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Growth of Psychrotolerant Pseudomonads and Achromobacteria on Various Chicken Tissues
SKIN BACTERIA
ogy, pp. 88-91. The Avi Publishing Company, Inc., Westport, Connecticut. Seekins, J. G., E. Guenther and W. G. Walter, 1958. A comparison of six methods for determining numbers of bacteria on poultry meat under three types of handling. Proc. Montana Acad. Sci. 18: 13-18. Sharf, J. M. (ed.), 1966. Recommended Methods for the Microbiological Examination of Foods, pp. 119-125. American Public Health Association, Inc., New York.
Growth of Psychrotolerant Pseudomonads and Achromobacteria on Various Chicken Tissues D . S. C L A R K
Division of Biology, National Research Council of Canada, Ottawa, Ontario, Canada (Received for publication April 21, 1970)
INTRODUCTION
E
ARLY work with laboratory-processed chickens indicated that bacteria-induced changes, leading to off-odour of whole birds during refrigerated storage, occur first on the surface of the skin (Lochhead and Landerkin, 1935). In modern commercial processing other tissues besides skin (gut-cavity lining, muscle in the neck area and connective tissue between the skin and muscle) are freely exposed to contamination; the surface layer of skin is removed by scalding at 59°C, and bacteria present are uniformly spread over all surfaces during washing and cooling processes carried out in continuously-operated tanks. There are therefore reasons to question whether or not skin is still the initial site of bacteriainduced off-odour in refrigerated whole poultry. This matter is particularly important in the case of cut-up poultry meat where larger areas of muscle and connective tissue surfaces are exposed to contamination. This paper compares the growth rates of Issued as N.R.C.C. No. 11611.
three groups of psychrotolerant bacteria (pigmented pseudomonads, non-pigmented pseudomonads, and achromobacteria) on the surfaces of five different tissues (skin, breast muscle, breast muscle lining, leg muscle, and gut-cavity lining). It also describes studies of the effect of the pH of the tissues on the growth rates of the test organisms. METHODS Test samples of the various tissues were obtained from 9-week-old broiler-type chickens (Ottawa Meat Control Strain) raised and processed in the laboratory. After slaughter (by cutting the jugular vein and carotid arteries) the chickens were scalded (1 min. at 59°C), plucked by hand and rubbed clean with a sterile damp cloth. All tissue samples (except gut-cavity lining) were taken prior to evisceration. Excised skin was washed and pieces 9 cm square were stretched onto circular stainless steel disks for inoculation as described previously (Clark, 1968). Breast and thigh muscles were sliced along the grain into pieces about £ inch thick and the pieces
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
mation of surface areas of cut-up chicken and use in microbiological analysis. Food Technol. 13: 241-243. Kotula, A. W., 1966. Variability in microbiological samplings of chickens by the swab method. Poultry Sci. 45: 233-236. Mailman, W. L., L. E. Dawson, B. M. Sultzer and H. S. Wright, 1958. Studies on microbiological methods for predicting shelf-life of dressed poultry. Food Technol. 12: 122-126. Mountney, G. J., 1966. Poultry Products Technol-
1315
1316
D. S. CLARK
phosphate or phosphate-citrate buffer) was comminuted in an Omni mixer (5 min.), filtered through cheese cloth, and the filtrate dispersed in 8 ml. volumes in culture tubes. The tubes were irradiated (500,000 rads) to kill vegetative cells of psychrotolerant PIGMENTED
PSEUDOMONADS
•I—^H—t—\—i—I—i—I—i—I—i—I—i—h | NONPIGMENTED PSEUDOMONADS |
o
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I
'
I '
I '
I '
I '
Li_
°
I
'
1 ' I
.
|ACHROMOBACTERIA |
INCUBATION
TIME,
DAYS
FIG. 1. Growth and production of off-odour by psychrotolerant bacteria on various chicken tissues incubated at 0°C.
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
arranged in Petri dishes to give a total flat surface area of about 9-10 sq. cm. in each dish. Portions of breast-muscle lining to be tested were obtained from the first slice taken from the outside of the breast muscle. To obtain samples of gutcavity lining, the eviscerated carcass was split in half front to back and pieces of the gut-cavity walls placed in Petri dishes with the cavitylining facing upwards. The upper surface of all test material so deployed was inoculated uniformly (104 cells/sq.cm.; Clark, 1963) with cells from 24-hour-old cultures grown at 20°C. on Standard Methods agar (SM agar, Difco). The inoculum was composed of a mixture of 10 strains of pigmented Pseudomonas, a mixture of 10 strains of non-pigmented Pseudomonas or a mixture of 10 strains of Achromobacter. The individual strains were grown separately and mixed uniformly prior to inoculation (Clark, 1968). All organisms were previously isolated from the surface of processed poultry (Clark and Lentz, 1969). The inoculated tissues were incubated at 0°C. in a water-saturated atmosphere and analysed for total count by the spray gun (Clark 1965a,b) and surface plating (Clark, 1967) techniques. In most tests the tissues were analysed for total count after 7 days of incubation when the organisms were presumed to be in the log phase of growth (Clark, 1968). For these tests 20 samples of each tissue were used for each inocula and 3 count determinations were made on each piece. In a few experiments the test material was examined at regular intervals (up to a total incubation time of 15 days) for both total count and development of offodour (detectable by sniffing). The effect of pH on growth rate was determined in extracts of skin or breast muscle buffered at pH values ranging from 4.57.0. A suspension of finely chopped skin or muscle (% wt./vol.) in buffer (0.1 M
1317
PSEUDOMONADS AND ACHROMOBACTEEIA ON TISSUES TABLE 1.—Bacterial counts on various tissues after 7 days of incubation at 0°C. (initial inoculation, 10* cells/sq.cm. Each figure is the average of 60 counts)
Tissue
Pigmented pseudomonads « . / Av. count / Standard sq. cm.XlO- 4 deviation 720 466 150 29 25
Achromobacteria
Av. count/ sq. cm.XlO - 4
Standard deviation
Av. count/ sq. cm.XlO- 4
Standard deviation
1448 1160 230 40 24
560 581 120 40 24
328 187 170 2 2
412 185 234 11 20
450 324 200 14 14
organisms, inoculated with 24-hr.-old cells of the mixed inocula (104 cells/ml.), and incubated at 0°C. Tests were made in triplicate. The resultant cultures were surfaceplated on SM agar after 7 days. The natural pH of the various tissues for birds kept 1 day in crushed ice was determined by measurements made on water extracts prepared by blending about % g. of tissue in 5 ml. of glass-distilled water in an Omni mixer. Gut-cavity and breast muscle linings used for these determinations were peeled away from the underlying tissues with the aid of scapel and tweezers; all tissues were finely chopped with scissors before blending. RESULTS AND DISCUSSION
The rate of growth of all three groups of bacteria was fastest on skin, based on comparisons of population densities and on the elapsed time from inoculation to onset of detectable off-odour (Figure 1). Extensive colony count after 7 days of incubation and statistical analysis of the results (t-test), showed that population differences between skin and the other tissues (Table 1) were significant at the 5% level. These differences among the tissues, however, were mainly due to differences in the length of the lag periods rather than to differences in generation time during the log phase of growth (Figure 1). This suggests that the faster relative accumulation of cells on skin is a function of the time required for the
inoculum to adjust to the substrate, rather than of the composition of particular tissues or of their ability to support rapid cell multiplication once this is initiated. Off-odour was detected in all tissues when the total bacterial count was about 108/sq.cm. Because of differences in the length of the lag period (Figure 1), offodour in skin occurred 48 hr. sooner than in breast muscle, 24 hr. sooner than in leg muscle and 6-10 hr. sooner than in the gutcavity lining. Because the lag period for these bacteria has been observed to increase with decrease in size of inoculum (Ayres et al., 1950) such time differences would probably be even greater under commercial conditions where the initial contamination of psychrotolerant bacteria is considerably less than 104/sq.cm. (Clark and Lentz, 1969). Any increase in storage temperature would probably shorten these differences (Ayres et al., 1950). Results of pH determinations indicated that the differences in the lag periods were not related to the pH of the tissues. Table 2 shows that the growth of all three inocula in extracts of skin and muscle was not markedly affected by pH values in the range 5.9-7.0, which covered the average values determined for the various tissues (Table 3). Below pH 5.9 growth was inhibited, particularly in the case of the pseudomonads. Possibly the inhibitory effect of low pH is more pronounced in surface than in submerged culture, in which case the ob-
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
Skin Cavity lining Leg muscle Breast muscle Breast muscle lining
Non-pigmented pseudomonads
1318
D. S. CLARK
TABLE 2.—Effect of pH on growth of psychrotolerant bacteria in extracts of chicken breast muscle and skin Logio of total count after 7 days of incubation pH
Non-pigmented pseudomonads
breast muscle
skin
breast muscle
skin
5.7 6.2 6.8 6.9 6.8
5.9 7.6 7.8 7.9 7.8
3.0 6.8 7.2 7.1 7.2
5.4 7.4 7.8 7.8 7.9
pH Tissue
•
Achromobacteria
served pH might explain the longer lag period for the more acid tissues (breast muscle and breast muscle lining). However, a marked difference in length of lag occurred between leg muscle and skin (Figure 1) but the pH of the two tissues were nearly the same (Table 3). It appears that one or more factors other than pH are involved. The results showed that skin is the preferred tissue to use in the study of factors affecting the rate of microbial spoilage of refrigerated whole poultry or cut-up poultry containing skin surfaces. Growth was nearly as rapid on the gut-cavity walls but this surface is less easy to obtain and handle. Skin is easy to prepare for experimental purposes and the growth rate of psychrotolerant organisms on it is independent on the location of the skin on the bird or the age of the bird (Clark, 1968). SUMMARY
Psychrotolerant pseudomonads and achromobacteria grew faster on skin than on other tissues taken from chicken carcasses. Observations were made on rates of population growth and detectable offodour. Differences in overall growth rates on the various tissues were attributed to differences in the duration of the lag period; cell generation times during the log period were
tissues
Skin B reast muscle Leg muscle Gut-cavity lining Breast-muscle lining
Average*
Range
6.5 5.9 6.4 6.5 6.0
6.4-6.6 5.7-6.2 6.3-6.5 6.2-6.6 5.7-6.1
* Values shown are the average of 24 determinations on material from 12 birds.
nearly the same on all tissues. Differences in the lag period were not related to differences in the pH among the tissues. ACKNOWLEDGMENT The author thanks Mr. N. U. Cholette for technical assistance. REFERENCES Ayres, J. C , W. S. Ogilvy and G. F. Stewart, 1950. Post mortem changes in stored meats. I. Microorganisms associated with development of slime on eviscerated cut-up poultry. Food Technol. 4 : 199-205. Clark, D. S., 1963. Uniform inoculation of nutrient surfaces. Biotech. Bioeng. 5: 123-129. Clark, D. S. 1965a. Method of estimating the bacterial population on surfaces. Can. J. Microbiol. 11: 407-413. Clark, D. S., 196Sb. Improvement of spray-gun method of estimating bacterial populations on surfaces. Can. J. Microbiol. 11: 1021-1022. Clark, D. S., 1967. Comparison of pour and surface plate methods for determination of bacterial counts. Can. J. Microbiol. 13: 1409-1412. Clark, D. S., 1968. Growth of psychrotolerant pseudomonads and achromobacter on chicken skin. Poultry Sci. 47: 1575-1578. Clark, D. S., and C. P. Lentz, 1969. Microbiological studies in poultry processing plants in Canada. Can. Instit. Food Technol. J. 2: 33-36. Lochhead, A. G., and G. B. Landerkin, 1935. Bacteriological studies of dressed poultry. I. Preliminary investigation of bacterial action at chill temperatures. Sci. Agric. 15: 765.
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
5.0 5.7 5.9 6.5 7.0
Pigmented pseudomonads
T A B L E 3.—Natural pH of fresh chicken
ogy, pp. 88-91. The Avi Publishing Company, Inc., Westport, Connecticut. Seekins, J. G., E. Guenther and W. G. Walter, 1958. A comparison of six methods for determining numbers of bacteria on poultry meat under three types of handling. Proc. Montana Acad. Sci. 18: 13-18. Sharf, J. M. (ed.), 1966. Recommended Methods for the Microbiological Examination of Foods, pp. 119-125. American Public Health Association, Inc., New York.
Growth of Psychrotolerant Pseudomonads and Achromobacteria on Various Chicken Tissues D . S. C L A R K
Division of Biology, National Research Council of Canada, Ottawa, Ontario, Canada (Received for publication April 21, 1970)
INTRODUCTION
E
ARLY work with laboratory-processed chickens indicated that bacteria-induced changes, leading to off-odour of whole birds during refrigerated storage, occur first on the surface of the skin (Lochhead and Landerkin, 1935). In modern commercial processing other tissues besides skin (gut-cavity lining, muscle in the neck area and connective tissue between the skin and muscle) are freely exposed to contamination; the surface layer of skin is removed by scalding at 59°C, and bacteria present are uniformly spread over all surfaces during washing and cooling processes carried out in continuously-operated tanks. There are therefore reasons to question whether or not skin is still the initial site of bacteriainduced off-odour in refrigerated whole poultry. This matter is particularly important in the case of cut-up poultry meat where larger areas of muscle and connective tissue surfaces are exposed to contamination. This paper compares the growth rates of Issued as N.R.C.C. No. 11611.
three groups of psychrotolerant bacteria (pigmented pseudomonads, non-pigmented pseudomonads, and achromobacteria) on the surfaces of five different tissues (skin, breast muscle, breast muscle lining, leg muscle, and gut-cavity lining). It also describes studies of the effect of the pH of the tissues on the growth rates of the test organisms. METHODS Test samples of the various tissues were obtained from 9-week-old broiler-type chickens (Ottawa Meat Control Strain) raised and processed in the laboratory. After slaughter (by cutting the jugular vein and carotid arteries) the chickens were scalded (1 min. at 59°C), plucked by hand and rubbed clean with a sterile damp cloth. All tissue samples (except gut-cavity lining) were taken prior to evisceration. Excised skin was washed and pieces 9 cm square were stretched onto circular stainless steel disks for inoculation as described previously (Clark, 1968). Breast and thigh muscles were sliced along the grain into pieces about £ inch thick and the pieces
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
mation of surface areas of cut-up chicken and use in microbiological analysis. Food Technol. 13: 241-243. Kotula, A. W., 1966. Variability in microbiological samplings of chickens by the swab method. Poultry Sci. 45: 233-236. Mailman, W. L., L. E. Dawson, B. M. Sultzer and H. S. Wright, 1958. Studies on microbiological methods for predicting shelf-life of dressed poultry. Food Technol. 12: 122-126. Mountney, G. J., 1966. Poultry Products Technol-
1315
1316
D. S. CLARK
phosphate or phosphate-citrate buffer) was comminuted in an Omni mixer (5 min.), filtered through cheese cloth, and the filtrate dispersed in 8 ml. volumes in culture tubes. The tubes were irradiated (500,000 rads) to kill vegetative cells of psychrotolerant PIGMENTED
PSEUDOMONADS
•I—^H—t—\—i—I—i—I—i—I—i—I—i—h | NONPIGMENTED PSEUDOMONADS |
o
<
I
'
I '
I '
I '
I '
Li_
°
I
'
1 ' I
.
|ACHROMOBACTERIA |
INCUBATION
TIME,
DAYS
FIG. 1. Growth and production of off-odour by psychrotolerant bacteria on various chicken tissues incubated at 0°C.
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
arranged in Petri dishes to give a total flat surface area of about 9-10 sq. cm. in each dish. Portions of breast-muscle lining to be tested were obtained from the first slice taken from the outside of the breast muscle. To obtain samples of gutcavity lining, the eviscerated carcass was split in half front to back and pieces of the gut-cavity walls placed in Petri dishes with the cavitylining facing upwards. The upper surface of all test material so deployed was inoculated uniformly (104 cells/sq.cm.; Clark, 1963) with cells from 24-hour-old cultures grown at 20°C. on Standard Methods agar (SM agar, Difco). The inoculum was composed of a mixture of 10 strains of pigmented Pseudomonas, a mixture of 10 strains of non-pigmented Pseudomonas or a mixture of 10 strains of Achromobacter. The individual strains were grown separately and mixed uniformly prior to inoculation (Clark, 1968). All organisms were previously isolated from the surface of processed poultry (Clark and Lentz, 1969). The inoculated tissues were incubated at 0°C. in a water-saturated atmosphere and analysed for total count by the spray gun (Clark 1965a,b) and surface plating (Clark, 1967) techniques. In most tests the tissues were analysed for total count after 7 days of incubation when the organisms were presumed to be in the log phase of growth (Clark, 1968). For these tests 20 samples of each tissue were used for each inocula and 3 count determinations were made on each piece. In a few experiments the test material was examined at regular intervals (up to a total incubation time of 15 days) for both total count and development of offodour (detectable by sniffing). The effect of pH on growth rate was determined in extracts of skin or breast muscle buffered at pH values ranging from 4.57.0. A suspension of finely chopped skin or muscle (% wt./vol.) in buffer (0.1 M
1317
PSEUDOMONADS AND ACHROMOBACTEEIA ON TISSUES TABLE 1.—Bacterial counts on various tissues after 7 days of incubation at 0°C. (initial inoculation, 10* cells/sq.cm. Each figure is the average of 60 counts)
Tissue
Pigmented pseudomonads « . / Av. count / Standard sq. cm.XlO- 4 deviation 720 466 150 29 25
Achromobacteria
Av. count/ sq. cm.XlO - 4
Standard deviation
Av. count/ sq. cm.XlO- 4
Standard deviation
1448 1160 230 40 24
560 581 120 40 24
328 187 170 2 2
412 185 234 11 20
450 324 200 14 14
organisms, inoculated with 24-hr.-old cells of the mixed inocula (104 cells/ml.), and incubated at 0°C. Tests were made in triplicate. The resultant cultures were surfaceplated on SM agar after 7 days. The natural pH of the various tissues for birds kept 1 day in crushed ice was determined by measurements made on water extracts prepared by blending about % g. of tissue in 5 ml. of glass-distilled water in an Omni mixer. Gut-cavity and breast muscle linings used for these determinations were peeled away from the underlying tissues with the aid of scapel and tweezers; all tissues were finely chopped with scissors before blending. RESULTS AND DISCUSSION
The rate of growth of all three groups of bacteria was fastest on skin, based on comparisons of population densities and on the elapsed time from inoculation to onset of detectable off-odour (Figure 1). Extensive colony count after 7 days of incubation and statistical analysis of the results (t-test), showed that population differences between skin and the other tissues (Table 1) were significant at the 5% level. These differences among the tissues, however, were mainly due to differences in the length of the lag periods rather than to differences in generation time during the log phase of growth (Figure 1). This suggests that the faster relative accumulation of cells on skin is a function of the time required for the
inoculum to adjust to the substrate, rather than of the composition of particular tissues or of their ability to support rapid cell multiplication once this is initiated. Off-odour was detected in all tissues when the total bacterial count was about 108/sq.cm. Because of differences in the length of the lag period (Figure 1), offodour in skin occurred 48 hr. sooner than in breast muscle, 24 hr. sooner than in leg muscle and 6-10 hr. sooner than in the gutcavity lining. Because the lag period for these bacteria has been observed to increase with decrease in size of inoculum (Ayres et al., 1950) such time differences would probably be even greater under commercial conditions where the initial contamination of psychrotolerant bacteria is considerably less than 104/sq.cm. (Clark and Lentz, 1969). Any increase in storage temperature would probably shorten these differences (Ayres et al., 1950). Results of pH determinations indicated that the differences in the lag periods were not related to the pH of the tissues. Table 2 shows that the growth of all three inocula in extracts of skin and muscle was not markedly affected by pH values in the range 5.9-7.0, which covered the average values determined for the various tissues (Table 3). Below pH 5.9 growth was inhibited, particularly in the case of the pseudomonads. Possibly the inhibitory effect of low pH is more pronounced in surface than in submerged culture, in which case the ob-
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
Skin Cavity lining Leg muscle Breast muscle Breast muscle lining
Non-pigmented pseudomonads
1318
D. S. CLARK
TABLE 2.—Effect of pH on growth of psychrotolerant bacteria in extracts of chicken breast muscle and skin Logio of total count after 7 days of incubation pH
Non-pigmented pseudomonads
breast muscle
skin
breast muscle
skin
5.7 6.2 6.8 6.9 6.8
5.9 7.6 7.8 7.9 7.8
3.0 6.8 7.2 7.1 7.2
5.4 7.4 7.8 7.8 7.9
pH Tissue
•
Achromobacteria
served pH might explain the longer lag period for the more acid tissues (breast muscle and breast muscle lining). However, a marked difference in length of lag occurred between leg muscle and skin (Figure 1) but the pH of the two tissues were nearly the same (Table 3). It appears that one or more factors other than pH are involved. The results showed that skin is the preferred tissue to use in the study of factors affecting the rate of microbial spoilage of refrigerated whole poultry or cut-up poultry containing skin surfaces. Growth was nearly as rapid on the gut-cavity walls but this surface is less easy to obtain and handle. Skin is easy to prepare for experimental purposes and the growth rate of psychrotolerant organisms on it is independent on the location of the skin on the bird or the age of the bird (Clark, 1968). SUMMARY
Psychrotolerant pseudomonads and achromobacteria grew faster on skin than on other tissues taken from chicken carcasses. Observations were made on rates of population growth and detectable offodour. Differences in overall growth rates on the various tissues were attributed to differences in the duration of the lag period; cell generation times during the log period were
tissues
Skin B reast muscle Leg muscle Gut-cavity lining Breast-muscle lining
Average*
Range
6.5 5.9 6.4 6.5 6.0
6.4-6.6 5.7-6.2 6.3-6.5 6.2-6.6 5.7-6.1
* Values shown are the average of 24 determinations on material from 12 birds.
nearly the same on all tissues. Differences in the lag period were not related to differences in the pH among the tissues. ACKNOWLEDGMENT The author thanks Mr. N. U. Cholette for technical assistance. REFERENCES Ayres, J. C , W. S. Ogilvy and G. F. Stewart, 1950. Post mortem changes in stored meats. I. Microorganisms associated with development of slime on eviscerated cut-up poultry. Food Technol. 4 : 199-205. Clark, D. S., 1963. Uniform inoculation of nutrient surfaces. Biotech. Bioeng. 5: 123-129. Clark, D. S. 1965a. Method of estimating the bacterial population on surfaces. Can. J. Microbiol. 11: 407-413. Clark, D. S., 196Sb. Improvement of spray-gun method of estimating bacterial populations on surfaces. Can. J. Microbiol. 11: 1021-1022. Clark, D. S., 1967. Comparison of pour and surface plate methods for determination of bacterial counts. Can. J. Microbiol. 13: 1409-1412. Clark, D. S., 1968. Growth of psychrotolerant pseudomonads and achromobacter on chicken skin. Poultry Sci. 47: 1575-1578. Clark, D. S., and C. P. Lentz, 1969. Microbiological studies in poultry processing plants in Canada. Can. Instit. Food Technol. J. 2: 33-36. Lochhead, A. G., and G. B. Landerkin, 1935. Bacteriological studies of dressed poultry. I. Preliminary investigation of bacterial action at chill temperatures. Sci. Agric. 15: 765.
Downloaded from http://ps.oxfordjournals.org/ at University of California Santa Barbara/Davidson Library on June 30, 2015
5.0 5.7 5.9 6.5 7.0
Pigmented pseudomonads
T A B L E 3.—Natural pH of fresh chicken