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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Techniques for the isolation of salmonellas from eggs a
T. J. Humphrey & A. Whitehead
a
a
The Food Laboratory , Public Health Laboratory , Church Lane, Exeter, EX2 5A, England Published online: 08 Nov 2007.
To cite this article: T. J. Humphrey & A. Whitehead (1992) Techniques for the isolation of salmonellas from eggs, British Poultry Science, 33:4, 761-768, DOI: 10.1080/00071669208417517 To link to this article: http://dx.doi.org/10.1080/00071669208417517
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1992) 33: 761-768
TECHNIQUES FOR THE ISOLATION OF SALMONELLAS FROM EGGS
T. J. HUMPHREY AND A. WHITEHEAD The Food Laboratory, Public Health Laboratory, Church Lane, Exeter EX2 5A, England
Downloaded by [New York University] at 11:22 21 May 2015
Received for publication 23rd May 1991
Abstract 1. When the contents of 4 or more naturally-contaminated intact eggs were combined, the isolation rate of Salmonella enteritidis was improved by extending incubation at 37°C from 24 to 48 h before sub-culture. 2. The isolation rate of salmonellas from raw homogenised whole egg was significantly increased by the inclusion of Novobiocin and Cefsulodin in the primary culture media. 3. Rappaport Vassiliadis broth was found to be superior to Selenite as a selective enrichment medium.
INTRODUCTION
The contamination of eggs with salmonellas, principally Salmonella enteritidis (Rodrigue et al., 1990), but including 5. sofia in Australia and Israel (Harrington et al., 1991), is of international concern, and has meant that the examination of eggs and products derived from them for salmonellas is of interest to a wide range of people. Previous research (Humphrey et al., 1989a, b) has indicated that approximately 3% of the eggs from an infected hen will be contaminated in the contents with S. enteritidis. There is also a tendency, although not statistically significant, for infected laying hens from the same flock to lay eggs with salmonella-positive contents at/or around the same time. This phenomenon has also been seen with broiler-breeder flocks (Lanning, personal communication). Thus, the efficacy of testing may be dependent on the timing of sampling in addition to the techniques used. The importance of the latter, however, should not be underestimated. Many thousands of intact eggs and samples of homogenised egg have been examined for salmonellas at the Exeter Public Health Laboratory. In so doing, techniques, which maximise the chances of salmonella-detection, have been developed. This paper describes the above work and gives details of techniques and procedures which, if used as indicated, will enable those examining either intact or homogenised eggs to be sure that satisfactory isolation methods are being used. 761
762
T. J. HUMPHREY AND A. WHITEHEAD
MATERIALS AND METHODS
Downloaded by [New York University] at 11:22 21 May 2015
Artificial contamination of egg contents
Dilutions of overnight Lemco broth (Oxoid) cultures of strains of S. enteritidis phage type (PT) 4, previously isolated from egg contents, were inoculated, using an automatic pipette (Finn pipette), into laboratory prepared samples of homogenised whole egg, yolk or albumen with or without the addition of up to three volumes of buffered peptone water (BPW). The concentration of the inoculum was approximately 10 cells per 100 ml of egg/BPW mix. The cultures were incubated for up to 48 h at 37°C and then sub-cultured on to XLD agar (Oxoid) which was incubated for 24 h at 37°C. While the above experiments can provide potentially useful information, there is a possibility that the experimental procedures did not fully reflect the growth characteristics of salmonellas occurring internally in egg contents. The albumen would appear to be the principal site of contamination in eggs from naturally infected hens (Humphrey et al., 1991) and, to assess the impact of exposure to this potentially hostile environment, dilutions of broth cultures were held for 24 h in egg albumen (pH 9-2) at either 20 or +4°C before inoculation into either egg yolk, albumen or homogenised whole egg. Culture, thereafter, was as described above.
Examination of intact eggs
Eggs were either purchased from flocks identified by the Ministry of Agriculture, Fisheries and Foods (MAFF) as being infected with S. enteritidis or examined as part of an investigation of egg-associated cases of human infection. On arrival at the laboratory, each egg was inspected individually under a strong light. For the purposes of the investigations reported in this paper, only eggs with clean, intact shells were examined for the presence of salmonellas in the contents. After initial screening, eggs were transferred to clean egg trays and stored in a locked room. The relative humidity was approximately 40% at a storage temperature of 20°C. Eggs shells were disinfected by immersion in Industrial Methylated Spirits for 5 min (Humphrey et al., 19896) and the contents removed, aseptically, to sterile containers. Egg contents were homogenised in a stomacher and either added to up to three volumes of BPW or were incubated with no further additions. Incubation was at 37°C with the samples being cultured on to XLD agar at 24 and 48 h. In addition, at 24 and 48 h, 0-1 ml of egg culture was inoculated into 10 ml of either Selenite or Rappaport Vassiliadis (RV) broth (Oxoid). Selenite was incubated at 37°C for 18 to 24 h and RV at 41°C for 48 h before culture on to selective agar.
Examination of homogenised whole egg
Volumes of 100 ml were removed from samples of commercially-produced
SALMONELLAS AND EGGS
763
homogenised raw whole egg and added to an equal volume of BPW. With some samples, the antibiotics Novobiocin (Sigma) and Cefsulodin (Ciba), at final concentration of 5 /xg/ml and 10 /Ug/ml, respectively, were also added. The egg samples were incubated for up to 48 h at 37°C and were sub-cultured at 24 and 48 h into either Rappaport Vassiliadis or Selenite broth. These media were incubated and sub-cultured as described above. On the majority of samples, total viable counts were performed on blood agar with plates being incubated for three days at 30°C before counting.
Downloaded by [New York University] at 11:22 21 May 2015
Identification of salmonellas
Salmonella-like colonies were confirmed by biochemical and serological testing and representative isolates were sent to the PHLS Division of Enteric Pathogens (DEP) for confirmation and phage typing.
RESULTS
Growth of Salmonella enteritidis in artificially contaminated egg contents
The addition of BPW has no significant effect on the growth of S. enteritidis in either artificially-contaminated homogenised whole egg or egg yolk. In contrast, the bacterium did not grow, at either 37°C or room temperature, in the albumen of fresh eggs and grew only relatively slowly when an equal volume of BPW was added. The addition of two or more volumes of BPW, however, permitted the rapid growth of the target organisms. Preexposure to albumen had no significant effects on growth rates in either egg yolk or homogenised whole egg. In a minority of experiments, pre-exposed cells of 5. enteritidis were capable of slow growth in albumen, but as above, however, growth was greatly enhanced by the addition of BPW. Isolation of salmonellas from the contents of intact shell eggs
In this study, S. enteritidis phage type (PT) 4 was the only salmonella isolated from the contents of intact eggs. In all cases, it was present in pure culture. Influence of length of incubation on the isolation of salmonellas from egg contents
When the contents of single eggs were homogenised and incubated individually, extending primary incubation from 24 to 48 h had no effect on the isolation rate of salmonellas (Table 1). In contrast, extended incubation increased the isolation rate when the contents of 4 or more eggs were combined (Table 1). The addition of an equal volume of BPW to homogenised egg contents had no effect on the isolation rate of S. enteritidis. It did, however, largely prevent the egg contents becoming semi-solid during incubation at
764
T. J. HUMPHREY AND A. WHITEHEAD
37°C. This, which was not the result of the growth of micro-organisms because the majority of samples were sterile, made sub-culture and plating difficult to perform, and also posed problems in obtaining a representative sample. TABLE 1
The isolation of salmonellas from the contents of naturally contaminated eggs
Number of eggs examined
Downloaded by [New York University] at 11:22 21 May 2015
Eggs examined in batches of four Eggs examined individually
Number salmonella-positive1 after incubation for 24 h 48 h
248
0
2
457
5
5
2
X = 2-01 />
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Techniques for the isolation of salmonellas from eggs a
T. J. Humphrey & A. Whitehead
a
a
The Food Laboratory , Public Health Laboratory , Church Lane, Exeter, EX2 5A, England Published online: 08 Nov 2007.
To cite this article: T. J. Humphrey & A. Whitehead (1992) Techniques for the isolation of salmonellas from eggs, British Poultry Science, 33:4, 761-768, DOI: 10.1080/00071669208417517 To link to this article: http://dx.doi.org/10.1080/00071669208417517
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1992) 33: 761-768
TECHNIQUES FOR THE ISOLATION OF SALMONELLAS FROM EGGS
T. J. HUMPHREY AND A. WHITEHEAD The Food Laboratory, Public Health Laboratory, Church Lane, Exeter EX2 5A, England
Downloaded by [New York University] at 11:22 21 May 2015
Received for publication 23rd May 1991
Abstract 1. When the contents of 4 or more naturally-contaminated intact eggs were combined, the isolation rate of Salmonella enteritidis was improved by extending incubation at 37°C from 24 to 48 h before sub-culture. 2. The isolation rate of salmonellas from raw homogenised whole egg was significantly increased by the inclusion of Novobiocin and Cefsulodin in the primary culture media. 3. Rappaport Vassiliadis broth was found to be superior to Selenite as a selective enrichment medium.
INTRODUCTION
The contamination of eggs with salmonellas, principally Salmonella enteritidis (Rodrigue et al., 1990), but including 5. sofia in Australia and Israel (Harrington et al., 1991), is of international concern, and has meant that the examination of eggs and products derived from them for salmonellas is of interest to a wide range of people. Previous research (Humphrey et al., 1989a, b) has indicated that approximately 3% of the eggs from an infected hen will be contaminated in the contents with S. enteritidis. There is also a tendency, although not statistically significant, for infected laying hens from the same flock to lay eggs with salmonella-positive contents at/or around the same time. This phenomenon has also been seen with broiler-breeder flocks (Lanning, personal communication). Thus, the efficacy of testing may be dependent on the timing of sampling in addition to the techniques used. The importance of the latter, however, should not be underestimated. Many thousands of intact eggs and samples of homogenised egg have been examined for salmonellas at the Exeter Public Health Laboratory. In so doing, techniques, which maximise the chances of salmonella-detection, have been developed. This paper describes the above work and gives details of techniques and procedures which, if used as indicated, will enable those examining either intact or homogenised eggs to be sure that satisfactory isolation methods are being used. 761
762
T. J. HUMPHREY AND A. WHITEHEAD
MATERIALS AND METHODS
Downloaded by [New York University] at 11:22 21 May 2015
Artificial contamination of egg contents
Dilutions of overnight Lemco broth (Oxoid) cultures of strains of S. enteritidis phage type (PT) 4, previously isolated from egg contents, were inoculated, using an automatic pipette (Finn pipette), into laboratory prepared samples of homogenised whole egg, yolk or albumen with or without the addition of up to three volumes of buffered peptone water (BPW). The concentration of the inoculum was approximately 10 cells per 100 ml of egg/BPW mix. The cultures were incubated for up to 48 h at 37°C and then sub-cultured on to XLD agar (Oxoid) which was incubated for 24 h at 37°C. While the above experiments can provide potentially useful information, there is a possibility that the experimental procedures did not fully reflect the growth characteristics of salmonellas occurring internally in egg contents. The albumen would appear to be the principal site of contamination in eggs from naturally infected hens (Humphrey et al., 1991) and, to assess the impact of exposure to this potentially hostile environment, dilutions of broth cultures were held for 24 h in egg albumen (pH 9-2) at either 20 or +4°C before inoculation into either egg yolk, albumen or homogenised whole egg. Culture, thereafter, was as described above.
Examination of intact eggs
Eggs were either purchased from flocks identified by the Ministry of Agriculture, Fisheries and Foods (MAFF) as being infected with S. enteritidis or examined as part of an investigation of egg-associated cases of human infection. On arrival at the laboratory, each egg was inspected individually under a strong light. For the purposes of the investigations reported in this paper, only eggs with clean, intact shells were examined for the presence of salmonellas in the contents. After initial screening, eggs were transferred to clean egg trays and stored in a locked room. The relative humidity was approximately 40% at a storage temperature of 20°C. Eggs shells were disinfected by immersion in Industrial Methylated Spirits for 5 min (Humphrey et al., 19896) and the contents removed, aseptically, to sterile containers. Egg contents were homogenised in a stomacher and either added to up to three volumes of BPW or were incubated with no further additions. Incubation was at 37°C with the samples being cultured on to XLD agar at 24 and 48 h. In addition, at 24 and 48 h, 0-1 ml of egg culture was inoculated into 10 ml of either Selenite or Rappaport Vassiliadis (RV) broth (Oxoid). Selenite was incubated at 37°C for 18 to 24 h and RV at 41°C for 48 h before culture on to selective agar.
Examination of homogenised whole egg
Volumes of 100 ml were removed from samples of commercially-produced
SALMONELLAS AND EGGS
763
homogenised raw whole egg and added to an equal volume of BPW. With some samples, the antibiotics Novobiocin (Sigma) and Cefsulodin (Ciba), at final concentration of 5 /xg/ml and 10 /Ug/ml, respectively, were also added. The egg samples were incubated for up to 48 h at 37°C and were sub-cultured at 24 and 48 h into either Rappaport Vassiliadis or Selenite broth. These media were incubated and sub-cultured as described above. On the majority of samples, total viable counts were performed on blood agar with plates being incubated for three days at 30°C before counting.
Downloaded by [New York University] at 11:22 21 May 2015
Identification of salmonellas
Salmonella-like colonies were confirmed by biochemical and serological testing and representative isolates were sent to the PHLS Division of Enteric Pathogens (DEP) for confirmation and phage typing.
RESULTS
Growth of Salmonella enteritidis in artificially contaminated egg contents
The addition of BPW has no significant effect on the growth of S. enteritidis in either artificially-contaminated homogenised whole egg or egg yolk. In contrast, the bacterium did not grow, at either 37°C or room temperature, in the albumen of fresh eggs and grew only relatively slowly when an equal volume of BPW was added. The addition of two or more volumes of BPW, however, permitted the rapid growth of the target organisms. Preexposure to albumen had no significant effects on growth rates in either egg yolk or homogenised whole egg. In a minority of experiments, pre-exposed cells of 5. enteritidis were capable of slow growth in albumen, but as above, however, growth was greatly enhanced by the addition of BPW. Isolation of salmonellas from the contents of intact shell eggs
In this study, S. enteritidis phage type (PT) 4 was the only salmonella isolated from the contents of intact eggs. In all cases, it was present in pure culture. Influence of length of incubation on the isolation of salmonellas from egg contents
When the contents of single eggs were homogenised and incubated individually, extending primary incubation from 24 to 48 h had no effect on the isolation rate of salmonellas (Table 1). In contrast, extended incubation increased the isolation rate when the contents of 4 or more eggs were combined (Table 1). The addition of an equal volume of BPW to homogenised egg contents had no effect on the isolation rate of S. enteritidis. It did, however, largely prevent the egg contents becoming semi-solid during incubation at
764
T. J. HUMPHREY AND A. WHITEHEAD
37°C. This, which was not the result of the growth of micro-organisms because the majority of samples were sterile, made sub-culture and plating difficult to perform, and also posed problems in obtaining a representative sample. TABLE 1
The isolation of salmonellas from the contents of naturally contaminated eggs
Number of eggs examined
Downloaded by [New York University] at 11:22 21 May 2015
Eggs examined in batches of four Eggs examined individually
Number salmonella-positive1 after incubation for 24 h 48 h
248
0
2
457
5
5
2
X = 2-01 />
