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Lysine-Iron-Cystine-Neutral Red (LICNR) broth
357
Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 1995 Elsevier Science B.V. All rights reserved
Lysine-Iron-Cystine-Neutral Red (LICNR) broth Description and history Lysine iron cystine neutral red broth was developed by Hargrove et al. (1971) as a presumptive test for salmonellae in dairy products. This medium was further investigated and modified by Hoben et al. (1973)who found it useful for a variety of foods and food ingredients. Salmonellae decarboxylate lysine but do not ferment salicin and hence produce an alkaline reaction. In addition they produce hydrogen sulphide from cystine which reacts with the ferric salt to give a black precipitate of ferric sulphide. D'Aoust (1977) and Morgan-Jones (1982) have both reported that the colour change could only be used as a guide and the presence of salmonellae should be confirmed by conventional methods.
Composition (grams) L-Lysine Tryptone Yeast extract Mannitol Glucose Salicin Iron (III) ammonium citrate (brown) Sodium thiosulphate L-Cystine Neutral red Distilled or deionized water
10.0 5.0 3.0 5.0 1.0 1.0 0.5 0.1 0.1 0.025 1000.0
Preparation Suspend all the ingredients in the water and heat to dissolve, mix well and adjust pH to 6.2. Dispense into tubes in appropriate quantities. Either use immediately or sterilize at 121~ for 15 min.
Physical properties Appearance pH
Red, clear. 6.2 + 0.2
358
Shelf life Ready to use medium
7 days at 4 + 2~ in the dark.
Inoculation method for samples Inoculate broths with liquid food or macerates of food in appropriate volumes of the appropriate decimal dilution. Using a Most Probable Number (MPN) method this medium can be used to enumerate salmonellae in a food, the accuracy required determining whether 3, 5 or 10 tubes are inoculated at each decimal dilution.
Incubation method At 37~ for 24-48 h in air.
Reading of results and interpretation The colour change of the medium to yellow together with a black deposit, provides presumptive evidence for the presence of salmonellae. Those broths showing the colour change should be confirmed by streaking a loopful of the broth on a medium selective for salmonellae and confirming characteristic colonies by biochemical and serological tests.
Quality assessment (i) Productivity Test strains
Salmonella enteritidis 50073 Salmonella typhimurium 50100 Salmonella virchow 50077
Inoculation method
Dilution to extinction.
Criteria
Recovery in LICNR should be within 1 titre unit of the recovery in tryptone soya broth with colour change to yellow and the formation of a black precipitate after 48 h at 37~
(ii) Selectivity Test strains
Inoculation method
Escherichia coli 50034 Pseudomonas aeruginosa 50067 Dilution to extinction.
359 Criteria
G r o w t h is u n i n h i b i t e d in L I C N R . A t 24 h n o c o l o u r change should be seen with either organism. After 48 h Escherichia coli m a y t u r n t h e i n d i c a t o r faintly o r a n g e b u t n o c h a n g e s h o u l d b e o b s e r v e d w i t h Pseudomonas aeruginosa. N o b l a c k p r e c i p i t a t e s h o u l d b e formed.
References D'Aoust, J.Y. (1977) Limitations on lysine-iron-cystine-neutral red broth in the presumptive identification of salmonella. Appl. Environ. Microbiol. 34, 595-596. Hargrove, R.C., McDonough, F.C. and Reomen, R.H. (1971) A selective medium and presumptive procedure for detecting salmonella in dairy products. J. Milk Food Technol. 34, 6-11. Hoben, D.A., Ashton, D.H. and Peterson, A.C. (1973) A rapid presumptive procedure for the detection of Salmonella in foods and food ingredients. Appl. Microbiol. 25, 123-129. Morgan-Jones, S.C. (1982) A method for enumerating salmonellas from environments in the poultry industry. In: Isolation and identification methods for food poisoning organisms, edited by J.E.L. Corry, D. Roberts and F.A. Skinner. Academic Press, London, pp. 83-90.
Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 1995 Elsevier Science B.V. All rights reserved
Lysine-Iron-Cystine-Neutral Red (LICNR) broth Description and history Lysine iron cystine neutral red broth was developed by Hargrove et al. (1971) as a presumptive test for salmonellae in dairy products. This medium was further investigated and modified by Hoben et al. (1973)who found it useful for a variety of foods and food ingredients. Salmonellae decarboxylate lysine but do not ferment salicin and hence produce an alkaline reaction. In addition they produce hydrogen sulphide from cystine which reacts with the ferric salt to give a black precipitate of ferric sulphide. D'Aoust (1977) and Morgan-Jones (1982) have both reported that the colour change could only be used as a guide and the presence of salmonellae should be confirmed by conventional methods.
Composition (grams) L-Lysine Tryptone Yeast extract Mannitol Glucose Salicin Iron (III) ammonium citrate (brown) Sodium thiosulphate L-Cystine Neutral red Distilled or deionized water
10.0 5.0 3.0 5.0 1.0 1.0 0.5 0.1 0.1 0.025 1000.0
Preparation Suspend all the ingredients in the water and heat to dissolve, mix well and adjust pH to 6.2. Dispense into tubes in appropriate quantities. Either use immediately or sterilize at 121~ for 15 min.
Physical properties Appearance pH
Red, clear. 6.2 + 0.2
358
Shelf life Ready to use medium
7 days at 4 + 2~ in the dark.
Inoculation method for samples Inoculate broths with liquid food or macerates of food in appropriate volumes of the appropriate decimal dilution. Using a Most Probable Number (MPN) method this medium can be used to enumerate salmonellae in a food, the accuracy required determining whether 3, 5 or 10 tubes are inoculated at each decimal dilution.
Incubation method At 37~ for 24-48 h in air.
Reading of results and interpretation The colour change of the medium to yellow together with a black deposit, provides presumptive evidence for the presence of salmonellae. Those broths showing the colour change should be confirmed by streaking a loopful of the broth on a medium selective for salmonellae and confirming characteristic colonies by biochemical and serological tests.
Quality assessment (i) Productivity Test strains
Salmonella enteritidis 50073 Salmonella typhimurium 50100 Salmonella virchow 50077
Inoculation method
Dilution to extinction.
Criteria
Recovery in LICNR should be within 1 titre unit of the recovery in tryptone soya broth with colour change to yellow and the formation of a black precipitate after 48 h at 37~
(ii) Selectivity Test strains
Inoculation method
Escherichia coli 50034 Pseudomonas aeruginosa 50067 Dilution to extinction.
359 Criteria
G r o w t h is u n i n h i b i t e d in L I C N R . A t 24 h n o c o l o u r change should be seen with either organism. After 48 h Escherichia coli m a y t u r n t h e i n d i c a t o r faintly o r a n g e b u t n o c h a n g e s h o u l d b e o b s e r v e d w i t h Pseudomonas aeruginosa. N o b l a c k p r e c i p i t a t e s h o u l d b e formed.
References D'Aoust, J.Y. (1977) Limitations on lysine-iron-cystine-neutral red broth in the presumptive identification of salmonella. Appl. Environ. Microbiol. 34, 595-596. Hargrove, R.C., McDonough, F.C. and Reomen, R.H. (1971) A selective medium and presumptive procedure for detecting salmonella in dairy products. J. Milk Food Technol. 34, 6-11. Hoben, D.A., Ashton, D.H. and Peterson, A.C. (1973) A rapid presumptive procedure for the detection of Salmonella in foods and food ingredients. Appl. Microbiol. 25, 123-129. Morgan-Jones, S.C. (1982) A method for enumerating salmonellas from environments in the poultry industry. In: Isolation and identification methods for food poisoning organisms, edited by J.E.L. Corry, D. Roberts and F.A. Skinner. Academic Press, London, pp. 83-90.