used as developed by the National Institute of Public Health and Environmental Protection with support of the European Communities Bureau of Reference (BCR).
Materials and Methods
Reference samples Listeria monocytogenes reference samples consisted of gelatin capsules with 0.25 g of spray-dried milk, artificially contaminated with L. monocytogenes strain Scott A; serotype 4b at a contamination level of approx. 104 cfu/capsule. The samples were prepared as described by In 't Veld et al. (1991). Media and reagents Blood agar plates prepared in one batch from Columbia Blood Agar Base (Oxoid CM331) and 5% defribinated sheep blood were distributed to the participants. Palcam agar with the addition of 2.5% egg yolk (Palcamy agar) was prepared by each participating laboratory according to Van Netten et al. (1989). Five laboratories also tested Palcam agar without the addition of egg yolk. Oxford agar, consisting of Listeria Selective Medium Base (Oxoid CM836) and Listeria Selective Supplement (Oxoid SR140), was prepared from one batch of dehydrated medium by the participating laboratories. Commercial media were prepared according to the manufacturer's instructions. Confirmation Typical colonies were inoculated on Tryptone Soya Agar (Oxoid CM131) plates. The plates were incubated for 24 h at 37 ° C. Single colonies were further tested for Gram-staining (positive), catalase (positive), glucose (positive slant and butt in Triple Sugar Iron Agar; Oxoid CM277) and hemolysis (positive). For the hemolysis test, a 2% sheep erythrocytes suspension was prepared by washing sterile defribinated sheep blood three times in PBS-buffer, pH 7.4. From this suspension 100 Izl was pipetted in duplicate in cups of a microtiter plate (round bottom). To the erythrocytes suspension 100/xl of a Listeria culture (grown 48 h at 37°C in Brain Heart Infusion Broth; Oxoid CM 225) was added. The microtiter plate was incubated for 45 rain at 37 ° C, followed by incubation for 2 h at 4 ° C. The presence of hemolysins was shown by a homogeneous red liquid. A L.
297 monocytogenes and a L. innocua strain were tested simultaneously as a positive
and negative control. Procedure
Each participating laboratory received a series of 10 test capsules. The capsules were added to tubes (¢ 26 cm) with 10 ml of physiological peptone salt solution, preheated to 37 °C. The tubes were incubated in a water bath at 37°C. The solution was mixed for 5 s. on a Vortex mixer after 5, 15 and 30 min of incubation. After the last mixing, 0.1 ml of solution from each tube was spread in duplicate on plates (~ 9 cm) with Palcam and Palcamy agar, Oxford agar and Blood agar. The plates were incubated at 3 7 ° C aerobically (Blood agar and Oxford agar) or microaerobically (Palcam and Palcamy agar). Typical colonies were counted after 1 and 2 days incubation and confirmed (3-5 colonies from each plate). Statistical analysis
For each laboratory the mean number of typical colonies after 24 and 48 h incubation and the ratio of the mean number of colony forming units (cfu) on a selective medium to that on blood agar were calculated. Differences between laboratories were calculated with analysis of variance, based on log transformed counts (Snedecor and Cochran, 1980). Using the standard error of difference between two means, it was calculated which pair of two participating laboratories obtained significantly different counts, by applying the following formula: log mean counts (lab x ) - l o g mean counts (lab y) standard error of difference of means
= t-partition
Using the Wilcoxon matched paired signed test (Wardlaw, 1985), differences in productivity between media were tested. The average counts per laboratory after 48 h incubation, as given in Table I, were used for ranking. Data from all laboratories were used, except for those laboratories which did not find colonies on Oxford agar.
Results and Discussion
The results are summarized in Table I. Counts on blood agar Counts of L. monocytogenes on blood agar after 24 h incubation were for most
laboratories 90% of the numbers found after 48 h incubation. The mean count after 48 h incubation was 90.3 (range 70.4-111.2) cfu/plate. Analysis of variance showed significant differences ( P < 0.01) between laboratories. In comparison to laboratory 5, the laboratory which counted nearest to the mean count of all laboratories, the laboratories 1, 3, 4 and 10 found significantly lower and labora-
298 TABLE I Mean counts after 48 h incubation for the tested media and the participating laboratories Laboratory
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 mean
colony counts Blood agar
Palcamy agar
Oxford agar
Palcam agar
70,9 89,3 74,8 73.5 90.6 107.8 77,9 91.3 111.2 70.4 95.7 94.9 98.4 97.0 99.7 101.3
52.2 (74) a 70.6 (79) 67.2 (90) 37.0 (44) 62.1 (69) 81.6 (76) 46.5 (60) 52.3 (57) 77.6 (70) 65.3 (92) 53.4 (56) 71.5 (75) 64.0 (65) 68.3 (70) 55.3 (56) 76.6 (76)
54.3 (77) 44.9 (50) 55.8 (75) 0.0 (0) 40.0 (44) 68.1 (63) 0.0 (0) 62.8 (69) 70.5 (63) 0.0 (0) 51.7 (54) 58.3 (61) 67.1 (68) 70.6 (73) 67.1 (67) 52.7 (52)
43.0 (61) 46.4 (52) _ c -
62.6 (69)
47,7 (53) 58.8 (65) t~
47.6 (53)
90.3
24.4 (25) 65.0 (67) 59.1 (58)
" Percentage mean count on selective medium with regard to that on blood agar after 48 h incubation. b Without the results of laboratories 4, 7 and 10. c Not done.
tory 9 significantly higher counts. The value of the standard error of differences b e t w e e n t w o m e a n s f o r t h i s m e d i u m w a s 0 . 0 3 3 5 7 o n t h e log s c a l e .
Counts on Palcam and Palcarny agar M e a n c o u n t s o f L. monocytogenes c o l o n i e s o n P a l c a m y a g a r o b t a i n e d a f t e r 24 h i n c u b a t i o n w e r e 5 7 % o f t h e n u m b e r o f c o l o n i e s f o u n d a f t e r 48 h i n c u b a t i o n . T h r e e l a b o r a t o r i e s (4, 10 a n d 16) c o u l d n o t c o u n t c o l o n i e s a f t e r 24 h i n c u b a t i o n . F r o m t h e s e l a b o r a t o r i e s o n l y l a b o r a t o r y 4 f o u n d a low n u m b e r o f c o l o n i e s a f t e r 48 h i n c u b a t i o n . T h e m e a n c o u n t o n P a l c a m y a g a r a f t e r 48 h i n c u b a t i o n w a s 62.6 cfu/plate, which was in average 69% (range 44-92%) of the counts on blood agar. A n a l y s i s o f v a r i a n c e s h o w e d s i g n i f i c a n t d i f f e r e n c e s ( P < 0.01) b e t w e e n l a b o r a t o ries. T h e v a l u e o f t h e s t a n d a r d e r r o r o f d i f f e r e n c e s b e t w e e n t w o m e a n s f o r t h i s m e d i u m w a s 0 . 0 3 6 1 2 o n t h e log s c a l e . L a b o r a t o r i e s 4 a n d 7 f o u n d s i g n i f i c a n t l y l o w e r n u m b e r s a n d l a b o r a t o r i e s 6, 9 a n d 16 s i g n i f i c a n t l y h i g h e r n u m b e r s w h e n c o m p a r e d t o l a b o r a t o r y 5. A s b o t h P a l c a m a n d P a l c a m y , in c o n t r a s t t o t h e commercial Oxford formulae, were prepared by each participating laboratory from i n g r e d i e n t s , d i f f e r e n c e s in p r o d u c t i v i t y o f t h e m e d i a p r e p a r e d c o u l d h a v e c o n tributed to the obtained variation.
299
The five laboratories which tested Palcam agar found lower counts on this medium than on Palcamy agar. Three of these five laboratories could not count colonies after 24 h incubation.
Counts on Oxford agar Counts of L. monocytogenes colonies on Oxford agar obtained after 24 h incubation were 79% of the number of colonies found after 48 h incubation, varying from 0% to 92%. T h r e e laboratories (4, 7 and 10) could not count colonies after 48 h incubation. Although no explanation for their negative results could be found, incorrect preparation of the medium will probably have been the cause. The mean counts on Oxford agar after 48 h incubation (exclusive of laboratories 4, 7 and 10) was 58.8 c f u / p l a t e , which was in average 65% of the counts on blood agar. Analysis of variance (exclusive of laboratories 4, 7 and 10) showed significant differences between laboratories. The value of the standard error of differences between two means for this medium was 0.03147 on the log scale. Laboratories 2 and 5 found significantly lower and laboratories 9 and 14 significantly higher counts, when compared to laboratory 12. In general, recovery of sublethally injured L. monocytogenes was higher after 48 h incubation. Differences between media Significant ( P < 0.05) differences were found between the counts on blood agar and Palcamy or Oxford agar. The counts (per laboratory) on blood agar were consistently higher than on Palcamy or Oxford agar. Although the average counts on Palcamy agar were higher than on Oxford agar, this difference was not significant. The Wilcoxon's test could not be used to test the difference between Palcamy and Palcam agar, because only five laboratories tested Palcam agar. However, the results on Palcamy were consistently higher than on Palcam agar, indicating that the addition of egg yolk may be beneficial to recover sublethally injured cells of L. monocytogenes.
Acknowledgements The authors gratefully acknowledge the participating laboratories: Food Inspection Services in Alkmaar, Amsterdam, Enschede, Goes, 's-Gravenhage, Groningen, 's-Hertogenbosch, Leeuwarden, Maastricht, Nijmegen, Rotterdam, Utrecht and Zutphen; C I V O - T N O , Zeist; R I K I L T , Wageningen; Agricultural University, Wageningen; RIVM, Bilthoven.
References Curtis, G.D.W., Mitchell, R.G., King, A.F. and Griffin, E.J. (1989) A selective medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 8, 95-98.
300 In 't Veld, P.H., Soentoro, P.S.S., Delfgou-Van Asch, E.H.M. and Notermans, S. (1991) Influence of reconstitution on isolation and enumeration of Listeria monocytogenes from milkpowder used for reference samples. J. Food Protect. 54, 124-126. Snedecor, G.W. and Cochran, W.G. (1980) Statistical Methods. The Iowa State University Press, Ames, IA. Van Netten, P., Perales, I., Van de Moosdijk, A., Curtis, G.D.W. and Mossel, D.A.A. (1989) Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8, 299-316. Wardlaw, A.C. (1985) Practical Statistics for Experimental Biologists. John Wiley & Sons, Chichester.
295
F O O D 00423
Recovery of Listeria monocytogenes on selective agar media in a collaborative study using reference samples P . H . in 't V e l d 1 a n d E. de Boer 2 1 Laboratory for Water and Food Microbiology, National Institute of Public Health and Enc,ironmental Protection, Bilthot~en, and 2 Inspectorate for Health Protection, Zutphen, The Netherlands" (Received 10 D e c e m b e r 1990; revision received 2 April 1991; accepted 22 May 1991)
Sixteen laboratories compared counts of Listeria monocytogenes in reference samples using Blood agar, Palcam(y) agar and Oxford agar. Significant differences were found between laboratories. T h e m e a n counts on Blood agar were significantly higher than on Palcam(y) or Oxford agar. The m e a n counts on Palcamy agar were somewhat higher than on Oxford agar (only after 48 h incubation), but no significant difference was found. Addition of egg yolk to Palcam agar seems to be benificial for the recovery of sublethally injured cells. Recovery of L. monocytogenes was higher after 48 h incubation for all media tested. Key words: L&teria monocytogenes; Recovery; Comparative study; Reference sample
Introduction
Recent outbreaks of listeriosis emphasized the need for effective methods for the isolation, enumeration and identification of Listeria monocytogenes. Though many Listeria isolation media are described, only a few of these are suitable for the selective isolation or enumeration of Listeria species, including L. monocytogenes from foods. Van Netten et al. (1989) described Palcam agar, a selective and differential agar for the isolation of Listeria. The selectivity of this medium is based on the inhibiting substances polymyxin B sulphate, acriflavin, lithium chloride and ceftazidime. Differential diagnosis is possible using the components aesculin, ferric ammonium citrate, mannitol and phenol red, resulting in gray-green coloured Listeria colonies with a black-brown halo. Recovery of sublethally injured ceils on Palcam agar can be improved by the addition of egg yolk.
Correspondence address." E. de Boer, Inspectorate for Health Protection, P.O. Box 9012, 7200 G N Zutphen, The Netherlands.
296
Oxford agar according to Curtis et al. (1989) contains colistin, cefotetan, fosfomycin, acriflavin, cycloheximide and lithium chloride as selective substances. The aesculin-ferric ammonium citrate indicator system results in Listeria colonies surrounded by black haloes. A collaborative study was conducted in 16 laboratories to evaluate the productivity of the Palcamy and Oxford media in relation to blood agar as enumeration media for L. monocytogenes. Also, five laboratories tested Palcam agar. For this purpose reference samples were used as developed by the National Institute of Public Health and Environmental Protection with support of the European Communities Bureau of Reference (BCR).
Materials and Methods
Reference samples Listeria monocytogenes reference samples consisted of gelatin capsules with 0.25 g of spray-dried milk, artificially contaminated with L. monocytogenes strain Scott A; serotype 4b at a contamination level of approx. 104 cfu/capsule. The samples were prepared as described by In 't Veld et al. (1991). Media and reagents Blood agar plates prepared in one batch from Columbia Blood Agar Base (Oxoid CM331) and 5% defribinated sheep blood were distributed to the participants. Palcam agar with the addition of 2.5% egg yolk (Palcamy agar) was prepared by each participating laboratory according to Van Netten et al. (1989). Five laboratories also tested Palcam agar without the addition of egg yolk. Oxford agar, consisting of Listeria Selective Medium Base (Oxoid CM836) and Listeria Selective Supplement (Oxoid SR140), was prepared from one batch of dehydrated medium by the participating laboratories. Commercial media were prepared according to the manufacturer's instructions. Confirmation Typical colonies were inoculated on Tryptone Soya Agar (Oxoid CM131) plates. The plates were incubated for 24 h at 37 ° C. Single colonies were further tested for Gram-staining (positive), catalase (positive), glucose (positive slant and butt in Triple Sugar Iron Agar; Oxoid CM277) and hemolysis (positive). For the hemolysis test, a 2% sheep erythrocytes suspension was prepared by washing sterile defribinated sheep blood three times in PBS-buffer, pH 7.4. From this suspension 100 Izl was pipetted in duplicate in cups of a microtiter plate (round bottom). To the erythrocytes suspension 100/xl of a Listeria culture (grown 48 h at 37°C in Brain Heart Infusion Broth; Oxoid CM 225) was added. The microtiter plate was incubated for 45 rain at 37 ° C, followed by incubation for 2 h at 4 ° C. The presence of hemolysins was shown by a homogeneous red liquid. A L.
297 monocytogenes and a L. innocua strain were tested simultaneously as a positive
and negative control. Procedure
Each participating laboratory received a series of 10 test capsules. The capsules were added to tubes (¢ 26 cm) with 10 ml of physiological peptone salt solution, preheated to 37 °C. The tubes were incubated in a water bath at 37°C. The solution was mixed for 5 s. on a Vortex mixer after 5, 15 and 30 min of incubation. After the last mixing, 0.1 ml of solution from each tube was spread in duplicate on plates (~ 9 cm) with Palcam and Palcamy agar, Oxford agar and Blood agar. The plates were incubated at 3 7 ° C aerobically (Blood agar and Oxford agar) or microaerobically (Palcam and Palcamy agar). Typical colonies were counted after 1 and 2 days incubation and confirmed (3-5 colonies from each plate). Statistical analysis
For each laboratory the mean number of typical colonies after 24 and 48 h incubation and the ratio of the mean number of colony forming units (cfu) on a selective medium to that on blood agar were calculated. Differences between laboratories were calculated with analysis of variance, based on log transformed counts (Snedecor and Cochran, 1980). Using the standard error of difference between two means, it was calculated which pair of two participating laboratories obtained significantly different counts, by applying the following formula: log mean counts (lab x ) - l o g mean counts (lab y) standard error of difference of means
= t-partition
Using the Wilcoxon matched paired signed test (Wardlaw, 1985), differences in productivity between media were tested. The average counts per laboratory after 48 h incubation, as given in Table I, were used for ranking. Data from all laboratories were used, except for those laboratories which did not find colonies on Oxford agar.
Results and Discussion
The results are summarized in Table I. Counts on blood agar Counts of L. monocytogenes on blood agar after 24 h incubation were for most
laboratories 90% of the numbers found after 48 h incubation. The mean count after 48 h incubation was 90.3 (range 70.4-111.2) cfu/plate. Analysis of variance showed significant differences ( P < 0.01) between laboratories. In comparison to laboratory 5, the laboratory which counted nearest to the mean count of all laboratories, the laboratories 1, 3, 4 and 10 found significantly lower and labora-
298 TABLE I Mean counts after 48 h incubation for the tested media and the participating laboratories Laboratory
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 mean
colony counts Blood agar
Palcamy agar
Oxford agar
Palcam agar
70,9 89,3 74,8 73.5 90.6 107.8 77,9 91.3 111.2 70.4 95.7 94.9 98.4 97.0 99.7 101.3
52.2 (74) a 70.6 (79) 67.2 (90) 37.0 (44) 62.1 (69) 81.6 (76) 46.5 (60) 52.3 (57) 77.6 (70) 65.3 (92) 53.4 (56) 71.5 (75) 64.0 (65) 68.3 (70) 55.3 (56) 76.6 (76)
54.3 (77) 44.9 (50) 55.8 (75) 0.0 (0) 40.0 (44) 68.1 (63) 0.0 (0) 62.8 (69) 70.5 (63) 0.0 (0) 51.7 (54) 58.3 (61) 67.1 (68) 70.6 (73) 67.1 (67) 52.7 (52)
43.0 (61) 46.4 (52) _ c -
62.6 (69)
47,7 (53) 58.8 (65) t~
47.6 (53)
90.3
24.4 (25) 65.0 (67) 59.1 (58)
" Percentage mean count on selective medium with regard to that on blood agar after 48 h incubation. b Without the results of laboratories 4, 7 and 10. c Not done.
tory 9 significantly higher counts. The value of the standard error of differences b e t w e e n t w o m e a n s f o r t h i s m e d i u m w a s 0 . 0 3 3 5 7 o n t h e log s c a l e .
Counts on Palcam and Palcarny agar M e a n c o u n t s o f L. monocytogenes c o l o n i e s o n P a l c a m y a g a r o b t a i n e d a f t e r 24 h i n c u b a t i o n w e r e 5 7 % o f t h e n u m b e r o f c o l o n i e s f o u n d a f t e r 48 h i n c u b a t i o n . T h r e e l a b o r a t o r i e s (4, 10 a n d 16) c o u l d n o t c o u n t c o l o n i e s a f t e r 24 h i n c u b a t i o n . F r o m t h e s e l a b o r a t o r i e s o n l y l a b o r a t o r y 4 f o u n d a low n u m b e r o f c o l o n i e s a f t e r 48 h i n c u b a t i o n . T h e m e a n c o u n t o n P a l c a m y a g a r a f t e r 48 h i n c u b a t i o n w a s 62.6 cfu/plate, which was in average 69% (range 44-92%) of the counts on blood agar. A n a l y s i s o f v a r i a n c e s h o w e d s i g n i f i c a n t d i f f e r e n c e s ( P < 0.01) b e t w e e n l a b o r a t o ries. T h e v a l u e o f t h e s t a n d a r d e r r o r o f d i f f e r e n c e s b e t w e e n t w o m e a n s f o r t h i s m e d i u m w a s 0 . 0 3 6 1 2 o n t h e log s c a l e . L a b o r a t o r i e s 4 a n d 7 f o u n d s i g n i f i c a n t l y l o w e r n u m b e r s a n d l a b o r a t o r i e s 6, 9 a n d 16 s i g n i f i c a n t l y h i g h e r n u m b e r s w h e n c o m p a r e d t o l a b o r a t o r y 5. A s b o t h P a l c a m a n d P a l c a m y , in c o n t r a s t t o t h e commercial Oxford formulae, were prepared by each participating laboratory from i n g r e d i e n t s , d i f f e r e n c e s in p r o d u c t i v i t y o f t h e m e d i a p r e p a r e d c o u l d h a v e c o n tributed to the obtained variation.
299
The five laboratories which tested Palcam agar found lower counts on this medium than on Palcamy agar. Three of these five laboratories could not count colonies after 24 h incubation.
Counts on Oxford agar Counts of L. monocytogenes colonies on Oxford agar obtained after 24 h incubation were 79% of the number of colonies found after 48 h incubation, varying from 0% to 92%. T h r e e laboratories (4, 7 and 10) could not count colonies after 48 h incubation. Although no explanation for their negative results could be found, incorrect preparation of the medium will probably have been the cause. The mean counts on Oxford agar after 48 h incubation (exclusive of laboratories 4, 7 and 10) was 58.8 c f u / p l a t e , which was in average 65% of the counts on blood agar. Analysis of variance (exclusive of laboratories 4, 7 and 10) showed significant differences between laboratories. The value of the standard error of differences between two means for this medium was 0.03147 on the log scale. Laboratories 2 and 5 found significantly lower and laboratories 9 and 14 significantly higher counts, when compared to laboratory 12. In general, recovery of sublethally injured L. monocytogenes was higher after 48 h incubation. Differences between media Significant ( P < 0.05) differences were found between the counts on blood agar and Palcamy or Oxford agar. The counts (per laboratory) on blood agar were consistently higher than on Palcamy or Oxford agar. Although the average counts on Palcamy agar were higher than on Oxford agar, this difference was not significant. The Wilcoxon's test could not be used to test the difference between Palcamy and Palcam agar, because only five laboratories tested Palcam agar. However, the results on Palcamy were consistently higher than on Palcam agar, indicating that the addition of egg yolk may be beneficial to recover sublethally injured cells of L. monocytogenes.
Acknowledgements The authors gratefully acknowledge the participating laboratories: Food Inspection Services in Alkmaar, Amsterdam, Enschede, Goes, 's-Gravenhage, Groningen, 's-Hertogenbosch, Leeuwarden, Maastricht, Nijmegen, Rotterdam, Utrecht and Zutphen; C I V O - T N O , Zeist; R I K I L T , Wageningen; Agricultural University, Wageningen; RIVM, Bilthoven.
References Curtis, G.D.W., Mitchell, R.G., King, A.F. and Griffin, E.J. (1989) A selective medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 8, 95-98.
300 In 't Veld, P.H., Soentoro, P.S.S., Delfgou-Van Asch, E.H.M. and Notermans, S. (1991) Influence of reconstitution on isolation and enumeration of Listeria monocytogenes from milkpowder used for reference samples. J. Food Protect. 54, 124-126. Snedecor, G.W. and Cochran, W.G. (1980) Statistical Methods. The Iowa State University Press, Ames, IA. Van Netten, P., Perales, I., Van de Moosdijk, A., Curtis, G.D.W. and Mossel, D.A.A. (1989) Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8, 299-316. Wardlaw, A.C. (1985) Practical Statistics for Experimental Biologists. John Wiley & Sons, Chichester.
