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Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods
International Journal of Food Microbiology 14 ( 1991 ) 43-50
43
© 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1605/91/$03.50 FOOD 00429
Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods Burton W. Blais and Hiroshi Yamazaki Department of Biology and Institute of Biochemistry, Carleton Unicersity, Ottawa, Ontario, Canada (Received 11 February 1991; accepted 12 July 1991)
A rapid and economical semi-quantitative test for Salmonella cells in foods is proposed. Food samples containing different levels of Salmonella cells were homogenized and serially diluted in enrichment broths and then incubated for about 20 h at 37 ° C. The presence of Salmonella cells in each dilution was assayed by capturing deoxycholate-extracted Salmonella lipopolysaccharides on a sheet of polymyxin-coated polyester cloth, followed by colorimetric detection with an anti-Salmonella antibodyenzyme conjugate. The minimum dilutions which resulted in no detectable growth were correlated with the extent of Salmonella contamination in the food samples. Key words: Food manufacturing; Monitoring; Salmonella; Polymyxin
Introduction
The extent of microbial contamination in processed foods largely depends on sanitation in food manufacturing processes. Monitoring sanitation requires quantitative assays of microorganisms in food samples or processing equipment at different stages of the processing operation. For such monitoring in commercial processes rapidity, reproducibility and economy are important concerns. We have previously demonstrated the advantages of polyester cloth as an immunoadsorbent for enzyme immunoassay (EIA) which allows rapid immunoreactions because of the cloth's large surface area and efficient washing because of the cloth's macroporosity (Blais and Yamazaki, 1989). We have recently demonstrated the capture of bacterial lipopolysaccharide (LPS) antigens on polymyxin B-coated polyester cloth for EIA (Blais and Yamazaki, 1991a,b). Since the polymyxin-coated cloth (polymyxin-cloth) has a large capacity for binding LPS, it is possible to detect Salmonella LPS even in the presence of excess non-Salmonella LPS. Polymyxin B is available in a purified form and stable, and is considerably Correspondence address: H. Yamazaki, Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6.
44
less expensive to use than antibodies. The present paper demonstrates the application of polymyxin-cloth as a commercially attractive alternative to antibody-coated cloth for the semi-quantitative assay of Salmonella LPS antigens in processed foods. Salmonella typhimurium strain LT2 was used as a model system for this study.
Materials and Methods
Chemicals and immunoreagents Polymyxin B sulfate (No. P-1004) and sodium deoxycholate (No. D-6750) were obtained from Sigma Chemical Co. CSA-1 anti-Salmonella antibody-peroxidase conjugate (No. 04-91-99), T M B microwell peroxidase substrate system (No. 50-7600) and T M B membrane peroxidase substrate system (No. 50-77-00) were from Kirkegaard and Perry Laboratories, Inc. The conjugate was stored as a 0.1 m g / m l stock in 0.01 M phosphate-buffered (pH 7.2) and 0.85% NaC1 (PBS) at - 2 0 ° C . For use, it was diluted 1:4000 in PBS containing 0.05% Tween 20 (PBST). Buffered peptone, yeast extract, selenite cysteine, tetrathionate broth base, and nutrient broth were from Difco.
Preparation of food samples A variety of food samples were obtained from a local supermarket. Before inoculation with Salmonella cells, meat samples such as chicken breast and lean beef were blended to a paste using 5 x 10 s high speed bursts in a standard food processor. Solid samples (1 g) such as cheese and baker's chocolate were first mixed with 2 ml of buffered peptone water (BPW) containing 0.5% ( w / v ) yeast extract (BPWYE) and then blended as above. Powdered, semi-solid and liquid samples (flour, mayonnaise and milk) were used without blending.
Preparation of Salmonella-inoculated food samples Salmonella typhimurium strain LT2 stored on a nutrient agar slant at 4°C was inoculated into B P W Y E and grown to a density of about 10 9 cells/ml (late log phase) by shaking at 37°C for 16 h, and then diluted in sterile B P W Y E to obtain various cell densities (confirmed by viable counts obtained by plating serial dilutions of the suspensions on nutrient agar and incubating the plates at 37°C for 20 h). 1 ml of the diluted culture was mixed with the food homogenates containing the equivalent of 1 g of original food sample as described above. BPWYE was added to the mixture to a final volume of 10 ml and vigorously mixed in a vortex mixer until a uniform suspension was obtained. The suspension was then serially diluted 10-fold by transferring 0.5 ml to 4.5 ml of BPWYE in 2.5 x 15 cm culture tubes. All of the tubes then shaken at 37 ° C for about 20 h. One-tenth volume of 1.1% (w/v) sodium deoxycholate in PBS was added to each tube, and the tubes were then autoclaved at 121°C for 5 min. Heating the cells in deoxycholate dissociates LPS antigens into non-sedimentable form suitable for assay on
45 polymyxin-cloth (Blais and Yamazaki, 1991b). The treated samples were cooled to room temperature and then assayed by the dot blot EIA on polymyxin-cloth.
Dot blot EIA on polymyxin-coated polyester cloth Polyester cloth (DuPont, Sontara 8100) was cut into 6 x 4 cm sheets, then saturated with 3 ml of polymyxin B sulfate solution (5 m g / m l in PBS). After incubation for 16 h at room temperature, each sheet was washed with a total of about 6 x 10 ml of PBST on a filter under suction. For comparison, a 6 x 4 cm sheet of nitrocellulose (Schleicher and Schuell, No. 33172) was similarly coated with polymyxin. 10 #1 of the deoxycholate-heat-treated samples were pipetted onto the polymyxin-cloth sheet at 1-cm intervals, so that each 6 x 4 cm sheet accommodated a total of 24 samples. After 30 rain incubation at room temperature, the sheets were washed with PBST as above, and then saturated with 2 ml of the CSA-1 antibody-peroxidase conjugate. After 30 min incubation at room temperature, the sheets were washed with PBST, then blotted and incubated with 2 ml of TMB membrane peroxidase substrate system for 10 rain. Wherever samples containing Salmonella LPS were dot blotted on the polymyxin-cloth insoluble blue spots appeared. Comparison of polyester cloth with a nitrocellulose membrane Suspensions of S. typhimurium cells in B P W Y E at various cell densities were heated in 0.1% deoxycholate and 10/zl of the treated cell samples were incubated for 30 rain at room temperature with either polyester cloth or nitrocellulose membrane sheets similarly coated with polymyxin (5 mg/ml). The sheets were then washed with PBST and Salmonella LPS antigens captured on the sheets were detected using the anti-Salmonella antibody-peroxidase conjugate and the peroxidase detection system as described above. Effect of selectit,e and non-selectiz,e media on the assay of LPS antigens S. typhimurium LPS was suspended at 0 . 1 / z g / m l in non-selective media (BPW, BPWYE and nutrient broth) and selective media (selenite cysteine broth and tetrathionate broth). The suspensions were then assayed by the quantitative EIA on polymyxin-cloth as described previously (Blais and Yamazaki, 1991b). Briefly, 10 izl of deoxycholate-heat-treated samples were incubated for 30 min at room temperature with 6 x 6 mm polymyxin-cloth squares. The squares were then washed with PBST and incubated with 50/zl of the anti-Salmonella antibody-peroxidase conjugate for 30 min at room temperature. After washing with PBST, the squares were transferred to 16 x 100 mm test tubes and shaken in I ml of of T M B microwell peroxidase substrate system for 30 min at room temperature. The developed substrate solution was then transferred to a 1-ml capacity cuvette (1-cm light path) and its absorbance at 370 nm was determined. Results and Discussion Nitrocellulose membrane is commonly used in dot blot EIAs because of its large binding surface (Tijssen, 1985). However, it is difficult to wash efficiently because
46 TABLE I Dot blot E1A on polyester cloth and a nitrocellulose membrane Solid
Salmonella cells/ml
phase
l0 s
107
106
+ a +
+ +
+
+ +_
_+ +
Polyester cloth Coated Uncoated Nitrocellulose Coated Uncoated
105
<1
a + . strong colour reaction (dark blue spot); _+, weak colour reaction (faint blue spot): - . no colour reaction.
of its microporosity, whereas polyester cloth is easy to wash because of its macroporosity. Both nitrocellulose membrane sheets and polyester cloth were similarly coated with polymyxin and their effectiveness in the dot blot E1A was compared. Table I shows that polymyxin-cloth enabled the detection of 106 Salmonella cells/ml, whereas the uncoated cloth detected 107 tO 106 cells/ml. Thus, polymyxin coating stimulates the binding of LPS to the cloth. Polymyxin-cioth produced smaller and more intense spots than the uncoated cloth, suggesting that polymyxin coating increases the affinity for the LPS antigens. The EIA on uncoated nitrocellulose membrane produced only faint colour spots at 107 to 108 cells/ml. Coating the nitrocellulose membrane with polymyxin only slightly increased the sensitivity of Salmonella detection. Since the polymyxin-coated polyester cloth showed at least 10 times greater sensitivity for the detection of Salmonella than the nitrocellulose membrane, the polymyxin-cloth was used in all subsequent experiments. The dot blot EIA on polymyxin-cloth was then applied in a semi-quantitative test for Salmonella ceils in foods. Various food samples were homogenized in BPWYE and then inoculated with different numbers of Salmonella cells, and then serially diluted 10-fold in BPWYE. The diluted cultures were shaken at 37 °C for about 20 h and were then assayed by the dot blot EIA on polymyxin-cloth as described above. Table II shows that for all of the foods this procedure could distinguish inoculated food samples originally containing 1-10 cells/ml from those containing 100-1000 cells/ml. These results show that samples containing higher levels of Salmonella cells require greater dilutions (designated 'endpoints') to obtain Salrnonella-negative cultures. However, the endpoints obtained for individual samples cannot be definite because they only signify probability in terms of the most probable number (MPN) distribution of cells throughout the dilution series. Hence, some sample suspensions could be diluted by a factor exceeding the original inoculated cell density and still yield a positive EIA signal (i.e., growth of Salmonella cells). The statistical reliability of the endpoints can be increased by repetitions of the assay. Alternatively, the use of smaller dilutions (e.g., 2-fold
47
TABLE
II
Dot blot EIA of serially 10-fold diluted food samples inoculated with different levels of Food sample
Cells/ml
None
1000
Chicken meat
a
Beef meat
Milk
Mayonnaise
Flour
10
10 2
10 3
10 4
+ c
+
+
+
+
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
Chocolate
.
. .
.
+
+
+
+
-
+
+
+
+
-
10 ]
+
. .
+ .
.
. . .
. . .
. . .
1000
+
+
+
+
-
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
.
. .
. .
. .
1000
+
+
+
+
-
100
+
+
+
-
-
10
+
.
1
+
0
.
.
. .
.
. .
.
. .
. .
1000
+
+
+
+
-
100
+
+
+
+
-
• 10
+
+
-
-
-
1
+
.
0
.
.
. .
. .
. w
.
1000
+
+
+
+
-
100
+
+
+
-
-
+ .
. .
. .
. .
I
.
+
+
+
+
+
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
. .
. .
.
+
+
+
+
+
100
+
+
+
+
-
10
+
+
-
-
-
0
.
a Final density of inoculated
Salmonella
.
.
.
c e l l s in t h e o r i g i n a l s a m p l e s u s p e n s i o n a f t e r a d j u s t m e n t o f t h e
v o l u m e to I0 ml. b D i l u t i o n o f t h e o r i g i n a l s u s p e n s i o n a f t e r a d j u s t m e n t o f t h e v o l u m e t o 10 m l . c +, colour reaction (blue spot); -,
i i
.
1000
.
w
.
1000
.
l0 t
.
100
0
Cheese
.
.
1000
10
cells
Dilution factor b None
0
Salmonella
no colour reaction (no spot).
48 T A B L E III Effects of various enrichment media on the dot blot E1A for Salmonella lipopolysaccharide (LPSI Medium ~
LPS (~g/ml)
A 37r~b
BPW
0 0.1 0 0.1 0 0.1 0 0.1 0 0.1
0.06±0.0 0.34±0.03 0.05±0.0 0.37±0.04 0.06±0.0 0.32±0.02 0.07±0.0 0.33±0.04 0.06±0.0 0.35±0.03
BPWYE NB SCB TBB
The enrichment media tested were buffered peptone water (BPW), BPW supplemented with 0.5~c ( w / v ) yeast extract (BPWYE), nutrient broth (NB), selenite cysteine broth (SCB) and tetrathionate broth base (TBB). b Mean A370 value_+SE (n = 4).
instead of 10-fold) should allow a finer distinction between differences in the extent of Salmonella contamination. Such assays would involve larger numbers of samples for the dot blot EIA and would thus benefit from the use of the inexpensive antigen-capturing agent, polymyxin. In these experiments, buffered peptone water supplemented with yeast extract was used as an enrichment medium. In this non-selective medium, all uninoculated food samples developed turbidity (due to bacterial growth confirmed by plating) until the food samples were diluted at least 103-fold, except chicken breast which showed turbidity even after a 106-fold dilution. Thus, the presence of bacteria other than Salmonella does not seem to have affected the present assay. This procedure (combining enrichment culture and the dot blot EIA) detected levels of Salmonella contamination as low as 1-10 cells/ml in the original inoculated sample suspension. In meats such as chicken, Salmonella contamination may comprise a very low percentage of the total microbial population. Some microorganisms of chicken faeces origin have been shown to inhibit the growth of Sahnonella (Pivnick and Nurmi, 1982). Furthermore, the LPS of non-Salmonella Gram-negative bacteria present in large excess would compete for binding sites on the polymyxin-cloth, thus reducing the detectability by this procedure. In such cases, it may be desirable to employ selective enrichment media in this procedure. However. these media may contain components which interfere with the present dot blot E1A. Table II1 shows that the assay was equally effective in all of these media. However, interference with the assay using other media which were not tested is a possibility. In some foods (particularly dry foods), Salmonella cells may be injured and may not be enriched by some selective media to which these cells are sensitive. If such foods contain a large excess of non-Salmonella organisms which interfere with the
40
present procedure, it will be necessary to use enrichment in non-selective media followed by enrichment in selective media. Alternatively, selective media which will allow the growth of injured Salmonella cells should be sought. We have demonstrated here that the polymyxin-cloth-based dot blot enzyme immunoassay can be combined with a simple dilution enrichment culture technique to semi-quantitatively distinguish different levels of Salmonella contamination in various foods. Since the procedure is rapid and economical, it has the potential to be useful in monitoring sanitation in food manufacturing processes.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n cil o f C a n a d a G r a n t A 4698.
References Blais, B.W. and Yamazaki, H. (1989) Rapid antibody assay on the basis of the initial rate of immunoreaction. Biotechnol. Tech. 3, 253-256. Blais, B.W. and Yamazaki, H. (1991a) Use of polymyxin-coated polyester cloth for the enzyme immunoassay of Salmonella lipopolysaccharide antigens. Int. J. Food Microbiol. 11, 195-204. Blais, B.W. and Yamazaki, H. (1991b) Use of detergents in the preparation of Salmonella antigens for enzyme immunoassay on polymyxin-coated polyester cloth. Int. J. Food Microbiol. 11,329-336. Pivnick, H. and Nurmi, E. (1982) The Nurmi concept and its role in the control of Salmonella in poultry. In: Davies, R. (Ed.) Developments in food microbiology-l, Applied Science Publishers, Limited, Essex, pp. 41-70. Tijssen, P. (1985) Practice and theory of enzyme immunoassays. In: R.H. Burdon and P.H. van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, N.Y. 549 pp.
43
© 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1605/91/$03.50 FOOD 00429
Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods Burton W. Blais and Hiroshi Yamazaki Department of Biology and Institute of Biochemistry, Carleton Unicersity, Ottawa, Ontario, Canada (Received 11 February 1991; accepted 12 July 1991)
A rapid and economical semi-quantitative test for Salmonella cells in foods is proposed. Food samples containing different levels of Salmonella cells were homogenized and serially diluted in enrichment broths and then incubated for about 20 h at 37 ° C. The presence of Salmonella cells in each dilution was assayed by capturing deoxycholate-extracted Salmonella lipopolysaccharides on a sheet of polymyxin-coated polyester cloth, followed by colorimetric detection with an anti-Salmonella antibodyenzyme conjugate. The minimum dilutions which resulted in no detectable growth were correlated with the extent of Salmonella contamination in the food samples. Key words: Food manufacturing; Monitoring; Salmonella; Polymyxin
Introduction
The extent of microbial contamination in processed foods largely depends on sanitation in food manufacturing processes. Monitoring sanitation requires quantitative assays of microorganisms in food samples or processing equipment at different stages of the processing operation. For such monitoring in commercial processes rapidity, reproducibility and economy are important concerns. We have previously demonstrated the advantages of polyester cloth as an immunoadsorbent for enzyme immunoassay (EIA) which allows rapid immunoreactions because of the cloth's large surface area and efficient washing because of the cloth's macroporosity (Blais and Yamazaki, 1989). We have recently demonstrated the capture of bacterial lipopolysaccharide (LPS) antigens on polymyxin B-coated polyester cloth for EIA (Blais and Yamazaki, 1991a,b). Since the polymyxin-coated cloth (polymyxin-cloth) has a large capacity for binding LPS, it is possible to detect Salmonella LPS even in the presence of excess non-Salmonella LPS. Polymyxin B is available in a purified form and stable, and is considerably Correspondence address: H. Yamazaki, Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6.
44
less expensive to use than antibodies. The present paper demonstrates the application of polymyxin-cloth as a commercially attractive alternative to antibody-coated cloth for the semi-quantitative assay of Salmonella LPS antigens in processed foods. Salmonella typhimurium strain LT2 was used as a model system for this study.
Materials and Methods
Chemicals and immunoreagents Polymyxin B sulfate (No. P-1004) and sodium deoxycholate (No. D-6750) were obtained from Sigma Chemical Co. CSA-1 anti-Salmonella antibody-peroxidase conjugate (No. 04-91-99), T M B microwell peroxidase substrate system (No. 50-7600) and T M B membrane peroxidase substrate system (No. 50-77-00) were from Kirkegaard and Perry Laboratories, Inc. The conjugate was stored as a 0.1 m g / m l stock in 0.01 M phosphate-buffered (pH 7.2) and 0.85% NaC1 (PBS) at - 2 0 ° C . For use, it was diluted 1:4000 in PBS containing 0.05% Tween 20 (PBST). Buffered peptone, yeast extract, selenite cysteine, tetrathionate broth base, and nutrient broth were from Difco.
Preparation of food samples A variety of food samples were obtained from a local supermarket. Before inoculation with Salmonella cells, meat samples such as chicken breast and lean beef were blended to a paste using 5 x 10 s high speed bursts in a standard food processor. Solid samples (1 g) such as cheese and baker's chocolate were first mixed with 2 ml of buffered peptone water (BPW) containing 0.5% ( w / v ) yeast extract (BPWYE) and then blended as above. Powdered, semi-solid and liquid samples (flour, mayonnaise and milk) were used without blending.
Preparation of Salmonella-inoculated food samples Salmonella typhimurium strain LT2 stored on a nutrient agar slant at 4°C was inoculated into B P W Y E and grown to a density of about 10 9 cells/ml (late log phase) by shaking at 37°C for 16 h, and then diluted in sterile B P W Y E to obtain various cell densities (confirmed by viable counts obtained by plating serial dilutions of the suspensions on nutrient agar and incubating the plates at 37°C for 20 h). 1 ml of the diluted culture was mixed with the food homogenates containing the equivalent of 1 g of original food sample as described above. BPWYE was added to the mixture to a final volume of 10 ml and vigorously mixed in a vortex mixer until a uniform suspension was obtained. The suspension was then serially diluted 10-fold by transferring 0.5 ml to 4.5 ml of BPWYE in 2.5 x 15 cm culture tubes. All of the tubes then shaken at 37 ° C for about 20 h. One-tenth volume of 1.1% (w/v) sodium deoxycholate in PBS was added to each tube, and the tubes were then autoclaved at 121°C for 5 min. Heating the cells in deoxycholate dissociates LPS antigens into non-sedimentable form suitable for assay on
45 polymyxin-cloth (Blais and Yamazaki, 1991b). The treated samples were cooled to room temperature and then assayed by the dot blot EIA on polymyxin-cloth.
Dot blot EIA on polymyxin-coated polyester cloth Polyester cloth (DuPont, Sontara 8100) was cut into 6 x 4 cm sheets, then saturated with 3 ml of polymyxin B sulfate solution (5 m g / m l in PBS). After incubation for 16 h at room temperature, each sheet was washed with a total of about 6 x 10 ml of PBST on a filter under suction. For comparison, a 6 x 4 cm sheet of nitrocellulose (Schleicher and Schuell, No. 33172) was similarly coated with polymyxin. 10 #1 of the deoxycholate-heat-treated samples were pipetted onto the polymyxin-cloth sheet at 1-cm intervals, so that each 6 x 4 cm sheet accommodated a total of 24 samples. After 30 rain incubation at room temperature, the sheets were washed with PBST as above, and then saturated with 2 ml of the CSA-1 antibody-peroxidase conjugate. After 30 min incubation at room temperature, the sheets were washed with PBST, then blotted and incubated with 2 ml of TMB membrane peroxidase substrate system for 10 rain. Wherever samples containing Salmonella LPS were dot blotted on the polymyxin-cloth insoluble blue spots appeared. Comparison of polyester cloth with a nitrocellulose membrane Suspensions of S. typhimurium cells in B P W Y E at various cell densities were heated in 0.1% deoxycholate and 10/zl of the treated cell samples were incubated for 30 rain at room temperature with either polyester cloth or nitrocellulose membrane sheets similarly coated with polymyxin (5 mg/ml). The sheets were then washed with PBST and Salmonella LPS antigens captured on the sheets were detected using the anti-Salmonella antibody-peroxidase conjugate and the peroxidase detection system as described above. Effect of selectit,e and non-selectiz,e media on the assay of LPS antigens S. typhimurium LPS was suspended at 0 . 1 / z g / m l in non-selective media (BPW, BPWYE and nutrient broth) and selective media (selenite cysteine broth and tetrathionate broth). The suspensions were then assayed by the quantitative EIA on polymyxin-cloth as described previously (Blais and Yamazaki, 1991b). Briefly, 10 izl of deoxycholate-heat-treated samples were incubated for 30 min at room temperature with 6 x 6 mm polymyxin-cloth squares. The squares were then washed with PBST and incubated with 50/zl of the anti-Salmonella antibody-peroxidase conjugate for 30 min at room temperature. After washing with PBST, the squares were transferred to 16 x 100 mm test tubes and shaken in I ml of of T M B microwell peroxidase substrate system for 30 min at room temperature. The developed substrate solution was then transferred to a 1-ml capacity cuvette (1-cm light path) and its absorbance at 370 nm was determined. Results and Discussion Nitrocellulose membrane is commonly used in dot blot EIAs because of its large binding surface (Tijssen, 1985). However, it is difficult to wash efficiently because
46 TABLE I Dot blot E1A on polyester cloth and a nitrocellulose membrane Solid
Salmonella cells/ml
phase
l0 s
107
106
+ a +
+ +
+
+ +_
_+ +
Polyester cloth Coated Uncoated Nitrocellulose Coated Uncoated
105
<1
a + . strong colour reaction (dark blue spot); _+, weak colour reaction (faint blue spot): - . no colour reaction.
of its microporosity, whereas polyester cloth is easy to wash because of its macroporosity. Both nitrocellulose membrane sheets and polyester cloth were similarly coated with polymyxin and their effectiveness in the dot blot E1A was compared. Table I shows that polymyxin-cloth enabled the detection of 106 Salmonella cells/ml, whereas the uncoated cloth detected 107 tO 106 cells/ml. Thus, polymyxin coating stimulates the binding of LPS to the cloth. Polymyxin-cioth produced smaller and more intense spots than the uncoated cloth, suggesting that polymyxin coating increases the affinity for the LPS antigens. The EIA on uncoated nitrocellulose membrane produced only faint colour spots at 107 to 108 cells/ml. Coating the nitrocellulose membrane with polymyxin only slightly increased the sensitivity of Salmonella detection. Since the polymyxin-coated polyester cloth showed at least 10 times greater sensitivity for the detection of Salmonella than the nitrocellulose membrane, the polymyxin-cloth was used in all subsequent experiments. The dot blot EIA on polymyxin-cloth was then applied in a semi-quantitative test for Salmonella ceils in foods. Various food samples were homogenized in BPWYE and then inoculated with different numbers of Salmonella cells, and then serially diluted 10-fold in BPWYE. The diluted cultures were shaken at 37 °C for about 20 h and were then assayed by the dot blot EIA on polymyxin-cloth as described above. Table II shows that for all of the foods this procedure could distinguish inoculated food samples originally containing 1-10 cells/ml from those containing 100-1000 cells/ml. These results show that samples containing higher levels of Salmonella cells require greater dilutions (designated 'endpoints') to obtain Salrnonella-negative cultures. However, the endpoints obtained for individual samples cannot be definite because they only signify probability in terms of the most probable number (MPN) distribution of cells throughout the dilution series. Hence, some sample suspensions could be diluted by a factor exceeding the original inoculated cell density and still yield a positive EIA signal (i.e., growth of Salmonella cells). The statistical reliability of the endpoints can be increased by repetitions of the assay. Alternatively, the use of smaller dilutions (e.g., 2-fold
47
TABLE
II
Dot blot EIA of serially 10-fold diluted food samples inoculated with different levels of Food sample
Cells/ml
None
1000
Chicken meat
a
Beef meat
Milk
Mayonnaise
Flour
10
10 2
10 3
10 4
+ c
+
+
+
+
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
Chocolate
.
. .
.
+
+
+
+
-
+
+
+
+
-
10 ]
+
. .
+ .
.
. . .
. . .
. . .
1000
+
+
+
+
-
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
.
. .
. .
. .
1000
+
+
+
+
-
100
+
+
+
-
-
10
+
.
1
+
0
.
.
. .
.
. .
.
. .
. .
1000
+
+
+
+
-
100
+
+
+
+
-
• 10
+
+
-
-
-
1
+
.
0
.
.
. .
. .
. w
.
1000
+
+
+
+
-
100
+
+
+
-
-
+ .
. .
. .
. .
I
.
+
+
+
+
+
100
+
+
+
-
-
10
+
+
-
-
-
1
+
.
0
.
. .
. .
.
+
+
+
+
+
100
+
+
+
+
-
10
+
+
-
-
-
0
.
a Final density of inoculated
Salmonella
.
.
.
c e l l s in t h e o r i g i n a l s a m p l e s u s p e n s i o n a f t e r a d j u s t m e n t o f t h e
v o l u m e to I0 ml. b D i l u t i o n o f t h e o r i g i n a l s u s p e n s i o n a f t e r a d j u s t m e n t o f t h e v o l u m e t o 10 m l . c +, colour reaction (blue spot); -,
i i
.
1000
.
w
.
1000
.
l0 t
.
100
0
Cheese
.
.
1000
10
cells
Dilution factor b None
0
Salmonella
no colour reaction (no spot).
48 T A B L E III Effects of various enrichment media on the dot blot E1A for Salmonella lipopolysaccharide (LPSI Medium ~
LPS (~g/ml)
A 37r~b
BPW
0 0.1 0 0.1 0 0.1 0 0.1 0 0.1
0.06±0.0 0.34±0.03 0.05±0.0 0.37±0.04 0.06±0.0 0.32±0.02 0.07±0.0 0.33±0.04 0.06±0.0 0.35±0.03
BPWYE NB SCB TBB
The enrichment media tested were buffered peptone water (BPW), BPW supplemented with 0.5~c ( w / v ) yeast extract (BPWYE), nutrient broth (NB), selenite cysteine broth (SCB) and tetrathionate broth base (TBB). b Mean A370 value_+SE (n = 4).
instead of 10-fold) should allow a finer distinction between differences in the extent of Salmonella contamination. Such assays would involve larger numbers of samples for the dot blot EIA and would thus benefit from the use of the inexpensive antigen-capturing agent, polymyxin. In these experiments, buffered peptone water supplemented with yeast extract was used as an enrichment medium. In this non-selective medium, all uninoculated food samples developed turbidity (due to bacterial growth confirmed by plating) until the food samples were diluted at least 103-fold, except chicken breast which showed turbidity even after a 106-fold dilution. Thus, the presence of bacteria other than Salmonella does not seem to have affected the present assay. This procedure (combining enrichment culture and the dot blot EIA) detected levels of Salmonella contamination as low as 1-10 cells/ml in the original inoculated sample suspension. In meats such as chicken, Salmonella contamination may comprise a very low percentage of the total microbial population. Some microorganisms of chicken faeces origin have been shown to inhibit the growth of Sahnonella (Pivnick and Nurmi, 1982). Furthermore, the LPS of non-Salmonella Gram-negative bacteria present in large excess would compete for binding sites on the polymyxin-cloth, thus reducing the detectability by this procedure. In such cases, it may be desirable to employ selective enrichment media in this procedure. However. these media may contain components which interfere with the present dot blot E1A. Table II1 shows that the assay was equally effective in all of these media. However, interference with the assay using other media which were not tested is a possibility. In some foods (particularly dry foods), Salmonella cells may be injured and may not be enriched by some selective media to which these cells are sensitive. If such foods contain a large excess of non-Salmonella organisms which interfere with the
40
present procedure, it will be necessary to use enrichment in non-selective media followed by enrichment in selective media. Alternatively, selective media which will allow the growth of injured Salmonella cells should be sought. We have demonstrated here that the polymyxin-cloth-based dot blot enzyme immunoassay can be combined with a simple dilution enrichment culture technique to semi-quantitatively distinguish different levels of Salmonella contamination in various foods. Since the procedure is rapid and economical, it has the potential to be useful in monitoring sanitation in food manufacturing processes.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n cil o f C a n a d a G r a n t A 4698.
References Blais, B.W. and Yamazaki, H. (1989) Rapid antibody assay on the basis of the initial rate of immunoreaction. Biotechnol. Tech. 3, 253-256. Blais, B.W. and Yamazaki, H. (1991a) Use of polymyxin-coated polyester cloth for the enzyme immunoassay of Salmonella lipopolysaccharide antigens. Int. J. Food Microbiol. 11, 195-204. Blais, B.W. and Yamazaki, H. (1991b) Use of detergents in the preparation of Salmonella antigens for enzyme immunoassay on polymyxin-coated polyester cloth. Int. J. Food Microbiol. 11,329-336. Pivnick, H. and Nurmi, E. (1982) The Nurmi concept and its role in the control of Salmonella in poultry. In: Davies, R. (Ed.) Developments in food microbiology-l, Applied Science Publishers, Limited, Essex, pp. 41-70. Tijssen, P. (1985) Practice and theory of enzyme immunoassays. In: R.H. Burdon and P.H. van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, N.Y. 549 pp.