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Lithium chloride phenylethanol moxalactam (LPM) agar
Handbook of Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 2003 Elsevier Science B.V. All rights reserved
501
Lithium chloride Phenylethanol Moxalactam (LPM) agar This monograph has been reviewed by members of the IUMS-ICFMH Working Party on Culture Media and given 'Proposed' status. Description and history LPM agar was developed in 1986 to recover Listeria spp. from very mixed microflora in UVM enrichment broths (q.v.) and it is more selective for listeria than other media developed previously (Lee and McClain, 1986). LPM's selective nature is used successfully by the USDA (McClain and Lee, 1988) and the FDA (Datta et al., 1988) to enumerate >100/g of Listeria spp. or Listeria monocytogenes from many kinds of contaminated foods by direct plating without enrichment. Listeria spp. will form colonies on LPM agar in 24 h at 30~ while most, but not all, of the competing microflora are suppressed. Listeria colonies can be recognised and selected for identification using the 45 ~ transillumination of Henry (1933). Listeria spp. which have been frozen grow well on LPM agar but heat injured cells do not. Composition (grams) Pancreatic digest of casein Peptone (peptic digest of animal tissue) Beef extract Sodium chloride Lithium chloride Glycine anhydride* (Sigma G-7251) 2-phenylethanol (Sigma P-6134) Sodium or ammonium moxalactam Agar Distilled or deionized water
5.0 5.0 3.0 5.0 5.0 10.0 2.5 0.02 15.0 1000.0
*Note: Do not substitute glycine anhydride with glycine, which makes the LPM very inhibitory to growth of Listeria spp.
502
Preparation Base Suspend the ingredients except the moxalactam in bottles or flasks containing a magnetic stirring bar and autoclave the medium for 12 min at 121~ Mix the medium gently after autoclaving. LPM is heat sensitive and so it is important to cool it promptly to 46~ in a water bath. Moxalactam solution Sodium or ammonium moxalactam (Eli Lilly or Sigma M-1900) 1.0 g Potassium buffer (0.1M) pH 6.0 100.0 ml (Made by adding 0.1 M mono-potassium phosphate solution to 0.1 M di-potassium hydrogen phosphate solution to reach pH 6.0). Sterilize by filtration through 0.2 ~tm filter. Store both the powder and 3-5 ml aliquots of the 1% moxalactam in a freezer at -60~ Complete medium Add 2 ml 1% moxalactam solution per litre of the autoclaved and cooled molten base while stirring with a magnetic mixer. Dispense 12 ml per 9 cm diameter Petri dish.
Physical properties Appearance pH
Almost clear and colourless. 7.3 + 0.2
Shelf life Dehydrated agar base Ready to use medium
Store at 2-8~ to prevent loss of selectivity. Use before manufacturer's expiry date. 2-3 weeks at 4 + 2~ It is essential to store plates in sealed plastic bags as the phenylethanol is volatile.
Inoculation method for samples Surface streaking or modified Miles-Misra. For rapid semiquantitative detection of >100/g of Listeria spp. in foods, swab the surface of solid samples with a moistened (dry for liquid samples) swab, and inoculate half of a LPM plate. Streak the other half of the plate with a sterile loop (McClain and Lee, 1988). Liquid samples can be concentrated by centrifugation before testing.
503
Incubation method LPM plates are usually placed in thin polyethylene plastic bags and incubated at 30~ in air for 20-24 h for isolation, and 40 h for counting colonies without magnification.
Reading of results and interpretation Under optimum 45 ~ transillumination the more isolated and larger (24h old) listeria colonies appear as whitish piles of crushed glass often showing mosaic-like internal structures occasionally having blue-grey iridescent areas. Smaller, more crowded, colonies have a more pronounced blue-grey iridescence that tends to sparkle. When growth becomes near confluent an even blue-grey iridescent sheen can be observed. For quantitative determination, count all the colonies that grow on LPM agar in 40 h and then identify the percentage of these colonies that are Listeria spp. or Listeria monocytogenes. For semiquantitative estimation, simply compare the density of all the colonies in the swabbed area with standard density photos of 102, 103, 104, 105, l0 6 a n d 10 7 cfu/ml of Listeria monocytogenes swabbed and streaked on LPM agar (similar to Oxoid Dip Slide) and then identify from the streaked area the percentage of colonies as Listeria spp. or Listeria monocytogenes. Alternatively, scan the swab and streak plates at 24 h using 45 ~ transillumination, and quantitatively enumerate the positive samples using the decimal dilution plating technique.
Quality assessment (i) Productivity Test strains
Inoculation method
(ii) Selectivity Test strains
Inoculation method
Listeria monocytogenes serovar 1/2a (ATCC 35152/ NCTC 7973) Listeria monocytogenes serovar 4b (ATCC 13932/ NCTC 10527) Listeria ivanovii serovar 5 NCIMB 50095 Modified Miles-Misra or spiral plating.
Enterococcus faecalis NCIMB 50030 Proteus mirabilis (ATCC 29906/CECT 4168/NCTC 11938) Modified Miles-Misra or spiral plating.
(iii) Characteristic appearance of colonies See above.
504 References Datta, A.R., Wentz, B.A., and Hill, W. E. (1988) Identification and enumeration of beta-haemolytic Listeria monocytogenes in naturally contaminated dairy products. J. Assoc. Off. Anal. Chem. 71, 673-675. Domjan Kovacs, H. and Ralovich, B. (1991) Model examination of selective media for isolation of Listeria strains. Acta Microbiol. Hung. 38, 141-145. Henry, B.S. (1933) Dissociation in the genus Brucella. J.Infect.Dis. 52, 374-402. Lee, W.H. and McClain, D. (1986) Improved Listeria monocytogenes selective agar. Appl. Environ. Microbiol. 52, 1215-1217. McClain, D. and Lee, W.H. (1988) Development of the USDA-FSIS method for the isolation of Listeria monocytogenes. J.Assoc.Off. Anal. Chem. 71, 660-664.
501
Lithium chloride Phenylethanol Moxalactam (LPM) agar This monograph has been reviewed by members of the IUMS-ICFMH Working Party on Culture Media and given 'Proposed' status. Description and history LPM agar was developed in 1986 to recover Listeria spp. from very mixed microflora in UVM enrichment broths (q.v.) and it is more selective for listeria than other media developed previously (Lee and McClain, 1986). LPM's selective nature is used successfully by the USDA (McClain and Lee, 1988) and the FDA (Datta et al., 1988) to enumerate >100/g of Listeria spp. or Listeria monocytogenes from many kinds of contaminated foods by direct plating without enrichment. Listeria spp. will form colonies on LPM agar in 24 h at 30~ while most, but not all, of the competing microflora are suppressed. Listeria colonies can be recognised and selected for identification using the 45 ~ transillumination of Henry (1933). Listeria spp. which have been frozen grow well on LPM agar but heat injured cells do not. Composition (grams) Pancreatic digest of casein Peptone (peptic digest of animal tissue) Beef extract Sodium chloride Lithium chloride Glycine anhydride* (Sigma G-7251) 2-phenylethanol (Sigma P-6134) Sodium or ammonium moxalactam Agar Distilled or deionized water
5.0 5.0 3.0 5.0 5.0 10.0 2.5 0.02 15.0 1000.0
*Note: Do not substitute glycine anhydride with glycine, which makes the LPM very inhibitory to growth of Listeria spp.
502
Preparation Base Suspend the ingredients except the moxalactam in bottles or flasks containing a magnetic stirring bar and autoclave the medium for 12 min at 121~ Mix the medium gently after autoclaving. LPM is heat sensitive and so it is important to cool it promptly to 46~ in a water bath. Moxalactam solution Sodium or ammonium moxalactam (Eli Lilly or Sigma M-1900) 1.0 g Potassium buffer (0.1M) pH 6.0 100.0 ml (Made by adding 0.1 M mono-potassium phosphate solution to 0.1 M di-potassium hydrogen phosphate solution to reach pH 6.0). Sterilize by filtration through 0.2 ~tm filter. Store both the powder and 3-5 ml aliquots of the 1% moxalactam in a freezer at -60~ Complete medium Add 2 ml 1% moxalactam solution per litre of the autoclaved and cooled molten base while stirring with a magnetic mixer. Dispense 12 ml per 9 cm diameter Petri dish.
Physical properties Appearance pH
Almost clear and colourless. 7.3 + 0.2
Shelf life Dehydrated agar base Ready to use medium
Store at 2-8~ to prevent loss of selectivity. Use before manufacturer's expiry date. 2-3 weeks at 4 + 2~ It is essential to store plates in sealed plastic bags as the phenylethanol is volatile.
Inoculation method for samples Surface streaking or modified Miles-Misra. For rapid semiquantitative detection of >100/g of Listeria spp. in foods, swab the surface of solid samples with a moistened (dry for liquid samples) swab, and inoculate half of a LPM plate. Streak the other half of the plate with a sterile loop (McClain and Lee, 1988). Liquid samples can be concentrated by centrifugation before testing.
503
Incubation method LPM plates are usually placed in thin polyethylene plastic bags and incubated at 30~ in air for 20-24 h for isolation, and 40 h for counting colonies without magnification.
Reading of results and interpretation Under optimum 45 ~ transillumination the more isolated and larger (24h old) listeria colonies appear as whitish piles of crushed glass often showing mosaic-like internal structures occasionally having blue-grey iridescent areas. Smaller, more crowded, colonies have a more pronounced blue-grey iridescence that tends to sparkle. When growth becomes near confluent an even blue-grey iridescent sheen can be observed. For quantitative determination, count all the colonies that grow on LPM agar in 40 h and then identify the percentage of these colonies that are Listeria spp. or Listeria monocytogenes. For semiquantitative estimation, simply compare the density of all the colonies in the swabbed area with standard density photos of 102, 103, 104, 105, l0 6 a n d 10 7 cfu/ml of Listeria monocytogenes swabbed and streaked on LPM agar (similar to Oxoid Dip Slide) and then identify from the streaked area the percentage of colonies as Listeria spp. or Listeria monocytogenes. Alternatively, scan the swab and streak plates at 24 h using 45 ~ transillumination, and quantitatively enumerate the positive samples using the decimal dilution plating technique.
Quality assessment (i) Productivity Test strains
Inoculation method
(ii) Selectivity Test strains
Inoculation method
Listeria monocytogenes serovar 1/2a (ATCC 35152/ NCTC 7973) Listeria monocytogenes serovar 4b (ATCC 13932/ NCTC 10527) Listeria ivanovii serovar 5 NCIMB 50095 Modified Miles-Misra or spiral plating.
Enterococcus faecalis NCIMB 50030 Proteus mirabilis (ATCC 29906/CECT 4168/NCTC 11938) Modified Miles-Misra or spiral plating.
(iii) Characteristic appearance of colonies See above.
504 References Datta, A.R., Wentz, B.A., and Hill, W. E. (1988) Identification and enumeration of beta-haemolytic Listeria monocytogenes in naturally contaminated dairy products. J. Assoc. Off. Anal. Chem. 71, 673-675. Domjan Kovacs, H. and Ralovich, B. (1991) Model examination of selective media for isolation of Listeria strains. Acta Microbiol. Hung. 38, 141-145. Henry, B.S. (1933) Dissociation in the genus Brucella. J.Infect.Dis. 52, 374-402. Lee, W.H. and McClain, D. (1986) Improved Listeria monocytogenes selective agar. Appl. Environ. Microbiol. 52, 1215-1217. McClain, D. and Lee, W.H. (1988) Development of the USDA-FSIS method for the isolation of Listeria monocytogenes. J.Assoc.Off. Anal. Chem. 71, 660-664.