An Evaluation of Techniques Commonly Used to Quantitatively Determine the Bacterial Population on Chicken Carcasses*

An Evaluation of Techniques Commonly Used to Quantitatively Determine the Bacterial Population on Chicken Carcasses*

GRADE STANDARDS AND CONSUMERS' PREFERENCES grade eggs higher than the A grade eggs. The difference between the mean ratings of the A and C grades was...

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GRADE STANDARDS AND CONSUMERS' PREFERENCES

grade eggs higher than the A grade eggs. The difference between the mean ratings of the A and C grades was not great. 6. Chalaza was considered by consumers as a negative factor in evaluating eggs. Consumers considering the chalaza rated the grade A egg lower than any other comment group. 7. There was close agreement in the mean rating scores of the various groups in St. Louis, Marshall, and Windsor. Exactly the same patterns were exhibited by the consumer groups in all areas. REFERENCES Bender, L. D., 19S8. A comparison of single- and multiple-stimulus techniques for visual preference of eggs, Paper presented annual meeting American Farm Economics Association, Winnipeg, Canada. Erdman, H. E., 1950. Problems in establishing grades for farm products, J. Farm Economics, 32(1): 15-29. Pond, T. H., and L. Kilpatrick, 1956. Grading and inspection of eggs and egg products, U.S.D.A., A.M.S. Agriculture Information Bulletin 159.

An Evaluation of Techniques Commonly Used to Quantitatively Determine the Bacterial Population on Chicken Carcasses* D A N I E L FROMM

Department of Poultry Science, North Carolina Agricultural Experiment Station, Raleigh, North Carolina (Received for publication January IS, 1959)

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IVE techniques are currently in use for the quantitative determinations of bacterial numbers on the surface of chicken carcasses. These methods are the cotton swab, alginate swab, spot plate, tissue sample and carcass rinse techniques. Gunderson et al. (1954) compared the cotton swab and spot plate techniques and * Published with the approval of the Director of Research as Paper No. 971 of the Journal Series.

found them to be quantitatively similarAyres et al. (1956) found that there was a consistent relationship between the bacterial numbers removed by the cotton swab technique and those recovered from the rinse technique. These workers made the assumption that either method was acceptable but preferred the cotton swab method because it was more convenient and did not give underestimates. Mall-

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or the presence of the chalaza cord. The relative evaluations of the A, B, and C grade eggs were greatly influenced by the criteria consumers used. 2. Consumers making any reference to albumen thickness as a quality criteria discriminated greatly between the A and B grades and even greater between the B and C grades. 3. Consumers using only the yolk as a criteria did not discriminate greatly between the A and B grades of eggs but discriminated greatly against the C grade eggs. 4. Consumers who did not make a comment on any of the major criteria rated the B grade eggs almost as high as the A grade and discriminated only slightly against C grade eggs. This group showed the smallest difference between the mean ratings of all grades. 5. Consumers who noted the presence of the chalaza, regardless of any other comments that were made, rated the B

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EXPERIMENTAL

General. The experiment was undertaken in 2 parts. In Part 1 the cotton swab, alginate swab, tissue sample and spot plate techniques were simultaneously evaluated. In Part 2 the alginate and rinse techniques were compared. Analyses were made from commercially processed 2 | pound broiler and 5 pound hen carcasses. The use of both broilers and hens was necessary because preliminary work showed that the bacterial recovery level was affected by the fatty condition of the carcass skin. The skin of hen carcasses are normally more fatty than that of broiler carcasses. The carcasses were chilled to 40°F. or less at the processing plant and transported to the Poultry Science Department laboratories within 20 minutes after removal from the chill tanks. Each carcass was halved on a band saw, packaged in a boat-type tray, covered with moisture proof, transparent and heat scaling (MTS) cellophane and held at a mean temperature of 36.5°F. Carcasses were analyzed at 0, 3, 6, and 9 days of storage in order to ascertain what effect the bacterial density would have on the efficiency of the various techniques as quantitative tools. All samples were plated in duplicate on heart infusion agar (Difco) and incubated at 25°C. for 72 hours.

Part 1. Four half carcasses were removed from refrigeration on each of the 4 analysis periods. Each half carcass was analyzed by the cotton swab, alginate swab, tissue sample and spot plate techniques in the pectoral region under the wing. Areas of one square inch immediately adjacent to each other were analyzed on each half carcass. The cotton swab technique consisted of scrubbing an area of one square inch with absorbent cotton on a wooden applicator. The swab was placed into 5 ml. of physiological saline from which it was originally moistened. The test tube containing the swab and diluent were slapped against the palm of the hand 50 times. Samples of the diluent were plated. In order to determine how effectively the microorganisms were dislodged from the swab, the swab was removed from the diluent, and excess diluent removed from the swab by pressing the swab against the sides of the test tube. The swab was placed in a 50 ml. Servall Omnimixer chamber containing 20 ml. of physiological saline solution and thoroughly comminuted for 30 seconds. The saline in the container was plated. Calcium alginate (Calgitex)1 was used in making the alginate analyses. Handling and sampling the chicken skin were the same as with the cotton swab. However, the diluent used consisted of 9 ml. 0.21% saline solution and 1 ml. of 10% sodium hexametaphosphate in a screw cap test tube. The alginate was rotated by hand until the swab went into suspension. Samples of the suspensoid were plated. Spot plates were prepared by pouring heart infusion agar in petri dishes 28.7 mm. in diameter and covered with a petri dish type covering. Analyses were made by pressing the uncovered petri dish against the chicken skin, removing the 1 Medical Alginates Limited, Wadsworth Road, Perivale, Middlesex, England.

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man el al. (1958) reported that cotton swab and rinse counts paralleled each other at the various levels of contamination but the cotton swab counts were more variable. From these results Mailman et al. concluded that the rinse technique is a more dependable research tool than the swab technique. The purpose of the present study is to compare the relative precision of the five techniques which are commonly used for quantitative determinations of bacterial contamination on eviscerated poultry carcasses.

BACTERIAL POPULATION OF CARCASSES

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agar plug from the dish and grinding the plug for 30 seconds in a 50 ml. Omnimixer chamber containing 40 ml. of physiological saline solution. The resultant solution in the chamber was plated. The tissue sample technique involved the aseptic removal of 1 square inch of skin, placing the sample in a 50 ml. Omnimixer chamber containing 20 ml. of physiological saline solution and grinding the material for 30 seconds. Samples of the comminuted material and diluent were plated. Part 1 was replicated twice (12 samples in each observation) for broilers and hens, and the data combined according to carcass type in the statistical analysis. Part 2. Eight half carcasses were removed from refrigeration on each of the 4 analysis periods. Each half carcass was analyzed by the alginate swab technique and the rinse technique. In order to determine the effectiveness of the rinse technique in removing bacteria from poultry carcasses, the skin was sampled in adFIG. 1. A comparison of the cotton swab, alginate jacent areas (1 square inch) by the algi- swab, spot plate and tissue sample techniques as nate technique before and after the rinsing quantitative methods for sampling broiler 1carcasses for bacteria after various storage periods. ' 2 operation. The rinse technique consisted 1 Each value represents a geometric mean for 12 of placing the half carcass of a broiler or observations. hen in a polyethylene bag containing 500 2 L.S.R. ml. or 1,000 ml. of physiological saline 0.05 = 1.1520 solution, respectively. The bag and con0.01 = 1.1750 tents were violently shaken 75 times (approximately 60 seconds). Samples of the shown in Figures 1 and 2 are the numbers of organisms remaining in the cotton saline solution were plated. Part 2 was replicated and the data com- swabs after sampling. The data show that bined according to carcass type in the the most consistently effective method of removing bacteria from broiler skins statistical analysis. (Figure 1) for quantitative measurements RESULTS was the tissue technique followed by the The data in Figures 1 and 2 show the alginate, cotton swab and spot plate techrelationships of the cotton swab, alginate niques. The numbers of organisms remainswab, spot plate and tissue sample tech- ing in the cotton swab after sampling were niques as effective quantitative methods of sufficient to approach the numbers detersampling broilers and hens with various mined with the spot plate technique on 0, levels of bacterial contamination. Also 3, and 6 days of storage. Data among hens

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D. FROMM

CARCASS

AGE

(DAYS!

FIG. 2. A comparison of the cotton swab, alginate swab, spot plate and tissue sample techniques as quantitative methods for sampling hen carcasses for bacteria after various storage periods.1' 2 1 Each value represents a geometric mean for 12 observations. 2 L.S.R. 0.05=1.2195 0.01 = 1.2860

(Figure 2) show that the alginate and tissue techniques were similar in efficiency in removing bacterial numbers from the skin of hens. The effectiveness of these techniques were followed by the cotton swab and spot plate techniques. The numbers of bacteria remaining in the cotton swabs after sampling was similar in magnitude to those isolated with the spot plate technique as was also indicative in the sampling of broiler carcasses (Figure 1). Statistical tests of the differences be-

The data in Table 1 present the coefficients of variation for various techniques employed in Part 1. The coefficients were consistently higher among hens than among broiler carcasses. The technique showing the least relative variability among broilers was the spot plate method. Among hen carcasses the lowest coefficient was found in the tissue sample technique. The relative variability within the various techniques was similar in the cotton swab, alginate swab and tissue methods among broiler carcasses, and the cotton swab, alginate swab and spot plate techniques among hen carcasses. The effectiveness of the rinse technique as a quantitative method for measuring microbial contamination of chicken carcasses is shown in Figures 3 and 4. There was a detectable reduction in bacterial TABLE 1.—Coefficients of variation of the cotton swab, alginate swab, spot plate and tissue sample techniques used on broiler and hen carcasses (original count basis) Technique Carcass type Broiler Hen

Cotton swab

Alginate swab

Spot plate

Tissue sample

70.61% 95.44%

75.43% 87.89%

59.41% 94.45%

74.30% 72.19%

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tween various techniques among broilers and hens were found to be highly significant. The statistical analyses were performed on the logarithms of the bacterial counts hence differences among means of the logarithms are in reality the logarithms of the ratios of the means on the original count. By the same token, the Least Significant Difference (L.S.D.) computed on the basis of logarithms becomes a Least Significant Ratio of means on the count basis when the antilogarithm of the L.S.D. is taken. The L.S.R.'s reported here refer to the least significant ratios between mean counts for the various determinations.

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BACTERIAL POPULATION OF CARCASSES TABLE 2.—Coefficients of variation of the alginate and rinse techniques used on broiler and hen carcasses {original count basis)

BEFORE

RINSE (SWAB) • —

CARCASS AFTER RINSE

Technique

RIN6E WATER

Carcass type Broiler Hen

CARCASS

Alginate swab

Rinse

76.48% 74.46%

73.42% 42.76%

(SWAB)

(RIN6E)

X°~

CARCASS BEFORE RINSE (SWAB)

• -

CARCASS AFTER RINSE (SWAB)

X~

RINSE WATER (RINSE)

o-

CARCASS

AGE (DAYS)

FIG. 4. A comparison of the alginate swab and rinse techniques as quantitative methods for sampling hen carcasses for bacteria after various storage periods.1' 2 1 Each value represents a geometric mean for 16 observations. 2 L.S.R. 0.05=1.2134 0.01 = 1.3092

ii

CARCASS

AGE (DAYS)

FIG. 3. A comparison of the alginate swab and rinse techniques as quantitative methods for sampling broiler carcasses for bacteria after various storage periods.1' 2 1 Each value represents a geometric mean for 16 observations. 2 L.S.R. 0.05 = 1.1262 0.01 = 1.1363

rinsing. The coefficients of variation presented in Table 2 show that the relative variability in bacterial counts was lower among rinse water solutions than in the alginate samples. When the data were separated into respective storage age groups, there were relationships among broilers between carcass contamination and rinse water bacterial numbers but this was not evident among hen carcasses (Table 3). DISCUSSION

The attributes of a desirable technique for quantitative sampling are primarily

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numbers on the carcasses after the rinsing operation among broilers and hens. However, this technique was not very effective in the removal of bacteria from the skin of broiler or hen carcasses as is evidenced by the numbers of organisms recovered by the alginate swab from the carcasses after

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D. FROMM

The results of this study tend to indicate that in order to remove bacteria from chicken carcass skin for quantitative determinations some sort of scrubbing action is necessary. The technique often used and recommended for the rinse method evidently does not furnish the friction necessary to effectively remove microorganisms from chicken skin. The presence of fat on the skin would inhibit the action of water in dislodging microorganisms from the skin. This phenomenon was evident in the lack of correlation among hens between carcass and rinse water bac-

TABLE 3.—Correlation coefficients of the relationship between bacterial load on chicken carcasses and the numbers of bacteria in the water of the rinse technique by storage groups Age of carcasses (days)

Carcass type

0

3

6

9

Broiler Hen

0.72** -0.06

0.38 0.40

0.65** 0.35

0.84** 0.14

**p=<0.01.

terial numbers for individual storage periods (Table 3). Comparative ease and speed of manipulation in making analyses were desirable attributes of the cotton and alginate swab techniques. Neither of these methods required special equipment, although a mechanical shaker could facilitate the cotton swab analysis. Since the accuracy of the cotton swab was inferior to the alginate swab technique and the differences in the degree of variability were slight, the results of this study show that the alginate swab technique would probably be the most efficient tool in analyzing chicken skin for microbial numbers. SUMMARY

A quantitative comparative analysis was made between the cotton swab, alginate swab, spot plate, tissue sample and rinse techniques in efficiency of removal of bacteria from the carcass skin of broilers and hens. I t was found that differences existed in the precision of these techniques. Considering accuracy, variability, and ease and speed of manipulation, the alginate swab technique was considered the method of choice. ACKNOWLEDGMENT The author wishes to express his appreciation to Messers David A. Allsbrook and Ebern T. Watson of Watson's Seafood and Poultry Company, Raleigh,

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accuracy of analysis with a minimum of variability, and secondly, ease and speed of manipulation. In this study the accuracy of analysis of the various techniques employed was determined on a relative basis. Therefore, that method which consistently produced the highest counts was considered the most accurate. According to the Recommended Methods for the Microbiological Examination of Foods (1958), it is suggested that the cotton swab, spot plate and rinse techniques be used in analyzing dressed eviscerated and cut-up poultry. In the present study the most accurate method among broilers was the tissue sample technique, although among hens no differences were found between the tissue sample and alginate techniques. The presence of a fatty film, which is normally found on hen carcasses, would probably inhibit the dislodging of microorganisms from a fatty cotton swab hence the greater differences between the alginate swabs and cotton swabs in hens than in broilers. The inferiority of the spot plate technique as a quantitative tool is evident (Figure 1 and 2). The inability of the agar to pick up bacteria might be caused by the moist, uneven and often fatty surface of chicken skin.

BACTERIAL POPULATION or CARCASSES

North Carolina, whose cooperation made this study possible.

Processing, Burgess Publishing Company, Minneapolis 15, Minn. Mailman, W. L., L. E. Dawson, B. M. Sultzer and H. S. Wright, 1958. Studies on the microbiological methods for predicting shelf-life of dressed poultry. Food Technol. 12: 122-126. Recommended Methods for the Microbiological Examination of Foods, 1958. American Public Health Association, Inc., New York 19, N. Y. 120-127.

Egg Production Characteristics and Adrenal Function in White Leghorns Confined at Different Floor Space Levels H . S. SlEGELf Virginia Polytechnic Institute, Blacksburg (Received for publication January 19,1959)

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MPROVED housing, equipment and disease control are making possible efficient management of more densely populated laying flocks. As a result, commercial poultrymen are turning to flock intensification as a means of achieving greater labor efficiency and reducing housing costs. Investigations dealing with the effect of space per bird on egg producing ability are numerous and often contradictory. Although Huttar et al. (1933) found egg production of White Leghorns not to be significantly reduced by decreased floor space, others have reported lower egg production in Barred Plymouth Rocks at less than 4.8 square feet per bird (Pearl and Surface, 1909), in White Leghorns at less than 4.0 square feet per bird (Anonymous, 1941) and in New Hampshire pullets at 2.76 square feet per bird (Hoffmann and Tomhave, 1945). Management and housing studies by Bressler and Walton (1957) have shown that large and

t This research was completed at Pennsylvania State University in partial fulfillment of requirements for the Doctor of Philosophy degree.

concentrated populations of White Leghorns could produce efficiently if suitable housing and equipment were used. However, Siegel (1957) found that rearrangement of equipment to provide more feeding, watering and roosting space per bird did not prevent lower egg production and higher mortality in small groups of White Leghorns confined at floor spaces of less than 2.40 square feet per bird. Several investigators have shown that reproductivity is reduced in mammals under conditions of over population (Christian, 1955, 1956; Christian and Davis, 1955, 1956; Louch, 1956). It has been hypothesized but not substantiated that this may result from stress stimulation of hypophyseal corticotropin secretion with a concomitant decrease in gonadotropin secretion. Investigations which indicate increased adrenal cortical activity in young male and adult female chickens when subjected to increased population densities (Siegel, 1958a,b) appear to support this suggestion. This report compares certain egg production characteristics of chickens confined at two different

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REFERENCES Ayres, J. C , H. W. Walker, M. J. Fanelli, A. W. King and F. Thomas, 1956. Use of antibiotics in prolonging storage life of dressed chicken. Food Technol. 10: 563-568. Gunderson, M. F., H. W. McFadden and J. S. Kyle, 1954. The Bacteriology of Commercial Poultry

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