- Email: [email protected]
Fraser broth — modified
Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.)
320
9 1995 Elsevier Science B.V. All rights reserved
Fraser broth - modified Description and history Fraser and Sperber (1988) developed the secondary enrichment broth (UVM Listeria Enrichment Broth II)used in the United States Department of Agriculture procedure for isolating Listeria monocytogenes from meats (McClain and Lee, 1988). The present formula is a subsequent modification with an increased amount of acriflavine. It gives a presumptive test for Listeria spp. as it contains an indicator for the hydrolysis of aesculin. This reaction is not exclusive to Listeria spp. so any microorganisms giving a positive reaction (blackening) must be isolated and further identified.
Composition (grams) Proteose p e p t o n e (peptic digest of animal tissue) Tryptone (pancreatic digest of casein) Beef extract Yeast extract Sodium chloride Di-sodium hydrogen o r t h o p h o s p h a t e " 2H2 O Potassium di-hydrogen o r t h o p h o s p h a t e Aesculin Lithium chloride Iron (III) a m m o n i u m citrate Nalidixic acid Acriflavine Distilled or deionized water
5.0 5.0 5.0 5.0 20.0 12.0 1.35 1.0 3.0 0.5 0.02 0.025 1000.0
Preparation Basal broth Suspend all of the ingredients, except the acriflavine and iron (III) ammonium citrate, in the water and heat to dissolve. Dispense 10 ml portions into 16 x 150 mm culture tubes with screw cap closures and sterilize at 121~ for 15 min.
321
Supplements To each tube of the cooled basal broth, immediately before use, add 0.1 ml portions of filter sterilized aqueous solutions of 0.25% of acriflavine and 5.0% iron (III) ammonium citrate.
Physical properties Appearance pH
Light straw colour, clear. 7.2 _+ 0.2
Shelf life Basal broth and supplement
14 days at 4 + 2~ Use complete medium immediately after addition of the supplements.
Inoculation method for samples Food or environmental samples are cultured in a primary listeria enrichment broth and incubated at 30~ for 24 h. These cultures are gently mixed and 0.1 ml portions are added to 10 ml of complete Fraser broth.
Incubation At 35~ for 24 and 48 h (Warburton et al., 1991) in air and in the dark.
Reading of results and interpretation After incubation the tubes are compared to an uninoculated control against a white background. Blackened cultures are considered as presumptively positive for Listeria spp. However, confirmatory tests are necessary on all positive cultures. Cultures which retain the original straw colour are considered to be negative.
Quality assessment (i) Productivity Test strains
Listeria monocytogenes serovar 1/2a (ATCC 35152 / NCTC 7973)
Listeria monocytogenes serovar 4b (ATCC 13932/ NCTC 10527)
Listeria ivanovii serovar 5 50095 Inoculation method
Dilution to extinction.
322 (ii) Selectivity Test strains
Enterococcus faecalis 50030 Proteus mirabilis ( A T C C 2 9 9 0 6 / C E C T 4 1 6 8 / N C T C 11938)
Inoculation method
Dilution to extinction.
Comments Selective p r o p e r t i e s of acriflavine may vary from lot to lot and m a n u f a c t u r e r to m a n u f a c t u r e r . E a c h new batch m u s t be assayed in c o m b i n a t i o n with o t h e r selective agents to be used in the m e d i u m to d e t e r m i n e the o p t i m u m c o n c e n t r a t i o n for use, with r e g a r d to the efficiency of selectivity and absence of inhibition of Listeria spp. S t o r a g e of stock solutions of acriflavine for p e r i o d s of m o r e t h a n o n e m o n t h is not recommended.
References Fraser, J.A. and Sperber, W.H. (1988) Rapid detection of Listeria spp. in food and environmental samples by esculin hydrolysis. J. Food Protect. 51, 762-765. McClain, D. and Lee, W.H. (1988) Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poulty. J. Assoc. Off. Anal. Chem. 71, 660-664. Warburton, D.W., Farber, J.M., Armstrong, A., Caldeira, R., Tiwari, N.P., Babiuk, T., Lacasse, P. and Read, S. (1991) A Canadian comparative study of modified versions of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes. J. Food Protect. 54, 669-676.
320
9 1995 Elsevier Science B.V. All rights reserved
Fraser broth - modified Description and history Fraser and Sperber (1988) developed the secondary enrichment broth (UVM Listeria Enrichment Broth II)used in the United States Department of Agriculture procedure for isolating Listeria monocytogenes from meats (McClain and Lee, 1988). The present formula is a subsequent modification with an increased amount of acriflavine. It gives a presumptive test for Listeria spp. as it contains an indicator for the hydrolysis of aesculin. This reaction is not exclusive to Listeria spp. so any microorganisms giving a positive reaction (blackening) must be isolated and further identified.
Composition (grams) Proteose p e p t o n e (peptic digest of animal tissue) Tryptone (pancreatic digest of casein) Beef extract Yeast extract Sodium chloride Di-sodium hydrogen o r t h o p h o s p h a t e " 2H2 O Potassium di-hydrogen o r t h o p h o s p h a t e Aesculin Lithium chloride Iron (III) a m m o n i u m citrate Nalidixic acid Acriflavine Distilled or deionized water
5.0 5.0 5.0 5.0 20.0 12.0 1.35 1.0 3.0 0.5 0.02 0.025 1000.0
Preparation Basal broth Suspend all of the ingredients, except the acriflavine and iron (III) ammonium citrate, in the water and heat to dissolve. Dispense 10 ml portions into 16 x 150 mm culture tubes with screw cap closures and sterilize at 121~ for 15 min.
321
Supplements To each tube of the cooled basal broth, immediately before use, add 0.1 ml portions of filter sterilized aqueous solutions of 0.25% of acriflavine and 5.0% iron (III) ammonium citrate.
Physical properties Appearance pH
Light straw colour, clear. 7.2 _+ 0.2
Shelf life Basal broth and supplement
14 days at 4 + 2~ Use complete medium immediately after addition of the supplements.
Inoculation method for samples Food or environmental samples are cultured in a primary listeria enrichment broth and incubated at 30~ for 24 h. These cultures are gently mixed and 0.1 ml portions are added to 10 ml of complete Fraser broth.
Incubation At 35~ for 24 and 48 h (Warburton et al., 1991) in air and in the dark.
Reading of results and interpretation After incubation the tubes are compared to an uninoculated control against a white background. Blackened cultures are considered as presumptively positive for Listeria spp. However, confirmatory tests are necessary on all positive cultures. Cultures which retain the original straw colour are considered to be negative.
Quality assessment (i) Productivity Test strains
Listeria monocytogenes serovar 1/2a (ATCC 35152 / NCTC 7973)
Listeria monocytogenes serovar 4b (ATCC 13932/ NCTC 10527)
Listeria ivanovii serovar 5 50095 Inoculation method
Dilution to extinction.
322 (ii) Selectivity Test strains
Enterococcus faecalis 50030 Proteus mirabilis ( A T C C 2 9 9 0 6 / C E C T 4 1 6 8 / N C T C 11938)
Inoculation method
Dilution to extinction.
Comments Selective p r o p e r t i e s of acriflavine may vary from lot to lot and m a n u f a c t u r e r to m a n u f a c t u r e r . E a c h new batch m u s t be assayed in c o m b i n a t i o n with o t h e r selective agents to be used in the m e d i u m to d e t e r m i n e the o p t i m u m c o n c e n t r a t i o n for use, with r e g a r d to the efficiency of selectivity and absence of inhibition of Listeria spp. S t o r a g e of stock solutions of acriflavine for p e r i o d s of m o r e t h a n o n e m o n t h is not recommended.
References Fraser, J.A. and Sperber, W.H. (1988) Rapid detection of Listeria spp. in food and environmental samples by esculin hydrolysis. J. Food Protect. 51, 762-765. McClain, D. and Lee, W.H. (1988) Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poulty. J. Assoc. Off. Anal. Chem. 71, 660-664. Warburton, D.W., Farber, J.M., Armstrong, A., Caldeira, R., Tiwari, N.P., Babiuk, T., Lacasse, P. and Read, S. (1991) A Canadian comparative study of modified versions of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes. J. Food Protect. 54, 669-676.