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Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods
International Journal ofFoodMicrobiology, 8 (1989) 219-223
219
Elsevier JFM90004
Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods R.E. Brackett
and L.R. Beuchat
Department of Food Science and Technology, University of Georgia, Griffin, ~ S.A.
Many methods and media currently exist for the detection and enumeration of Listeria monocytogenes. However, the suitability of any specific method or media is influenced by the purpose of the analysis and the type of food being analyzed. Food which are likely to contain high populations of contaminating microorganisms require highly selective media for enumeration. Moreover, media which contain indicator systems are also helpful in distinguishing L. monocytogenes colonies. In contrast, less selective media may be adequate for less contaminated foods. Key words: Listeria monocytogenes, methods and media for isolation; Listeria monocytogenes, enumeration
Introduction Listeria monocytogenes has in recent years e m e r g e d as one of the m o s t i m p o r t a n t f o o d b o r n e p a t h o g e n s . T h e serious c o n s e q u e n c e of h u m a n listeriosis m a k e s it of u t m o s t i m p o r t a n c e to quickly a n d a c c u r a t e l y d e t e r m i n e the p r e s e n c e of L. monocytogenes in foods. M e t h o d s designed for isolating a n d e n u m e r a t i n g L. monocytogenes have existed for m a n y years. M e d i a were originally f o r m u l a t e d for isolating L. monocytogenes f r o m relatively u n c o n t a m i n a t e d clinical samples. H o w e v e r , these m e d i a were of o n l y limited usefulness for s a m p l e s such as foods a n d feeds which often c o n t a i n high p o p u l a t i o n s of a wide range of c o n t a m i n a t i n g m i c r o f l o r a . This s i t u a t i o n has led to the d e v e l o p m e n t of a variety of selective m e d i a d e s i g n e d to isolate L. monocytogenes f r o m foods. A l t h o u g h m o r e m e d i a a n d m e t h o d s are n o w a v a i l a b l e for this p u r p o s e , c o n f u s i o n has also resulted as to which of the p l e t h o r a of m e d i a are m o s t a p p r o p r i a t e . T h e p u r p o s e of this p a p e r is to p r e s e n t s o m e p e r s p e c t i v e s o n h o w s o m e of the various m e d i a p e r f o r m for isolating L. monocytogenes f r o m different types of foods.
O n e of the m o s t i m p o r t a n t factors affecting the p e r f o r m a n c e of an analysis m e t h o d for L. monocytogenes is the t y p e of food f r o m which one is trying to isolate
* Correspondence address: R.E. Brackett, Department of Food Science and Technology, University of
Georgia, Griffin, GA 30223-1797, U.S.A. 0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
220 the organism. Foods differ in their suitability to support growth and survival of L. monocytogenes as well as other contaminating flora that may be present. Thus, methods designed to repress growth of contaminants typical of one type of food may or may not be suitable for a different type of food harboring different microflora. Moreover, physical characteristics of foods, such as particle size and pH, can also affect the ease and accuracy of detecting and identifying colonies as L. monocytogenes.
Another important consideration in comparing methods for recovering L. monocytogenes from foods is to specify the purpose of the analysis. Most methods that have been developed are designed primarily to determine presence of the organism in a food. These methods tend to be the most sensitive but do not allow accurate quantification. In contrast, direct plating methods are intended to quantify the population of L. monocytogenes in foods but usually lack sensitivity. Thus, each method has limitations and will be affected by various intrinsic and extrinsic factors.
Detection methods Two detection methods are used most extensively in the United States. These are the U.S. Food and Drug Administration (FDA) method (Lovett et al., 1987) and the U.S. Department of Agriculture Food Sanitation Inspection Service (FSIS) method (Lee and McClain, 1987). Both methods employ a primary enrichment procedure followed by an isolation procedure. The main difference between the two methods is the choice of enrichment broths and isolation agars. The FDA method uses the so-called EB enrichment broth for enrichment and modified McBride Listeria Agar (MMLA) as an isolation agar. In contrast, the FSIS method uses UVM broth for enrichment and LiCl-phenylethanol-moxalactam (LPMA) agar as an isolation agar. The FDA and FSIS methods were originally developed for analyzing different types of food. The FDA method was designed for dairy products, and the FSIS method was designed and is officially recommended primarily for meat and poultry products. In general, the FSIS procedure is more sensitive, especially in situations where the food contains a large number of microflora other than L. monocytogenes. Rapid methods employing gene probes or enzyme linked immunosorbant assays are also increasing being developed and used for dairy products and environmental samples. These methods usually require a pre-enrichment (UVM) step prior to the actual analysis.
Direct plating methods Extensive research has been done to compare the efficacy of media used in direct plating for recovering L. monocytogenes from various foods (Hao et al., 1987; Golden et al., 1988a,b, 1989). Information resulting from this research has also been valuable in selecting isolation agars used in other methods.
221 TABLE I Subjective comparison of ease of recognition and counting of L. monocytogenes from foods analyzed on test media incubated at 30 ° C for 48 h Test medium
Ease of recognition
Ease of counting
References
McBride Listeria Agar (MLA)
Fair
Fair
McBride and Gerard (1960)
Gum Base Nalidixic Acid Tryptone Agar (GBNTSA)
Fair
Excellent
Martin et al. (1984)
Dominguez Rodriguez Isolation Agar (DRIA)
Excellent
Good
Dominguez Rodriguez et al. (1984)
Modified Despierres Agar (MDA)
Good
Excellent
Despierres (1971
Donnelly's Listeria Enrichment Agar
Poor
Fair
Golden et al. (1988b)
LiC1-PhenylethanolMoxalactam Agar (LPMA)
Good
Fair
Lee and McClain (1987)
Modified Vogel Johnson Agar (MVJA)
Excellent
Excellent
Golden et al. (1988b)
Modified McBride Listeria Agar (MMLA)
Poor
Poor
Lovett et al. (1987)
Thus far, we have c o m p a r e d 17 different direct p l a t i n g m e d i a for their s u i t a b i l i t y to recover heat- a n d freeze-injured as well as u n i n j u r e d L. monocytogenes f r o m seven different foods. D e t a i l s of c o m p a r i s o n s with f o u r of these foods have b e e n r e p o r t e d p r e v i o u s l y ( G o l d e n et al., 1988a,b,c). Subjective c h a r a c t e r i s t i c s a n d relative a b i l i t y of the four best m e d i a to recover L. monocytogenes f r o m these f o o d s are s u m m a r i z e d in T a b l e s I a n d II, respectively. There were s u b s t a n t i a l differences in the p e r f o r m a n c e of media, d e p e n d i n g u p o n the type of food u n d e r analysis. Differences were p r i m a r i l y a t t r i b u t e d to the n u m b e r s of L. monocytogenes cells which c o u l d be recovered f r o m i n o c u l a t e d foods. However, other factors such as the t y p e a n d p o p u l a t i o n s of n a t u r a l l y o c c u r r i n g microflora, a n d ease of c o u n t i n g a n d recognizing Listeria c o l o n i e s were also important. In general, n o n - c u l t u r e d d a i r y foods ( p a s t e u r i z e d milk a n d ice c r e a m ) c o n t a i n e d relatively few c o m p e t i t i v e m i c r o o r g a n i s m s , c o m p r i s e d m a i n l y of lactic a c i d b a c t e r i a . I n a d d i t i o n , few of the c o n t a m i n a t i n g m i c r o o r g a n i s m s that grew on the d i r e c t p l a t i n g m e d i a listed in T a b l e II h a d c o l o n y m o r p h o l o g i e s similar to L. monocytogenes. R e c o v e r y of L. monocytogenes f r o m Brie cheese a n d c h o p p e d r a w c a b b a g e was h a m p e r e d b y the presence of large p o p u l a t i o n s of m i c r o f l o r a o t h e r t h a n L. monocytogenes. U n l i k e milk or ice cream, m a n y of the non-Listeria c o n t a m i n a n t s h a d
222 TABLE II Relative efficacy of the four best direct plating media for recovering uninjured and injured L.
monocytogenes cells from various foods Recovery
L. rnonocytogenes cells
medium
Uninjured
Injured
Pasteurized milk
GBNTSA a LPMA MVJA MLA
+ + + + b + + + + + + + +++
+ + + + + + + + + + ++++
Ice cream mix (chocolate)
GBNTSA LPMA MVJA MLA
+ + + + + + + + + + + +++
+ + + + + + + + + + ++++
Brie cheese
DRIA LPMA MVJA MLA
+ + + +
+ + + + + + + + +
+ + + + + + + +
Cabbage
MDA LPMA DLEA MVJA
+ + + +
+ + + +
+ + + +
Oysters
MLA GBNTSA DRIA MMLA
+ + + +
+ + + + + +
+ + + + + + +
Country cured ham
DRIA DLEA LPMA MVJA
+ + + +
+ + + + + + +
+ + + + + + +
Dry cured ham
MDA MMLA LPMA MVJA
+ + + +
+ + + + + +
+ + + + + + + +
Food
+ + + + +
+ + + + +
For acronym of media see Table I. b + + + + = excellent, + + + = good, + + = fair, + = acceptable under certain circumstances. a
morphologies s i m i l a r t o L. monocytogenes. T h i s m a d e e n u m e r a t i o n o f L. monocytogenes d i f f i c u l t o r i m p o s s i b l e o n a l l b u t t h e m o s t s e l e c t i v e m e d i a . O f t h e s e ,
colony LPMA
gave
enumerate
the highest
on
Gram-positive
MVJ.
recovery
Contaminating
and occasionally,
but
colonies
were
much
microflora
from
cabbage
Gram-negative
rods. The major
Brie cheese were molds, yeasts, and lesser amounts Country
and dry-cured
flora. However,
hams
to identify
consisted
and
largely
contaminants
of Gram-positive
also had high populations
most colonies which developed
easier
of
from
cocci.
of contaminating
micro-
on isolation agars were distinguisha-
223
ble from L. monocytogenes and consisted of Gram-positive cocci. MVJ agar recovered fewer L. monocytogenes cells than did some of the other media but yielded the most easily identifiable L. monocytogenes colonies. Oysters were the most difficult food to analyze for L. monocytogenes. Samples were routinely contaminated with high populations of microorganisms which appeared indistinguishable from L. monocytogenes on all but MVJ agar. These contaminants were primarily gram-positive rods but also included pseudomonads. Most selective media, e.g., LPMA and MVJA, resulted in fewest contaminants but also gave poorest recoveries of L. monocytogenes. In contrast, less selective media recovered more L. monocytogenes but were plagued with extensive contamination from other microorganisms. Thus, none of the media tested was considered to be satisfactory as a direct plating medium for analyzing oysters.
Conclusions
The type of food, populations and types of contaminating microflora, and likelihood of injured L. monocytogenes cells should be considered when choosing media and methods. In general, foods likely to contain low populations or injured L. monocytogenes are best analyzed using enrichment procedures. High populations of contaminating microflora necessitate using highly selective media (e.g., LPMA or MVJA). Undoubtedly, new media will be developed and compared with those listed in this paper. It is likely that these media will lead to further refinement and optimization for various types of foods.
References Despierres, M., (1971) Isolation of Listeria monocvtogenes in a medium inhibitory to Streptococcus faecalis. Ann. Inst. Pasteur 121,493-501. Dominguez Rodriguez, L., Suarez Fernandez, G., Fernandez, J., Garayzabal, F. and Rodriguez Ferri, E. (1984) New methodology for the isolation of Listeria monocytogenes from heavily contaminated environments. Appl. Environ. Microbiol. 47, 1188-1190. Golden, D.A., Beuchat, L.R. and Brackett, R.E. (1988a) Evaluation of selective direct-plating media for their suitability to recover uninjured, heat-injured and freeze-injured Listeria monocytogenes from foods. Appl. Environ. Microbiol. 54, 1451-1456. Golden, D.A., Beuchat, L.R. and Brackett, R.E. (1988b) Direct plating technique for enumeration of Listeria monocytogenes in foods. J. Assoc. Off. Anal. Chem. 71,647-650. Golden, D.A., Brackett, R.E. and Beuchat, L.R. (1989) Efficacy of direct plating media for recovering Listeria rnonocytogenes from foods. Intl. J. Food Microbiol., in press. Hao, D.D.Y., Beuchat, L.R. and Brackett, R.E. (1987) Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage. Appl. Environ. Microbiol. 53, 955-957. Lee, W.H. and McClain, D. (1987) Improved Listeria rnonocvtogenes selective agar. Appl. Environ. Microbiol. 52, 1215-1217. Lovett, J., Francis, D.W. and Hung, J.M. (1987) Listeria monocytogenes in raw milk, Detection, incidence and pathogenicity. J. Food Protect. 50, 188-192. Martin, R.S., Sumarah, R.K. and MacDonald, M.A. (1984) A synthetic based medium for the isolation of Listeria monocytogenes. Clin. Invest. Med. 7, 233-237. McBride, M.E. and Gerard, K.F. (1960) A selective method for isolations of Listeria monocytogenes from mixed bacterial populations, J. Lab. Clin. Med. 55, 153-157.
219
Elsevier JFM90004
Methods and media for the isolation and cultivation of Listeria monocytogenes from various foods R.E. Brackett
and L.R. Beuchat
Department of Food Science and Technology, University of Georgia, Griffin, ~ S.A.
Many methods and media currently exist for the detection and enumeration of Listeria monocytogenes. However, the suitability of any specific method or media is influenced by the purpose of the analysis and the type of food being analyzed. Food which are likely to contain high populations of contaminating microorganisms require highly selective media for enumeration. Moreover, media which contain indicator systems are also helpful in distinguishing L. monocytogenes colonies. In contrast, less selective media may be adequate for less contaminated foods. Key words: Listeria monocytogenes, methods and media for isolation; Listeria monocytogenes, enumeration
Introduction Listeria monocytogenes has in recent years e m e r g e d as one of the m o s t i m p o r t a n t f o o d b o r n e p a t h o g e n s . T h e serious c o n s e q u e n c e of h u m a n listeriosis m a k e s it of u t m o s t i m p o r t a n c e to quickly a n d a c c u r a t e l y d e t e r m i n e the p r e s e n c e of L. monocytogenes in foods. M e t h o d s designed for isolating a n d e n u m e r a t i n g L. monocytogenes have existed for m a n y years. M e d i a were originally f o r m u l a t e d for isolating L. monocytogenes f r o m relatively u n c o n t a m i n a t e d clinical samples. H o w e v e r , these m e d i a were of o n l y limited usefulness for s a m p l e s such as foods a n d feeds which often c o n t a i n high p o p u l a t i o n s of a wide range of c o n t a m i n a t i n g m i c r o f l o r a . This s i t u a t i o n has led to the d e v e l o p m e n t of a variety of selective m e d i a d e s i g n e d to isolate L. monocytogenes f r o m foods. A l t h o u g h m o r e m e d i a a n d m e t h o d s are n o w a v a i l a b l e for this p u r p o s e , c o n f u s i o n has also resulted as to which of the p l e t h o r a of m e d i a are m o s t a p p r o p r i a t e . T h e p u r p o s e of this p a p e r is to p r e s e n t s o m e p e r s p e c t i v e s o n h o w s o m e of the various m e d i a p e r f o r m for isolating L. monocytogenes f r o m different types of foods.
O n e of the m o s t i m p o r t a n t factors affecting the p e r f o r m a n c e of an analysis m e t h o d for L. monocytogenes is the t y p e of food f r o m which one is trying to isolate
* Correspondence address: R.E. Brackett, Department of Food Science and Technology, University of
Georgia, Griffin, GA 30223-1797, U.S.A. 0168-1605/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
220 the organism. Foods differ in their suitability to support growth and survival of L. monocytogenes as well as other contaminating flora that may be present. Thus, methods designed to repress growth of contaminants typical of one type of food may or may not be suitable for a different type of food harboring different microflora. Moreover, physical characteristics of foods, such as particle size and pH, can also affect the ease and accuracy of detecting and identifying colonies as L. monocytogenes.
Another important consideration in comparing methods for recovering L. monocytogenes from foods is to specify the purpose of the analysis. Most methods that have been developed are designed primarily to determine presence of the organism in a food. These methods tend to be the most sensitive but do not allow accurate quantification. In contrast, direct plating methods are intended to quantify the population of L. monocytogenes in foods but usually lack sensitivity. Thus, each method has limitations and will be affected by various intrinsic and extrinsic factors.
Detection methods Two detection methods are used most extensively in the United States. These are the U.S. Food and Drug Administration (FDA) method (Lovett et al., 1987) and the U.S. Department of Agriculture Food Sanitation Inspection Service (FSIS) method (Lee and McClain, 1987). Both methods employ a primary enrichment procedure followed by an isolation procedure. The main difference between the two methods is the choice of enrichment broths and isolation agars. The FDA method uses the so-called EB enrichment broth for enrichment and modified McBride Listeria Agar (MMLA) as an isolation agar. In contrast, the FSIS method uses UVM broth for enrichment and LiCl-phenylethanol-moxalactam (LPMA) agar as an isolation agar. The FDA and FSIS methods were originally developed for analyzing different types of food. The FDA method was designed for dairy products, and the FSIS method was designed and is officially recommended primarily for meat and poultry products. In general, the FSIS procedure is more sensitive, especially in situations where the food contains a large number of microflora other than L. monocytogenes. Rapid methods employing gene probes or enzyme linked immunosorbant assays are also increasing being developed and used for dairy products and environmental samples. These methods usually require a pre-enrichment (UVM) step prior to the actual analysis.
Direct plating methods Extensive research has been done to compare the efficacy of media used in direct plating for recovering L. monocytogenes from various foods (Hao et al., 1987; Golden et al., 1988a,b, 1989). Information resulting from this research has also been valuable in selecting isolation agars used in other methods.
221 TABLE I Subjective comparison of ease of recognition and counting of L. monocytogenes from foods analyzed on test media incubated at 30 ° C for 48 h Test medium
Ease of recognition
Ease of counting
References
McBride Listeria Agar (MLA)
Fair
Fair
McBride and Gerard (1960)
Gum Base Nalidixic Acid Tryptone Agar (GBNTSA)
Fair
Excellent
Martin et al. (1984)
Dominguez Rodriguez Isolation Agar (DRIA)
Excellent
Good
Dominguez Rodriguez et al. (1984)
Modified Despierres Agar (MDA)
Good
Excellent
Despierres (1971
Donnelly's Listeria Enrichment Agar
Poor
Fair
Golden et al. (1988b)
LiC1-PhenylethanolMoxalactam Agar (LPMA)
Good
Fair
Lee and McClain (1987)
Modified Vogel Johnson Agar (MVJA)
Excellent
Excellent
Golden et al. (1988b)
Modified McBride Listeria Agar (MMLA)
Poor
Poor
Lovett et al. (1987)
Thus far, we have c o m p a r e d 17 different direct p l a t i n g m e d i a for their s u i t a b i l i t y to recover heat- a n d freeze-injured as well as u n i n j u r e d L. monocytogenes f r o m seven different foods. D e t a i l s of c o m p a r i s o n s with f o u r of these foods have b e e n r e p o r t e d p r e v i o u s l y ( G o l d e n et al., 1988a,b,c). Subjective c h a r a c t e r i s t i c s a n d relative a b i l i t y of the four best m e d i a to recover L. monocytogenes f r o m these f o o d s are s u m m a r i z e d in T a b l e s I a n d II, respectively. There were s u b s t a n t i a l differences in the p e r f o r m a n c e of media, d e p e n d i n g u p o n the type of food u n d e r analysis. Differences were p r i m a r i l y a t t r i b u t e d to the n u m b e r s of L. monocytogenes cells which c o u l d be recovered f r o m i n o c u l a t e d foods. However, other factors such as the t y p e a n d p o p u l a t i o n s of n a t u r a l l y o c c u r r i n g microflora, a n d ease of c o u n t i n g a n d recognizing Listeria c o l o n i e s were also important. In general, n o n - c u l t u r e d d a i r y foods ( p a s t e u r i z e d milk a n d ice c r e a m ) c o n t a i n e d relatively few c o m p e t i t i v e m i c r o o r g a n i s m s , c o m p r i s e d m a i n l y of lactic a c i d b a c t e r i a . I n a d d i t i o n , few of the c o n t a m i n a t i n g m i c r o o r g a n i s m s that grew on the d i r e c t p l a t i n g m e d i a listed in T a b l e II h a d c o l o n y m o r p h o l o g i e s similar to L. monocytogenes. R e c o v e r y of L. monocytogenes f r o m Brie cheese a n d c h o p p e d r a w c a b b a g e was h a m p e r e d b y the presence of large p o p u l a t i o n s of m i c r o f l o r a o t h e r t h a n L. monocytogenes. U n l i k e milk or ice cream, m a n y of the non-Listeria c o n t a m i n a n t s h a d
222 TABLE II Relative efficacy of the four best direct plating media for recovering uninjured and injured L.
monocytogenes cells from various foods Recovery
L. rnonocytogenes cells
medium
Uninjured
Injured
Pasteurized milk
GBNTSA a LPMA MVJA MLA
+ + + + b + + + + + + + +++
+ + + + + + + + + + ++++
Ice cream mix (chocolate)
GBNTSA LPMA MVJA MLA
+ + + + + + + + + + + +++
+ + + + + + + + + + ++++
Brie cheese
DRIA LPMA MVJA MLA
+ + + +
+ + + + + + + + +
+ + + + + + + +
Cabbage
MDA LPMA DLEA MVJA
+ + + +
+ + + +
+ + + +
Oysters
MLA GBNTSA DRIA MMLA
+ + + +
+ + + + + +
+ + + + + + +
Country cured ham
DRIA DLEA LPMA MVJA
+ + + +
+ + + + + + +
+ + + + + + +
Dry cured ham
MDA MMLA LPMA MVJA
+ + + +
+ + + + + +
+ + + + + + + +
Food
+ + + + +
+ + + + +
For acronym of media see Table I. b + + + + = excellent, + + + = good, + + = fair, + = acceptable under certain circumstances. a
morphologies s i m i l a r t o L. monocytogenes. T h i s m a d e e n u m e r a t i o n o f L. monocytogenes d i f f i c u l t o r i m p o s s i b l e o n a l l b u t t h e m o s t s e l e c t i v e m e d i a . O f t h e s e ,
colony LPMA
gave
enumerate
the highest
on
Gram-positive
MVJ.
recovery
Contaminating
and occasionally,
but
colonies
were
much
microflora
from
cabbage
Gram-negative
rods. The major
Brie cheese were molds, yeasts, and lesser amounts Country
and dry-cured
flora. However,
hams
to identify
consisted
and
largely
contaminants
of Gram-positive
also had high populations
most colonies which developed
easier
of
from
cocci.
of contaminating
micro-
on isolation agars were distinguisha-
223
ble from L. monocytogenes and consisted of Gram-positive cocci. MVJ agar recovered fewer L. monocytogenes cells than did some of the other media but yielded the most easily identifiable L. monocytogenes colonies. Oysters were the most difficult food to analyze for L. monocytogenes. Samples were routinely contaminated with high populations of microorganisms which appeared indistinguishable from L. monocytogenes on all but MVJ agar. These contaminants were primarily gram-positive rods but also included pseudomonads. Most selective media, e.g., LPMA and MVJA, resulted in fewest contaminants but also gave poorest recoveries of L. monocytogenes. In contrast, less selective media recovered more L. monocytogenes but were plagued with extensive contamination from other microorganisms. Thus, none of the media tested was considered to be satisfactory as a direct plating medium for analyzing oysters.
Conclusions
The type of food, populations and types of contaminating microflora, and likelihood of injured L. monocytogenes cells should be considered when choosing media and methods. In general, foods likely to contain low populations or injured L. monocytogenes are best analyzed using enrichment procedures. High populations of contaminating microflora necessitate using highly selective media (e.g., LPMA or MVJA). Undoubtedly, new media will be developed and compared with those listed in this paper. It is likely that these media will lead to further refinement and optimization for various types of foods.
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