Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods.

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

Sensitivity of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline

The sensitivity of four strains of Listeria monocytogenes to irradiation on poultry meat and in phosphate-buffered saline was investigated. The D₁₀ values on poultry meat were 0.417–0.553 kGy depending on strain and plating medium used. Lower values were obtained in phosphate-buffered saline. Generally tryptone soya yeast extract and McBride agars gave a better recovery (higher D₁₀ value) than listeria selective agar. The D₁₀ values for L. monocytogenes were similar to those reported for Salmonella spp. irradiated under similar conditions. Therefore irradiation doses suggested to eliminate salmonellas from poultry carcasses would also be sufficient to remove L. monocytogenes.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Comparison of seven plating media for enumeration of Listeria spp.

The suitability of seven media for the enumeration of Listeria spp. was evaluated at 30 degrees C for 48 h. The media tested were (i) the original McBride Listeria agar formulation (with glycine); (ii) modified McBride agar containing glycine anhydride; (iii) LiCl-phenylethanol-moxalactam (LPM) agar; (iv) acriflavine-ceftazidime agar; (v) Rodriguez isolation agar (RISA); (vi) modified Vogel-Johnson (MVJ) agar; (vii) cyclohexanedione-nalidixic acid-phenylethanol agar; and tryptose agar as control. A total of 66 organisms were used including 11 Listeria monocytogenes strains and 5 other Listeria spp. For L. monocytogenes strains only, all media performed highly similarly. Of the other Listeria spp., only two grew on MVJ agar and three each grew on LPM and RISA. Only LPM agar inhibited the 50 non-listeriae, including five yeasts, while MVJ agar inhibited all but one yeast. The McBride Listeria agar formulation that contained glycine anhydride was less selective than the original. When pure cultures of 10 bacteria (including one L. monocytogenes strain) were combined and plated on four media, L. monocytogenes colonies were easiest to enumerate on MVJ agar, followed by LPM and RISA. These media ranked in the same order when plated with homogenates of various foods to which was added L. monocytogenes Scott A, but LPM agar was the best overall since Scott A was inhibited by MVJ. Upon microscopic examination of listerial colonies from the plating media, atypical cell morphology was noted with cells being about twofold in size on LPM, MVJ, and acriflavine-ceftazidime agars. Overall, LPM agar was the most suitable of the media tested even though it was inhibitory to Listeria grayi and Listeria murrayi.     

Methods for improved recovery of Listeria monocytogenes from cheese.

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Methods for improved recovery of Listeria monocytogenes from cheese

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Selective-enrichment procedure for isolation of Listeria monocytogenes from fecal and biologic specimens.

A selective-enrichment procedure (SEP) was developed to isolate Listeria monocytogenes from fecal and biologic specimens. This procedure was compared with direct plating with McBride listeria agar and 2-, 4-, and 8-week cold-enrichment procedures in recovering L. monocytogenes from mouse fecal, liver, and brain specimens. Although the SEP occasionally did not isolate the organism from specimens proved positive by the other procedures, the SEP isolated L. monocytogenes from about two and five times as many specimens as the cold-enrichment and direct-plating procedures, respectively.     

Selective-enrichment procedure for isolation of Listeria monocytogenes from fecal and biologic specimens

A selective-enrichment procedure (SEP) was developed to isolate Listeria monocytogenes from fecal and biologic specimens. This procedure was compared with direct plating with McBride listeria agar and 2-, 4-, and 8-week cold-enrichment procedures in recovering L. monocytogenes from mouse fecal, liver, and brain specimens. Although the SEP occasionally did not isolate the organism from specimens proved positive by the other procedures, the SEP isolated L. monocytogenes from about two and five times as many specimens as the cold-enrichment and direct-plating procedures, respectively.     

The rapid demonstration and presumptive identification of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluorescence test (DIFT)

Two monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) were successfully used in a direct immunofluorescence test (DIFT) to demonstrate listeria in seven samples of soft cheese where Listeria monocytogenes had been cultured by conventional techniques. Using DIFT, listeria was not detected in 20 cheese samples from which L. monocytogenes had not been isolated, or was present in low numbers (< 10²/g). The DIFT was also used to presumptively identify > 90% of strains of L. monocytogenes isolated from food and cultured on Modified McBride agar or Blood agar. Less than 10% of strains of other species of listeria would be misidentified when grown on these media. All tests were carried out within 2 h.     

A COLLABORATIVE STUDY FOR DIRECT ENUMERATION OF LOW LEVELS OF LISTERIA MONOCYTOGENES FROM VARIOUS FOODS

A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 10² and 10³/g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa-lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes.

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

Listeria spp. found on fresh market produce

From October 1987 to August 1988, 1,000 tests were conducted on 10 types of fresh produce from two Minneapolis area supermarkets to detect Listeria spp. The produce included broccoli, cabbage, carrots, cauliflower, cucumbers, lettuce, mushrooms, potatoes, radishes, and tomatoes. The vegetables were tested by the Food and Drug Administration method for isolation of Listeria spp., with the addition of LiCl-phenylethanol-moxalactam agar in the last 280 tests; 8.6 and 11.4% of these tests were positive by modified McBride and LiCl-phenylethanol-moxalactam agars, respectively. Listeria monocytogenes was isolated from cabbage, cucumbers, potatoes, and radishes; L. innocua was isolated from cucumbers, lettuce, mushrooms, potatoes, and radishes; L. seeligeri was isolated from cabbage and radishes; and L. welshimeri was isolated from cucumbers, potatoes, and radishes. The isolates were of various serotypes; however, the L. monocytogenes isolates were predominantly serotype 1 (82%). Only potatoes (25.8% positive) and radishes (30.3% positive) showed significant amounts of L. monocytogenes contamination.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage.

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Listeria spp. found on fresh market produce.

From October 1987 to August 1988, 1,000 tests were conducted on 10 types of fresh produce from two Minneapolis area supermarkets to detect Listeria spp. The produce included broccoli, cabbage, carrots, cauliflower, cucumbers, lettuce, mushrooms, potatoes, radishes, and tomatoes. The vegetables were tested by the Food and Drug Administration method for isolation of Listeria spp., with the addition of LiCl-phenylethanol-moxalactam agar in the last 280 tests; 8.6 and 11.4% of these tests were positive by modified McBride and LiCl-phenylethanol-moxalactam agars, respectively. Listeria monocytogenes was isolated from cabbage, cucumbers, potatoes, and radishes; L. innocua was isolated from cucumbers, lettuce, mushrooms, potatoes, and radishes; L. seeligeri was isolated from cabbage and radishes; and L. welshimeri was isolated from cucumbers, potatoes, and radishes. The isolates were of various serotypes; however, the L. monocytogenes isolates were predominantly serotype 1 (82%). Only potatoes (25.8% positive) and radishes (30.3% positive) showed significant amounts of L. monocytogenes contamination.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).