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The Effect of Washing Eggs under Commercial Conditions on Bacterial Loads on Egg Shells
The Effect of Washing Eggs under Commercial Conditions on Bacterial Loads on Egg Shells w. A. MOATS USDA, SEA, AR, Agricultural Research Center, Beltsville, Maryland 20705 (Received for publication November 20, 1978)
1979 Poultry Science 58:1228-1233
INTRODUCTION
The practice of cleaning eggs by washing was at one time widely condemned. At the present time, however, most eggs marketed commercially in the United States are cleaned by washing. A review of the literature on egg washing has been prepared separately (Moats, 1978a). Much of the published work has been concerned with the effect of washing on spoilage during long term storage. Long term storage-life of washed eggs is of little concern at present since few shell eggs are stored for extended periods. Eggs not immediately required for the fresh market are ordinarily diverted to breakers rather than to cold storage. Also, much of the work reported on washing was done with equipment and cleaning compounds different from those used by modern commercial egg washers. The most recent studies of washing eggs under commercial conditions are those of Brant et al, (1966) and March (1969). Today many commercial washers use a sanitizing rinse following washing, and such a rinse is required in plants operating under the voluntary shell egg grading service administrated by the USDA (1974). There is insufficient documentation, however, as to the value of such a rinse. The present study was undertaken to obtain
data on shells of mercial effect of
the bacteriological condition of the eggs washed under present day comconditions and also to evaluate the the sanitizing rinse following washing.
MATERIALS AND METHODS
Collection of Samples. Eggs were sampled from four commercial egg grading plants in Maryland and southeastern Pennsylvania. The eggs were collected from the processing lines with sterile metal tongs and placed in sterile, plastic-coated wire baskets enclosed in plastic bags for transport back to the laboratory. Each sample consisted of 10 unwashed and 10 washed eggs collected at random. In plants using a sanitizing rinse, the flow of sanitizer to the rinse water was temporarily cut off and 10 washed eggs were collected. Washwater samples were collected at the same time in sterile bottles and immediately chilled in ice. Samples were taken near the end of the morning run, and a number were also taken at the beginning of the afternoon run, shortly after the washwater had been completely changed. All washwater samples were tested bacteriologically on the same day they were collected. Egg samples were refrigerated and tested within a day or two of collection.
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ABSTRACT Thirty-three sample sets, each consisting of washed and unwashed eggs, and washwater were collected from four commercial egg grading plants in Maryland and southeastern Pennsylvania. The four plants used different combinations of washing compounds and sanitizing or water rinses. Bacterial loads on eggs were determined both by a surface rinse method and by a method involving evacuating and blending the shells (EB). Total plate counts at 22 and 37 C were not significantly different. Surface counts on washed eggs in plants using sanitizing rinses were frequently very low «50/shell) and were significantly lower than in one plant using water rinse. However, when the sanitizer was temporarily cut off in plants using a sanitizing rinse, surface counts on washed eggs were as low as before, indicating that any beneficial effect of the sanitizer was indirect. Correlations between bacterial counts of washwater and of washed eggs were low but significant. Further examination of the data indicated that little if any contamination of eggs by washwater occurred in most cases. Bacterial counts on selective and differential agars showed generally low but variable numbers of coliforms and enterococci and substantial numbers of micrococci on the shells. Salmonellae were isolated from two lots of unwashed eggs and one of washed eggs, by all the EB method.
BACTERIA ON WASHED EGGS
RESULTS AND DISCUSSION
Each of the four egg grading plants studied used a different combination of cleaning and sanitizing compounds (Table 1). Plant A used a chlorinated triazine sanitizer, Plant B a quaternary ammonium formulation, and Plant C sodium hypochlorite. Plant D did not add sanitizer to the rinse water. The cleaning com-
TABLE 1. Types of egg washing and sanithers used in plants studied Cleaner
Sanitizing rinse
Plant A Chlorinated (Brand A) Chlorinated (Brand B) Plant B Non-sanitizing Quaternary ammonium Plant C Chlorinated (Brand C) Chlorinated (sodium hypochlorite) Plant D Non-sanitizing None
pounds used all gave a highly alkaline reaction, usually above pH 11, when freshly made up in the washwater. However, the pH quickly fell below 11 during operation as the washwater was diluted by overflow and became mixed with dirt and egg solids, and washwaters at time of sampling were usually in the pH range 10 to 11. Washwater temperatures were 39 to 50 C. Results with washed and unwashed eggs are summarized in Table 2. The unwashed eggs were clean to lightly soiled in appearance. The data shown are bacterial counts on TSA at 37 C. Counts at 22 C were not significantly different from those at 37 C, but this does not necessarily mean that the bacteria found are the same at the two temperatures. Both R and EB methods were used so that both the total numbers of bacteria present and their distribution on the shells could be determined. Bacteria on the shell surface can contaminate the egg meat when the egg is broken out which is of particular concern in commercial egg-breaking operations. Shaking in liquid was selected for the surface rinse method since any bacteria not dislodged from the shell by this procedure are unlikely to contaminate the contents when the eggs are broken out. Surface counts (R) were extremely low for washed eggs from Plants A, B, and C which used sanitizing rinses following washing. Counts for 1 ml of rinse solution placed in duplicate were frequently 0, equivalent to <50 bacteria/ shell surface. In these plants, the sanitizer was temporarily cut off and an additional sample of washed eggs collected. The surface counts were not significantly different from those of eggs rinsed with sanitizer. However, in Plant D, which did not use a sanitizing rinse at all, surface counts of washed eggs were somewhat, but not excessively, higher than those of washed eggs from the other three. The sanitizing rinse may therefore be indirectly beneficial. The rinse waters drained into the washwater reser-
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Microbiological Tests. In each egg sample five eggs of the same treatment were tested by the evacuation and blending method (EB) and the five remaining by the surface rinse method (R). In R, the eggs were placed individually in 8 oz jars containing 100 ml of Tryptic-soy broth (TSB; Difco) and the jars were shaken on a mechanical rotary shaker for 15 min. The TSB from the jars was composited for bacteriological testing. For EB each egg was placed on a small flame-sterilized beaker and a small hole was carefully cut in the end with a sterile scalpel. A tube connected to a suction flask was inserted into the hole, and the contents of the egg were removed as completely as possible by suction. The shells of five eggs were composited in a 500 ml blender jar and blended with 200 ml of TSB for 2 min. Plating Method. Dilutions of the washwater and of the composited TSB's from the R and EB procedures for the eggs were prepared in . 1 % peptone. These were pour plated on Tryptic-soy agar (TSA; Difco) and duplicate plates were incubated at both 22 and 37 C. The sample sets were also pour plated on violet redbile agar (VRB; BBL) and surface plated on menterococcus agar (BBL), Staphylococcus 110 agar (BBL), and Baird-Parker agar (BBL) and incubated at 37 C. The TSA plates incubated at 22 C were counted after 5 days, VRB and menterococcus after 24 hr and others after 48 hr. Salmonella Tests. The remaining TSB from the R and EB procedures was incubated 24 hr at 37 C and streaked on Tryptic-soy xyloselysine (TSXL) and Tryptic-soy brilliant green (TSBG) agars (Moats and Kinner, 1976). Also, .5 ml portions of the 24 hr incubated TSB were added to 10 ml each of selenite-cystine and tetrathionate broths which were incubated 24 hr and then streaked on TSBG and TSXL plates. Salmonella suspect colonies from the plates were confirmed biochemically as described by Moats (1978b).
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MOATS TABLE 2. Mean logv
0
bacterial counts on egg shells at 37 C, (Counts/shell) Washed Sanitizing rinse
Unwashed Plant
Rinse
A B C D All plants
9 8 6 10 33
4.96c"g 407h-n 4.51 e _ 1 4.23g" m 4.44 d
Evacuationblending 5.55*^ 4.62e'k 4.82 d ^ 4.60e"J 4.92bc
Evacuationblending
Rinse 9
8 6
Washed Water-rinse
2.820"1 1.93 st 1.80'
23
3.86J-° 3 37nop
4 4 6 10 24
2.32q" t 3.29 f
Rinse
Evacuationblending
1.701 2.00 r s t l^O' 3.08°P<5 2.338
3.61k"P 3.21 n< l 2.7lP- c 4.541e-k 3.70',ef
N, number of samples.
voirs, perhaps maintaining a level of sanitizer in the washwaters. The sanitizing rinse also continually rinsed the egg conveyor with sanitizer which might reduce bacterial loads on the conveyor surfaces or other equipment surfaces which came in contact with the rinsed eggs. Bacterial counts of washwaters are summarized in Table 3. Counts for Plant C were lower than for the other three plants. Since counts for Plant D, which did not use a sanitizing rinse, were no higher than for Plants A and B, which did, it appears that the sanitizing rinse did not necessarily lower counts in the washwater significantly. In Plants A, B, and D, washwater samples were taken at 1100 hr, near the end of the morning run, and at 1300 hr, shortly after the washwater had been changed. Although counts were substantially lower at 1300 hr (Table 4) than at 1100 hr, the differences
were not statistically significant when considered on an individual plant basis but were significantly different when all data were combined. Bacterial counts of washwater and washed eggs were significantly correlated (Table 5) when the combined data were considered, although the coefficients of correlation were rather low. No such correlation was evident within the individual plants. The correlation between bacterial counts of washwater and washed eggs suggested the possibility of contamination of eggs from the washwater. The counts on eggs washed at 1100 hr and at 1300 hr were not significantly different, however (Table 6). If contamination by washwater had been appreciable, bacterial counts would have been lower for the eggs washed in
TABLE 4. Effect of time of sampling washwaters on bacterial counts on Tryptic-soy agar at 22 and 37 C (combined data)
TABLE 3. Mean logl 0 bacterial counts of washwaters on Tryptic-soy agar
Plant A B C D
Plant
Temperature of incubation
Log 1 0 counts/ml
22 37 22 37 22 37 22 37
5.64* 5.77* 5.60* 5.99* 3.67 b 4.18 b 5.50* 5.77*
ab ' Numbers followed by the same letter are not significantly different according to Duncan's multiple range test (1955).
A B D Combined data (Plants A, B, andD)
Time of sampling
N1
Mean bacterial counts/ml (X 10 3 )
1100 1300 1100 1300 1100 1300
hr hr hr hr hr hr
9 6 12 4 10 10
1120* 610* 2320* 376* 2100* 425*
1100 hr 1300 hr
31 20
1900* 4 7 jb
Number of samples. ab ' N u m b e r s followed by the same letter are not significantly different according to Duncan's multiple range test (1955).
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Numbers followed by the same letter are not significantly different according to Duncan's multiple range test (1955). Individual plants and all plants data are considered separately.
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BACTERIA ON WASHED EGGS TABLE 5. Correlations between log^ 0 bacterial counts on Tryptic-soy agar in wasbwaters and on eggs Egg treatment and microbiological test method
All data 22 and 37 C combined
Individual plants
Correlation coefficients No significant correlations
-.17675 -.15925 .34150* .46558** .43688** .42817**
'Significant at the 5% level. **Significant at the 1% level.
fresh washwater. Data from individual sample sets (Table 7) suggest that contamination may occur but is not necessarily related to bacterial loads in washwater. In set B-6, relatively clean eggs were washed in water with the highest bacterial count observed. Yet, the bacterial counts of the washed eggs were low, showing no evidence of contamination from the washwater. Set D-ll is an example of washed eggs having higher bacterial counts than the unwashed eggs. The bacterial counts of the washwater was not exceptionally high, although the pH of the washwater was only 8.6, indicating that the cleaning compound was nearly exhausted. Ten sample sets were also plated on selective
TABLE 6. Effect of time of sampling on logt „ bacterial counts of eggs1 Washed-sanitizing rinse
Washed water rinse
Time
N2
Rinse method
Evacuation blending method
1100 hr 1300 hr
22 12
2.55 a 2.15 a
3.65 a 3.76 a
2
N
Rinse method
Evacuation blending method
22 14
2.63 a 2.80 a
3.95 a 4.38 b
Plated on Tryptic-soy agar — combined data from 22 and 37 C incubation. Number of samples.
a ' Numbers in each column followed by the same letter are not significantly different by Duncan's multiple range test (1955).
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Unwashed Rinse Evacuation-blending Washed-sanitized Rinse Evacuation-blending Washed-no sanitizer Rinse Evacuation-blending
and differential media to obtain information as to the types of bacteria present on the shells (Tables 8, 9, and 10). Coliform and enterococcus counts of washwater were variable but fairly low. Fairly high counts were observed on Staphylococcus 110 and Baird-Parker media. However, bacterial colonies were not typical of staphylococci and examination of a number of isolates indicated that micrococci predominated on the plates. Results were similar with unwashed eggs. With washed eggs, surface counts (R) were very low, and nothing was found on any of the selective and differential media. Total shell counts (EB method) were also lower than those of the unwashed eggs but otherwise followed a similar pattern. All washed and unwashed eggs were also tested for salmonellae. Enrichment in selenitecystine and tetrathionate broths did not improve recoveries of salmonellae over those obtained by streaking from the TSB suspensions of bacteria after incubation 24 hr at 37 C. This was true with both naturally and artificially contaminated eggs. Salmonellae were recovered from two lots of unwashed eggs and one of washed eggs, all by the EB method. The washed eggs had been rinsed in chlorine and had a count of only 20 per shell. This demonstrates that a chlorine rinse may not necessarily free egg shells of salmonellae. The results show that washing under commercial conditions was highly effective in reducing surface bacterial counts on egg shells to low levels. In most cases, there was little evidence that eggs were contaminated to any significant degree by bacteria in the washwater. The effect of the sanitizing rinse following washing is unclear. It did not directly affect
MOATS
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TABLE 7. Relation between bacterial counts of washwater and egg shells Sample series
B-61 D-ll2
Egg treatment Washwater
Unwashed eggs
counts/ml
R*
EB*
9,800,000 500,000
1,200 21,000
5,480 76,000
Washed sanitizing rinse R <50
Washed water-rinse
EB
EB
R <50 850,000
2000
1,300 448,000
TABLE 8. Plate counts of washwater on selective and nonselective media (10 samples) Counts/ml Mean Tryptic-soy agar (37 C) Violet-red agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 medium
Range 178,000 <1' 5 210 50
1,030,000 176 1255 199,000 10,200
7,420,000 1000 3750 1,350,000 60,500
— -
<1 = No colonies found on duplicate plates.
counts on the shells as indicated by the tests in which the sanitizer was temporarily cut off. There is no clear evidence that the sanitizing rinse reduced bacterial loads in the washwater since bacterial counts of washwaters from Plants A and B were similar to those from Plant D. In Plant C additional active chlorine (BK powder) was added together with the chlorinated washing compound at the beginning of each half-day run and at the mid-run break which probably accounts for the lower counts in the washwater. The sanitizing rinse of the eggs would also
have sanitized the egg conveyor, and this might account for the slightly lower surface counts on eggs when a sanitizing rinse was used. Further investigations on this point are planned. Antimicrobial chemicals have been consistently found to be ineffective in destroying bacteria embedded in the shell (Moats, 1978a). The alkaline pH of the egg washing compounds used is unfavorable for growth of most bacteria and many types die off rapidly. A study of the survival of various types of bacteria under conditions of temperature and pH present in egg washers will be published sepa-
TABLE 9. Plate counts of unwashed eggs on selective and nonselective media Counts/shell (10 samples) Rinsed Mean Tryptic-soy agar 37 C Violet red bile agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 agar
1,100,000 23,000 4000 169,000 1,050,000
Evacuation-blending Range
2100 <50' <250 500 <250
5,110,000 205,000 22,000 945,000 7,550,000
<50, <250, etc. — no colonies were found on duplicate plates.
Mean 690,000 90 5220 45,700 380,000
Range 11,000 <20 <100 2000 4400
-
4,160,000 480 30,400 190,000 3,020,000
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Bacterial counts at 37 C on Tryptic-soy agar incubated 48 hr. Bacterial counts at 27 C on Tryptic-soy agar incubated 72 hr. *R = rinse method; EB = evacuation-blending method.
BACTERIA ON WASHED EGGS
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TABLE 10. Plate counts of washed eggs on selective and nonselective media Counts/shell (10 samples) Rinse Mean Tryptic-soy agar (37 C) Violet-red bile agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 agar 1
2110 <50' <250 <250 <250
Range
Mean
<50 - ]15,000 <50 <250 <250 <250
63,000 16 100 860 5660
Evacuation-blending Range 600 <20 <100 <100 <100
-
290,000 160 400 2000 26,000
<50, <250, etc., means no colonies were found on duplicate plates.
ACKNOWLEDGMENTS The author wishes to thank T. M. Brennan and J. I. Shultz, Jr., for technical assistance; E. J. Koch, SEA Biometrical Services, for preparing the statistical analyses of the data; R. H. Greenfield, USDA, Food Safety and Quality Service, for helpful discussions; and the following commercial firms which participated in this study: Maryland Fresh Eggs, Hampstead, MD, Pennfield Farms, Ephrata, PA, R. W. Souder, Lititz, PA, and Southern States Cooperative, Baltimore, MD.
REFERENCES Brant, A. W., P. B. Starr, and J. A. Hamann, 1966. The bacteriological, chemical, and physical requirements for commercial egg cleaning. USDA, ARS, Marketing Res. Rep. 770. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11:1—42. March, B. E., 1969. Bacterial infection of washed and unwashed eggs with respect to salmonellae. Appl. Microbiol. 1 7 : 9 8 - 1 0 1 . Moats, W. A., 1978a. Egg washing — A review. J. Food Protect. 41:919-925. Moats, W. A., 1978b. A comparison of four plating media with and without added novobiocin for isolation of salmonellae. Appl. Environ. Microbiol. 36:747-751. Moats, W. A., and J. A. Kinner, 1976. Observations on brilliant green agar with an H2 S indicator. Appl. Environ. Microbiol. 31:380-384. United States Department of Agriculture, Agricultural Marketing Service, 1974. Regulations governing the grading of shell eggs and United States standards, grades, and weight classes for shell eggs. (7CFR, part 56).
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rately. Indications are that buildup of bacteria in the washwaters can be controlled to a considerable extent by the addition of sufficient cleaning compound to maintain the pH at 10 or higher.
1979 Poultry Science 58:1228-1233
INTRODUCTION
The practice of cleaning eggs by washing was at one time widely condemned. At the present time, however, most eggs marketed commercially in the United States are cleaned by washing. A review of the literature on egg washing has been prepared separately (Moats, 1978a). Much of the published work has been concerned with the effect of washing on spoilage during long term storage. Long term storage-life of washed eggs is of little concern at present since few shell eggs are stored for extended periods. Eggs not immediately required for the fresh market are ordinarily diverted to breakers rather than to cold storage. Also, much of the work reported on washing was done with equipment and cleaning compounds different from those used by modern commercial egg washers. The most recent studies of washing eggs under commercial conditions are those of Brant et al, (1966) and March (1969). Today many commercial washers use a sanitizing rinse following washing, and such a rinse is required in plants operating under the voluntary shell egg grading service administrated by the USDA (1974). There is insufficient documentation, however, as to the value of such a rinse. The present study was undertaken to obtain
data on shells of mercial effect of
the bacteriological condition of the eggs washed under present day comconditions and also to evaluate the the sanitizing rinse following washing.
MATERIALS AND METHODS
Collection of Samples. Eggs were sampled from four commercial egg grading plants in Maryland and southeastern Pennsylvania. The eggs were collected from the processing lines with sterile metal tongs and placed in sterile, plastic-coated wire baskets enclosed in plastic bags for transport back to the laboratory. Each sample consisted of 10 unwashed and 10 washed eggs collected at random. In plants using a sanitizing rinse, the flow of sanitizer to the rinse water was temporarily cut off and 10 washed eggs were collected. Washwater samples were collected at the same time in sterile bottles and immediately chilled in ice. Samples were taken near the end of the morning run, and a number were also taken at the beginning of the afternoon run, shortly after the washwater had been completely changed. All washwater samples were tested bacteriologically on the same day they were collected. Egg samples were refrigerated and tested within a day or two of collection.
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ABSTRACT Thirty-three sample sets, each consisting of washed and unwashed eggs, and washwater were collected from four commercial egg grading plants in Maryland and southeastern Pennsylvania. The four plants used different combinations of washing compounds and sanitizing or water rinses. Bacterial loads on eggs were determined both by a surface rinse method and by a method involving evacuating and blending the shells (EB). Total plate counts at 22 and 37 C were not significantly different. Surface counts on washed eggs in plants using sanitizing rinses were frequently very low «50/shell) and were significantly lower than in one plant using water rinse. However, when the sanitizer was temporarily cut off in plants using a sanitizing rinse, surface counts on washed eggs were as low as before, indicating that any beneficial effect of the sanitizer was indirect. Correlations between bacterial counts of washwater and of washed eggs were low but significant. Further examination of the data indicated that little if any contamination of eggs by washwater occurred in most cases. Bacterial counts on selective and differential agars showed generally low but variable numbers of coliforms and enterococci and substantial numbers of micrococci on the shells. Salmonellae were isolated from two lots of unwashed eggs and one of washed eggs, by all the EB method.
BACTERIA ON WASHED EGGS
RESULTS AND DISCUSSION
Each of the four egg grading plants studied used a different combination of cleaning and sanitizing compounds (Table 1). Plant A used a chlorinated triazine sanitizer, Plant B a quaternary ammonium formulation, and Plant C sodium hypochlorite. Plant D did not add sanitizer to the rinse water. The cleaning com-
TABLE 1. Types of egg washing and sanithers used in plants studied Cleaner
Sanitizing rinse
Plant A Chlorinated (Brand A) Chlorinated (Brand B) Plant B Non-sanitizing Quaternary ammonium Plant C Chlorinated (Brand C) Chlorinated (sodium hypochlorite) Plant D Non-sanitizing None
pounds used all gave a highly alkaline reaction, usually above pH 11, when freshly made up in the washwater. However, the pH quickly fell below 11 during operation as the washwater was diluted by overflow and became mixed with dirt and egg solids, and washwaters at time of sampling were usually in the pH range 10 to 11. Washwater temperatures were 39 to 50 C. Results with washed and unwashed eggs are summarized in Table 2. The unwashed eggs were clean to lightly soiled in appearance. The data shown are bacterial counts on TSA at 37 C. Counts at 22 C were not significantly different from those at 37 C, but this does not necessarily mean that the bacteria found are the same at the two temperatures. Both R and EB methods were used so that both the total numbers of bacteria present and their distribution on the shells could be determined. Bacteria on the shell surface can contaminate the egg meat when the egg is broken out which is of particular concern in commercial egg-breaking operations. Shaking in liquid was selected for the surface rinse method since any bacteria not dislodged from the shell by this procedure are unlikely to contaminate the contents when the eggs are broken out. Surface counts (R) were extremely low for washed eggs from Plants A, B, and C which used sanitizing rinses following washing. Counts for 1 ml of rinse solution placed in duplicate were frequently 0, equivalent to <50 bacteria/ shell surface. In these plants, the sanitizer was temporarily cut off and an additional sample of washed eggs collected. The surface counts were not significantly different from those of eggs rinsed with sanitizer. However, in Plant D, which did not use a sanitizing rinse at all, surface counts of washed eggs were somewhat, but not excessively, higher than those of washed eggs from the other three. The sanitizing rinse may therefore be indirectly beneficial. The rinse waters drained into the washwater reser-
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Microbiological Tests. In each egg sample five eggs of the same treatment were tested by the evacuation and blending method (EB) and the five remaining by the surface rinse method (R). In R, the eggs were placed individually in 8 oz jars containing 100 ml of Tryptic-soy broth (TSB; Difco) and the jars were shaken on a mechanical rotary shaker for 15 min. The TSB from the jars was composited for bacteriological testing. For EB each egg was placed on a small flame-sterilized beaker and a small hole was carefully cut in the end with a sterile scalpel. A tube connected to a suction flask was inserted into the hole, and the contents of the egg were removed as completely as possible by suction. The shells of five eggs were composited in a 500 ml blender jar and blended with 200 ml of TSB for 2 min. Plating Method. Dilutions of the washwater and of the composited TSB's from the R and EB procedures for the eggs were prepared in . 1 % peptone. These were pour plated on Tryptic-soy agar (TSA; Difco) and duplicate plates were incubated at both 22 and 37 C. The sample sets were also pour plated on violet redbile agar (VRB; BBL) and surface plated on menterococcus agar (BBL), Staphylococcus 110 agar (BBL), and Baird-Parker agar (BBL) and incubated at 37 C. The TSA plates incubated at 22 C were counted after 5 days, VRB and menterococcus after 24 hr and others after 48 hr. Salmonella Tests. The remaining TSB from the R and EB procedures was incubated 24 hr at 37 C and streaked on Tryptic-soy xyloselysine (TSXL) and Tryptic-soy brilliant green (TSBG) agars (Moats and Kinner, 1976). Also, .5 ml portions of the 24 hr incubated TSB were added to 10 ml each of selenite-cystine and tetrathionate broths which were incubated 24 hr and then streaked on TSBG and TSXL plates. Salmonella suspect colonies from the plates were confirmed biochemically as described by Moats (1978b).
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MOATS TABLE 2. Mean logv
0
bacterial counts on egg shells at 37 C, (Counts/shell) Washed Sanitizing rinse
Unwashed Plant
Rinse
A B C D All plants
9 8 6 10 33
4.96c"g 407h-n 4.51 e _ 1 4.23g" m 4.44 d
Evacuationblending 5.55*^ 4.62e'k 4.82 d ^ 4.60e"J 4.92bc
Evacuationblending
Rinse 9
8 6
Washed Water-rinse
2.820"1 1.93 st 1.80'
23
3.86J-° 3 37nop
4 4 6 10 24
2.32q" t 3.29 f
Rinse
Evacuationblending
1.701 2.00 r s t l^O' 3.08°P<5 2.338
3.61k"P 3.21 n< l 2.7lP- c 4.541e-k 3.70',ef
N, number of samples.
voirs, perhaps maintaining a level of sanitizer in the washwaters. The sanitizing rinse also continually rinsed the egg conveyor with sanitizer which might reduce bacterial loads on the conveyor surfaces or other equipment surfaces which came in contact with the rinsed eggs. Bacterial counts of washwaters are summarized in Table 3. Counts for Plant C were lower than for the other three plants. Since counts for Plant D, which did not use a sanitizing rinse, were no higher than for Plants A and B, which did, it appears that the sanitizing rinse did not necessarily lower counts in the washwater significantly. In Plants A, B, and D, washwater samples were taken at 1100 hr, near the end of the morning run, and at 1300 hr, shortly after the washwater had been changed. Although counts were substantially lower at 1300 hr (Table 4) than at 1100 hr, the differences
were not statistically significant when considered on an individual plant basis but were significantly different when all data were combined. Bacterial counts of washwater and washed eggs were significantly correlated (Table 5) when the combined data were considered, although the coefficients of correlation were rather low. No such correlation was evident within the individual plants. The correlation between bacterial counts of washwater and washed eggs suggested the possibility of contamination of eggs from the washwater. The counts on eggs washed at 1100 hr and at 1300 hr were not significantly different, however (Table 6). If contamination by washwater had been appreciable, bacterial counts would have been lower for the eggs washed in
TABLE 4. Effect of time of sampling washwaters on bacterial counts on Tryptic-soy agar at 22 and 37 C (combined data)
TABLE 3. Mean logl 0 bacterial counts of washwaters on Tryptic-soy agar
Plant A B C D
Plant
Temperature of incubation
Log 1 0 counts/ml
22 37 22 37 22 37 22 37
5.64* 5.77* 5.60* 5.99* 3.67 b 4.18 b 5.50* 5.77*
ab ' Numbers followed by the same letter are not significantly different according to Duncan's multiple range test (1955).
A B D Combined data (Plants A, B, andD)
Time of sampling
N1
Mean bacterial counts/ml (X 10 3 )
1100 1300 1100 1300 1100 1300
hr hr hr hr hr hr
9 6 12 4 10 10
1120* 610* 2320* 376* 2100* 425*
1100 hr 1300 hr
31 20
1900* 4 7 jb
Number of samples. ab ' N u m b e r s followed by the same letter are not significantly different according to Duncan's multiple range test (1955).
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Numbers followed by the same letter are not significantly different according to Duncan's multiple range test (1955). Individual plants and all plants data are considered separately.
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BACTERIA ON WASHED EGGS TABLE 5. Correlations between log^ 0 bacterial counts on Tryptic-soy agar in wasbwaters and on eggs Egg treatment and microbiological test method
All data 22 and 37 C combined
Individual plants
Correlation coefficients No significant correlations
-.17675 -.15925 .34150* .46558** .43688** .42817**
'Significant at the 5% level. **Significant at the 1% level.
fresh washwater. Data from individual sample sets (Table 7) suggest that contamination may occur but is not necessarily related to bacterial loads in washwater. In set B-6, relatively clean eggs were washed in water with the highest bacterial count observed. Yet, the bacterial counts of the washed eggs were low, showing no evidence of contamination from the washwater. Set D-ll is an example of washed eggs having higher bacterial counts than the unwashed eggs. The bacterial counts of the washwater was not exceptionally high, although the pH of the washwater was only 8.6, indicating that the cleaning compound was nearly exhausted. Ten sample sets were also plated on selective
TABLE 6. Effect of time of sampling on logt „ bacterial counts of eggs1 Washed-sanitizing rinse
Washed water rinse
Time
N2
Rinse method
Evacuation blending method
1100 hr 1300 hr
22 12
2.55 a 2.15 a
3.65 a 3.76 a
2
N
Rinse method
Evacuation blending method
22 14
2.63 a 2.80 a
3.95 a 4.38 b
Plated on Tryptic-soy agar — combined data from 22 and 37 C incubation. Number of samples.
a ' Numbers in each column followed by the same letter are not significantly different by Duncan's multiple range test (1955).
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Unwashed Rinse Evacuation-blending Washed-sanitized Rinse Evacuation-blending Washed-no sanitizer Rinse Evacuation-blending
and differential media to obtain information as to the types of bacteria present on the shells (Tables 8, 9, and 10). Coliform and enterococcus counts of washwater were variable but fairly low. Fairly high counts were observed on Staphylococcus 110 and Baird-Parker media. However, bacterial colonies were not typical of staphylococci and examination of a number of isolates indicated that micrococci predominated on the plates. Results were similar with unwashed eggs. With washed eggs, surface counts (R) were very low, and nothing was found on any of the selective and differential media. Total shell counts (EB method) were also lower than those of the unwashed eggs but otherwise followed a similar pattern. All washed and unwashed eggs were also tested for salmonellae. Enrichment in selenitecystine and tetrathionate broths did not improve recoveries of salmonellae over those obtained by streaking from the TSB suspensions of bacteria after incubation 24 hr at 37 C. This was true with both naturally and artificially contaminated eggs. Salmonellae were recovered from two lots of unwashed eggs and one of washed eggs, all by the EB method. The washed eggs had been rinsed in chlorine and had a count of only 20 per shell. This demonstrates that a chlorine rinse may not necessarily free egg shells of salmonellae. The results show that washing under commercial conditions was highly effective in reducing surface bacterial counts on egg shells to low levels. In most cases, there was little evidence that eggs were contaminated to any significant degree by bacteria in the washwater. The effect of the sanitizing rinse following washing is unclear. It did not directly affect
MOATS
1232
TABLE 7. Relation between bacterial counts of washwater and egg shells Sample series
B-61 D-ll2
Egg treatment Washwater
Unwashed eggs
counts/ml
R*
EB*
9,800,000 500,000
1,200 21,000
5,480 76,000
Washed sanitizing rinse R <50
Washed water-rinse
EB
EB
R <50 850,000
2000
1,300 448,000
TABLE 8. Plate counts of washwater on selective and nonselective media (10 samples) Counts/ml Mean Tryptic-soy agar (37 C) Violet-red agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 medium
Range 178,000 <1' 5 210 50
1,030,000 176 1255 199,000 10,200
7,420,000 1000 3750 1,350,000 60,500
— -
<1 = No colonies found on duplicate plates.
counts on the shells as indicated by the tests in which the sanitizer was temporarily cut off. There is no clear evidence that the sanitizing rinse reduced bacterial loads in the washwater since bacterial counts of washwaters from Plants A and B were similar to those from Plant D. In Plant C additional active chlorine (BK powder) was added together with the chlorinated washing compound at the beginning of each half-day run and at the mid-run break which probably accounts for the lower counts in the washwater. The sanitizing rinse of the eggs would also
have sanitized the egg conveyor, and this might account for the slightly lower surface counts on eggs when a sanitizing rinse was used. Further investigations on this point are planned. Antimicrobial chemicals have been consistently found to be ineffective in destroying bacteria embedded in the shell (Moats, 1978a). The alkaline pH of the egg washing compounds used is unfavorable for growth of most bacteria and many types die off rapidly. A study of the survival of various types of bacteria under conditions of temperature and pH present in egg washers will be published sepa-
TABLE 9. Plate counts of unwashed eggs on selective and nonselective media Counts/shell (10 samples) Rinsed Mean Tryptic-soy agar 37 C Violet red bile agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 agar
1,100,000 23,000 4000 169,000 1,050,000
Evacuation-blending Range
2100 <50' <250 500 <250
5,110,000 205,000 22,000 945,000 7,550,000
<50, <250, etc. — no colonies were found on duplicate plates.
Mean 690,000 90 5220 45,700 380,000
Range 11,000 <20 <100 2000 4400
-
4,160,000 480 30,400 190,000 3,020,000
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Bacterial counts at 37 C on Tryptic-soy agar incubated 48 hr. Bacterial counts at 27 C on Tryptic-soy agar incubated 72 hr. *R = rinse method; EB = evacuation-blending method.
BACTERIA ON WASHED EGGS
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TABLE 10. Plate counts of washed eggs on selective and nonselective media Counts/shell (10 samples) Rinse Mean Tryptic-soy agar (37 C) Violet-red bile agar M-enterococcus agar Baird-Parker agar Staphylococcus 110 agar 1
2110 <50' <250 <250 <250
Range
Mean
<50 - ]15,000 <50 <250 <250 <250
63,000 16 100 860 5660
Evacuation-blending Range 600 <20 <100 <100 <100
-
290,000 160 400 2000 26,000
<50, <250, etc., means no colonies were found on duplicate plates.
ACKNOWLEDGMENTS The author wishes to thank T. M. Brennan and J. I. Shultz, Jr., for technical assistance; E. J. Koch, SEA Biometrical Services, for preparing the statistical analyses of the data; R. H. Greenfield, USDA, Food Safety and Quality Service, for helpful discussions; and the following commercial firms which participated in this study: Maryland Fresh Eggs, Hampstead, MD, Pennfield Farms, Ephrata, PA, R. W. Souder, Lititz, PA, and Southern States Cooperative, Baltimore, MD.
REFERENCES Brant, A. W., P. B. Starr, and J. A. Hamann, 1966. The bacteriological, chemical, and physical requirements for commercial egg cleaning. USDA, ARS, Marketing Res. Rep. 770. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11:1—42. March, B. E., 1969. Bacterial infection of washed and unwashed eggs with respect to salmonellae. Appl. Microbiol. 1 7 : 9 8 - 1 0 1 . Moats, W. A., 1978a. Egg washing — A review. J. Food Protect. 41:919-925. Moats, W. A., 1978b. A comparison of four plating media with and without added novobiocin for isolation of salmonellae. Appl. Environ. Microbiol. 36:747-751. Moats, W. A., and J. A. Kinner, 1976. Observations on brilliant green agar with an H2 S indicator. Appl. Environ. Microbiol. 31:380-384. United States Department of Agriculture, Agricultural Marketing Service, 1974. Regulations governing the grading of shell eggs and United States standards, grades, and weight classes for shell eggs. (7CFR, part 56).
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rately. Indications are that buildup of bacteria in the washwaters can be controlled to a considerable extent by the addition of sufficient cleaning compound to maintain the pH at 10 or higher.