"Land and Food Systems, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Sahasrabudhe, Jyoti Madhu"@en . "2010-03-26T03:34:16Z"@en . "1981"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Evaluation of several methods of fixing chicken skin for scanning electron microscopy (SEM) indicated standard chemical fixation using glutaraldehyde and osmium tetroxide followed by chemical dehydration with 2,2-dimethoxypropane to be the method of choice. SEM revealed that chicken skin has a convoluted surface. Two types of chicken skin, distinguishable on the basis of chemical composition and appearance were observed. Type I has a filamentous surface with 55% moisture and 25% fat, whereas Type II skin has a globular appearance, 52% fat and 33% moisture. The fatty acid profiles of Types I and II skin are the same. Bacteria have greater affinity for Type II than Type I skin. Attachment studies indicated that Salmonella typhimurium quickly attach to the skin surface and cannot be removed easily by washing with water or with water containing a surfactant."@en . "https://circle.library.ubc.ca/rest/handle/2429/22588?expand=metadata"@en . "A Scanning Electron Microscopic, Chemical and Microbiological Study of Two Types of Chicken Skin by J y o t i Madhu Sahasrabudhe B.Sc. (Hons.) Queen's Uni v e r s i t y , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1981 c) J y o t i Madhu Sahasrabudhe, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f /iool S and the samples were allowed to freeze by contact with the cold block (Figure 5). B. Proximate Analysis Chicken skin samples were obtained from Site A (before scalding) and Si t e C ( c h i l l tank) (see Figure 6). Five samples were taken from each s i t e . 1. Moisture determination Moisture content was determined by l y o p h i l i z a t i o n of f i n e l y chopped pieces of chicken skin. Analyses were carried out i n duplicate on f i v e samples of each skin type from carcasses taken at Site A and C. 2. Crude fat determination Crude fat was determined, after l y o p h i l i z a t i o n of the skin samples, by the use of a Goldfish extractor (AOCS, 1975). Petroleum ether was used as the extracting solvent and the extraction was continued for 6 h. Following extraction, the petroleum ether was allowed to evaporate from the extraction flasks at room temperature overnight. /23 S A M P L E F R E E Z E I N I S O P E N T A N E C O O L E D I N L I Q U I D N 2 F R E E Z E D R Y \u00E2\u0080\u00A2 G O L D C O A T E X A M I N E FIGURE 4: Flow sheet of the freeze-drying procedure. /24 FIGURE 5: Aluminum block used i n the f r e e z i n g of chicken ski n samples. /25 3. Protein determination Protein content of the skin samples was determined by the rapid micro-Kjeldahl method of Concon and Soltess (1973), using the Technicon Auto Analyzer (Technicon Instruments Corp., Tarrytown, NY). Analyses were carried out i n duplicate on t r i p l i c a t e 10 mg lyo p h i l i z e d samples. Results were expressed as percent protein wet weight, using a nitrogen to protein conversion factor of 6.25 (Coleman, 1968). 4. Total carbohydrate Total carbohydrate content of the skin samples was determined by the modified phenol-sulfuric acid method (Dubois et al., 1956). Analyses were performed on 10 mg of lyo p h i l i z e d skin. The skin samples were mixed with 2 mL of d i s t i l l e d deionized water i n a test tube. The tubes were stoppered and placed i n a 100\u00C2\u00B0C water bath for 1 min. Upon cooling to room temperature, 0.05 mL of 80% (w/v) phenol was added. Five mL of concentrated s u l f u r i c acid was then added rapidly. In order to complete hydrolysis of the skin tissue, vortexing of the mixture was required immediately after the addition of s u l f u r i c acid. After standing at room temperature for 10 min, the tubes were again vortexed, and subsequently incubated at 25\u00C2\u00B0C for 15 min. The absorbance at 485 nm was then measured with a Unicam SP 800B spectrophotometer. Quadruplicate analyses were carried out on t r i p l i c a t e skin samples. Total carbohydrate /26 was estimated from a standard curve for glucose. Total carbohydrate was expressed as percent wet weight. C. Fatty Acid Composition 1. Preparation of methyl esters i) Purification of crude lipid The crude l i p i d was p u r i f i e d by the method of Sahasrabudhe et al. (1979) using a biphasic separation of chloroform, methanol and water (2:1:0.8). The chloroform layer was then f i l t e r e d through phase separating f i l t e r paper (Whatman, Ltd., England) to remove any traces of water and then concentrated with a rotary evaporator at room temperature. This extract was stored under N^, at 4\u00C2\u00B0C, overnight. i i ) Transesterification P u r i f i e d l i p i d (50 mg) was refluxed with 40 mL of H^SO^methanol solution (2.0 mL concentrated ^SG^ i n 230 mL methanol:benzene 3:1) i n a flask placed i n a sand bath at 140\u00C2\u00B0C for 4 h. The solution was stored under N^, at 4\u00C2\u00B0C, overnight. Then 50 mL of water was added and the mixture extracted twice with 50 mL portions of petroleum ether. The combined extracts were washed with d i s t i l l e d deionized water u n t i l free from acid as evidenced by an external methyl red indicator. The extract was then f i l t e r e d through anhydrous sodium sulfate to remove any remaining moisture. The petroleum Ill ether was then removed under \] with a rotary evaporator at room temperature (Dr. M. R. Sahasrabudhe, Food Research I n s t i t u t e , personal communication). 2. Gas chromatography Fatty acid methyl esters were analysed with a Tracor model MT 200 gas chromatograph (Tracor, Austin, TX) equipped with dual flame ion i z a t i o n detectors. The column was 6 f t x 1/8 i n stainless steel packed with GP 5% DEGS-PS on a 110/120 mesh support (Supelco Inc., Bellefonte, PA) and operated on a temperature program sta r t i n g at 150\u00C2\u00B0C and ending at 200\u00C2\u00B0C, increasing by 4\u00C2\u00B0/min. Fatty acids were i d e n t i f i e d by retention times and percentages of each f a t t y acid were calculated using response factors determined on known standards (Supelco Inc., Bellefonte, PA). Peaks were recorded and analysed using a Hewlett Packard Model 3390A Reporting Integrator (Hewlett Packard, Avondale, PA). D. Bacterial Load of Chicken Carcasses Chickens were obtained from Site B (after defeathering) and Site C ( c h i l l tank, see Figure 6). The chickens were placed i n s t e r i l e polyethylene bags (1 Mrad y radiation) and transported on ice to the laboratory. Samples of c h i l l water were taken by dipping a s t e r i l e beaker into the c h i l l tank. The c h i l l water was then transferred to a s t e r i l e Whirl-pak bag (Arnold Nasco Ltd., Guelph, Ont.) and transported on ice to the laboratory. FIGURE 6: Sampling sites in the poultry processing plant. DEFEATHERING MACHINES EVISCERATING LINE ORGAN REMOVAL BLOOD TUNNEL BEHEADING and DEFOOTING SPIN CHILLER S I T E C If LIVE HANGING t t SLAUGHTERING A R E A FURTHER PROCESSING and PACKAGING /30 Bacterial enumeration was performed on a per cm''' basis. 2 This was done by cutting a 12.95 cm area frcm locations 1 and 2 (see Figure 7). The area was inscribed by the use of a round metal cookie cutter (radius - 2 cm). This area was measured by the use of a electronic planimeter Model EDGC (Nemonics Corp, USA). Each skin sample was placed i n a s t e r i l e Stomacher bag to which 50 mL of s t e r i l e 0.1% peptone broth (Difco, Detroit, MI) containing 1% Tween 80 (Difco, Detroit, MI) was added. Tween 80 was added as an aid i n homogenizing fat i n the chicken skin (Emswiler et al., 1977). The sample was then stomached for 2 min i n a Colworth stomacher Lab blender 400. (A.J. Seward, London, England). Appropriate s e r i a l decimal d i l u t i o n s of the macerated sample were prepared with 0.1% peptone broth and 0.02 ml aliquots, i n duplicate were plated on separate sectors of B r i l l i a n t green agar (BGA, Difco. Detroit, MI) and nutrient agar (NA, Difco, Detroit, MI) using the modified drop plate technique (ICMSF, 1978) (Figure 8). E. Attachment Studies 1. Preparation of inoculum A freeze dried culture of Salmonella typhimurium (ATCC 14028) was prepared as described by American Type Culture Collection (1976). The culture was maintained on NA slants at 4\u00C2\u00B0C. P r i o r to inoculation of the chicken samples, a loopful of S. typhimurium was transferred from the NA slant to several tubes containing 10 mL nutrient broth (NB) (Difco, Detroit, MI) and grown for 18-24 h at 35\u00C2\u00B0C. Subseqently, 2 mL of t h i s culture was /31 /32 FIGURE 8: B r i l l i a n t t>reen agar (lef t ) a m i n u t r i c - M a g a r f r i g h t ) p l a t e s . Each plate shows t h r o e d i h i t i o n s , e a c h d o n e i n d u p l i c a t e , u s i n g t h e d r o p p i n t o t e c h n i q u e . /33 transferred to each of several bottles containing 200 mL NB. The cultures were grown at 35\u00C2\u00B0C i n a shaking water bath for 18-24 h. This was done i n order to obtain maximum c e l l densities. Approximate concentrations of the bacte r i a l suspension were determined using an HF turbidimeter (Model DRT-1000, H.F. Instruments, Bolton, Ont.). Appropriate al-iquots were then taken and centrifuged for 10 min at 5000 g, i n order to remove the nutrient broth. The p e l l e t was resuspended i n 50 ml of s t e r i l e attachment medium (pH 7.2) consisting of 0.150 M NaCl, 0.0062 M Na\u00E2\u0080\u009EHP0., 0.0021 M NaHJPO. and 0.001 M EDTA (Notermans 2 4 2 4 and Kampelmacher, 1974). This was shaken vigorously i n order to obtain a uniform suspension. The solution was centrifuged for 7 min at 5000 g. The centrifugation and resuspension i n s t e r i l e attachment medium was repeated twice to ensure complete removal of the nutrient broth. After completion of the washing, the p e l l e t was resuspended i n 10 mL of s t e r i l e attachment medium and then added to the inoculating bath. 2. Preparation of skin samples Chicken legs were a s e p t i c a l l y removed from chicken carcasses obtained from Site C of a l o c a l poultry processing plant and transported on ice to the laboratory. These legs were t i e d with s t e r i l e s t r i n g , then hung from a rin g stand (Figure 9a). FIC U R E 9 A p p a r a t u s f o r t h e a t t a c h m e n t s t u d i e s . C h i c k e n legs hanging f r o m rin:\ s t a n d s i n a s t e r i l e h o o d f a ) . Chicken legs i m m e r s e d in a t t a c h m e n t m e d i u m f b ) . /35 They were then immersed for 15 min i n 3 L of attachment medium, containing approximately 10 S. typhimurium cells/mL. Control samples were also prepared by dipping the chicken legs into 3 L of s t e r i l e attachment medium (Figure 9b). After 15 min immersion i n the attachment medium, the chicken legs were removed from the media and allowed to hang. After each of the desired time intervals (0, 5, 10 and 15 min), two legs were removed for each of the following procedures (Figure 10). i) Inoculated samples Skin samples were cut from locations 1 and 2 (see Figure 11) d i r e c t l y after hanging for the desired length of time. i i ) Washed inoculated samples The inoculated legs were dipped quickly 15 times into 1.5 L of s t e r i l e attachment medium before skin samples were taken. iii) Surfactant-washed inoculated samples The inoculated legs were dipped quickly 15 times into 1.5 L s t e r i l e attachment medium containing 1% Tween 80. 3. Bacteriological analysis Skin samples were placed i n 100 mL of s t e r i l e 0.1% (w/v) peptone broth containing 1% Tween 80 i n a s t e r i l e stomacher bag. It was then stomached for 2 min i n a Colworth Lab blender 400. /36 C H I C K E N L E G CONTROL / \ T E S T D I P I N P H Y S I O L O G I C A L S A L I N E f o r 1 5 min D R I P F O R 0 , 5 , 1 0 , 1 5 m i n I N O C U L A T E D D I P I N P H Y S I O L O G I C A L S A L I N E + 1 x 1 0 8 Salmonella / m l f o r 1 5 mm D R I P F O R 0 , 5 , 1 0 , 1 5 min N ^ A S H E D W A S H I N P H Y S I O L O G I C A L S A L I N E SURFACTANT W A S H I N P H Y S I O L O G I C A L S A L I N E A N D 1 % T W E E N 8 0 M I C R O B I O L O G Y A N D S C A N N I N G E L E C T R O N M I C R O S C O P Y FIGURE 10: Flow sheet of the attachment procedure. /37 URP. 11: M e t a l t e m p l a t e u s e d t o i n s c r i b e c h i c k e n s k i n shown a t l o c a t i o n 1. L o c a t i o n 2 i s i n d i c a t e d by b v a r r o w . /38 Appropriate s e r i a l decimal d i l u t i o n s of the macerated sample were prepared with s t e r i l e 0.1% peptone broth and duplicate 0.02 ml aliquots were plated on separate sectors of BGA and NA using the modified drop plate technique (ICMSF, 1978). Bacterial enumeration was also performed on the attachment medium by preparation of s e r i a l decimal d i l u t i o n s i n 0.1% peptone broth and drop-plated on BGA and NA. A l l BGA plates were incubated for 24 h at 35\u00C2\u00B0C and the nutrient agar plates were incubated for 48 h at 35\u00C2\u00B0C. 4. S t a t i s t i c a l analysis The data was f i r s t analyzed by analysis of variance for a balanced 4 x 4 x 2 f a c t o r i a l design (Mendenhall, 1968; Londgren and McElrath, 1969) for each t r i a l . Each of the variables was f i r s t expressed as a logarithm to the base ten. There were four treatment levels: control, inoculated, washed and surfactant and four time l e v e l s : 0, 5, 10 and 15 min after removal from attachment media. There were two skin types: Type I and Type I I . Each c e l l had four observations giving a t o t a l of 128 observations for each t r i a l . The data from the two t r i a l s was then combined and analyzed a s a 4 x 4 x 2 x 2 balanced f a c t o r i a l design. Replication was the fourth factor, with 2 lev e l s : T r i a l 1 and T r i a l 2. This resulted i n a t o t a l of 256 observations. The data was analyzed on Amdahl V8 computer (Amdahl, Sunnyvale, CA) using UBC MFAV /39 (Le, 1980a) which c a l c u l a t e s a n a l y s i s o f v a r i a n c e , Neuman-Keuls 's m u l t i p l e range t e s t and UBC BMD 02V (Le, 1980b) which a l s o c a l c u l a t e s marg ina l means and i n t e r a c t i o n e f f e c t s . /40 RESULTS AND DISCUSSION A. Scanning Electron Microscopy Scanning electron microscopy can provide valuable information on the micro-topography of chicken skin, a substrate for food-borne bacteria. Poultry skin structure may play a role in the attachment of bacteria to poultry carcasses (McMeekin et al., 1979). In order to examine the involvement of the surface microstructure in the adhesion of bacteria to poultry skin, several methods of preparation of chicken skin were evaluated to determine the best method to visualise the microstructure of poultry skin. The micrograph of chicken skin prepared by standard chemical fixation and dehydration (Figure 12) reveals that chicken skin does not have a smooth surface, but rather i t is convoluted with many crevices which may serve to entrap bacteria. The structures shown are comparable to those reported by McMeekin et al. (1979), Thomas and McMeekin (1980), and Suderman and Cunningham (1980). McMeekin et al. (1979) and Thomas and McMeekin (1980) used osmium tetroxide vapors for fixation, whereas Suderman and Cunningham (1980) used phosphate buffered glutaraldehyde. Similar structures (Figure 13) can also be observed using light microscopy. The second method of fixation to be investigated was the thiocarbohydrazide method. Most biological specimens cannot conduct electrons and therefore must be treated specifically for SEM. In the standard method, a coating of gold-palladium is evaporated or FIGURE 12: SEM micrograph of chicken breast s k i n prepared by standard chemical f i x a t i o n and conventional ethanol dehydration and amyl acetate i n f i l t r a t i o n . [\u00E2\u0080\u00A2' IQJRF: 13: C h i c k e n b r e a s t mi c r o s c o p e . s k i n viewed under the li,s>ht /43 sputtered onto the surface to be examined. Since b i o l o g i c a l specimens have complex topographies, uneven coating frequently r e s u l t s . I f the coating i s inadequate, charging effects w i l l occur. These are evidenced by either abnormally bright areas or as discharges which produce dark areas lacking i n d e t a i l . In addition, the use of a o coating adds 100 - 200 A to the surface being examined (Kelley et al., 1973; Sweeny and Shapiro, 1977). This additional layer could mask the f i n e r d e t a i l of the surface structure (Hayat, 1978). The thiocarbohydrazide method employs the use of thiocarbo-hydrazide and osmium tetroxide i n addition to the routine glutaraldehyde f i x a t i o n and osmium tetroxide post-fixation. TCH i s used to render the tissue conductive and acts by bridging one molecule of OsO^ to another molecule of OsO^ (Figure 14). This enhances contrast (Hayat, 1978). McCowan et al. (1978) have used the thiocarbohydrazide procedure i n the f i x a t i o n of rumenal e p i t h e l i a l c e l l s and found that the thiocarbohydrazide treatment reduced charging while the absence of a gold layer gave clearer micrographs as compared to the standard method. In the f i x a t i o n of chicken skin, however, the TCH method did not y i e l d superior r e s u l t s . This procedure i s very tedious and time-consuming. A comparison of specimens prepared by the standard method and the thiocarbohydrazide method (Figure 15) reveals that the standard method i s better. The same stringy structures are again evident but the resolution and d e f i n i t i o n are not as clear i n the TCH fixed samples. The cracks and crevices are again present on the surface of the skin samples. 3 to L I G A N D 3 to '\u00E2\u0080\u00A2I M - L I G A N D M 3 tO to .. M - L I G A N D - M M = O s 0 4 L I G A N D = H 2 N N H C N H N H 2 S II MUUKfc 14: Bridging reaction between thiocarbohydrazide and osmium tetroxide for the enhancement of contrast i n SEM. 4^ /46 In order to shorten the preparation time, 2,2-dimethoxy-propane (DMP) was used for rapid dehydration. This step eliminated several hours of preparation time since ethanol dehydration and amyl acetate i n f i l t r a t i o n could be eliminated. A c i d i f i e d 2,2-dimethoxypropane combines with water to form methanol and acetone (Figure 16). This reaction i s endothermic. The advantages of th i s technique have been l i s t e d by Maser and Trimble (1976). DMP i s less expensive than either ethanol, acetone or amyl acetate and the procedure i s rapid since i t does not require physical exchange with water as does conventional dehydration. The products of the reaction, acetone and methanol, are solvents commonly used i n c r i t i c a l point drying. DMP i s miscible with l i q u i d CG^ and i s therefore compatible with CG^ exchange pr i o r to c r i t i c a l point drying. When one compares the micrographs of the specimens prepared by the two methods of dehydration (Figure 17), i t i s apparent that chemical dehydration did not induce alterations i n the surface structure of poultry skin. The same cracks and crevices were again evident. The fibrous structures (Figure 17, arrow) tended to crop up i n most of the sections, and similar structures have been reported by McMeekin et al. (1979). This technique has been used with success by Kahn et al. (1977) i n t h e i r study of c e l l s i n culture. They found that 2,2-dimethoxypropane i s a useful alternative to ethanol i n that \"it is fast, inexpensive, reduces the chance of air drying and insures complete removal of water\". / 4 7 O C H o O i J H 2 0 i. C H 3 - C - C H 3 \u00E2\u0080\u00A2 2 C H 3 O H + C H 3 C C H 3 O C H 3 2 , 2 - D M P M E T H A N O L A C E T O N E FIGURE 16: Reaction of 2,2 dimethoxypropane with water during dehydration of t i s s u e samples f or SEM. FIGURE 17 SEM micrographs of chicken breast skin prepared by (a) conventional dehydration and (bi by chemical dehydration /49 The t h i r d method under investigation was freeze-drying. Freeze-drying would be r e l a t i v e l y rapid and would bypass the \"washing\" effect of the buffers which are used i n the other two methods. When comparing, the micrographs of specimens prepared by the standard method and those prepared by freeze-drying (Figure 18) i t i s apparent that the standard method of f i x a t i o n i s better. It appears that the surface of the freeze-dried sample i s obscured by a coating of what may be melted f a t . This may be due to the use of isopentane, which could dissolve the l i p i d on i n i t i a l contact and cause i t to spread over the sample surface. Due to the rapid rate of freezing, the l i p i d may be frozen as an i c e - l i k e sheet on the skin surface. Several problems were encountered i n the process of freezing. Large samples of skin were required since the skin curled and cracked on contact with the cold isopentane. Other studies i n the laboratory on beef muscle, did not y i e l d positive r e s u l t s . A surface f i l m was deposited on the samples during the freezing process (R. Yada, personal communication). Therefore, freeze-drying as a method of preparation of chicken skin for SEM was discarded. When comparing these methods as to cost and time (Table I ) , as well as to resolution and c l a r i t y of d e t a i l , i s apparent that the standard chemical method i s approximately two-thirds the cost of the thiocarbohydrazide method. I t i s also much quicker (5 h compared with 13.5 h). Chemical dehydration using 2,2-dimethoxypropane i s considerably faster than conventional methods and shortens the procedure by several hours. TABLE I: Time and cost estimates for the preparation of ten samples for Scanning electron microscopy. A. Fixation Chemical Standard Fixation Thiocarbo-hydrazide Fixation Freeze drying Glutaraldehyde $4.20 $4.20 -Osmium tetroxide $2.85 $9.50 -Thiocarbohydrazide - $0.95 -Gold $2.50 - $2.50 Isopentane - - $1.50 Liquid nitrogen - - $2.00 Total cost $9.55 $14.65 $6.00 Time 5h 13.5h 0.2h B. Dehydration Chemical Standard Dehydration Chemical Dehydration Ethanol $0.13 -Amyl acetate $1.00 -2,2 DMP - $2.25 Total cost $1.13 $2.25 Time 3.5h 0.25h /52 From these preliminary i n v e s t i g a t i o n s , i t was determined that the standard chemical f i x a t i o n using glutaraldehyde and osmium tetroxide followed by chemical dehydration using 2,2-dimethoxypropane was the best method for preparing chicken skin f o r SEM. This combination gave reproducible r e s u l t s i n a short period of time. During the i n i t i a l methodology t r i a l s , skin samples were taken from chicken breasts. Several types of microstructures were observed i n the scanning electron micrographs. Two skin types could be c l e a r l y d i f f e r e n t i a t e d at higher magnifications (Figure 20) though at low magnifications (Figure 19), they were not discernable. At a magnification of 7000x, breast skin f a l l s into two categories. Micrographs (Figure 20) i n d i c a t e Type I skin has the filamentous, convoluted structures discussed e a r l i e r . Type II skin has a smoother, globular surface appearance. There are also crevices in which b a c t e r i a may become entrapped. The existence of Type II skin has not been previously reported i n the l i t e r a t u r e . In order to determine whether Types I and II skin are present at other locations on the chicken carcass, samples were taken from the leg and back regions of the chicken carcass. The samples were prepared by the standard chemical method and chemical dehydration. Sections from the leg (Figure 21) reveal once again, two c l e a r l y discernable types of chicken skin. Figure 21 i s at a lower f a ) I-'TfllJRli 20: SHM micrograph chicken breast Typo I and (b) Type II s k i n obtained from FIGURF. 21: SEM micrographs o f (a) Type I and (b) Type I I s k i n o b t a i n e d from ch i c k e n 1egs. /56 m a g n i f i c a t i o n than Figure 20 to demonstrate that the d i f f e r e n c e i s observable at a somewhat lower m a g n i f i c a t i o n . Two s k i n types were a l s o found on the back s k i n s e c t i o n s (Figure 22). Comparison of the micrographs of Type I s k i n from l e g , breast and back s k i n (Figure 23), i n d i c a t e that l e g and breast s k i n are q u i t e s i m i l a r i n surface appearance. Ski n from both of these l o c a t i o n s d i s p l a y the filamentous, convoluted m i c r o s t r u c t u r e described e a r l i e r . The back s k i n , however, has a somewhat coarser topography, which i s c l e a r l y d i f f e r e n t from e i t h e r l e g or breast s k i n . Comparison of micrographs of Type I I s k i n (Figure 24) r e v e a l s again, the congruence of l e g and breast s k i n and a d i s s i m i l a r i t y of back s k i n . The d i f f e r e n c e s may be due to the f a c t that back s k i n has d i f f e r e n t subcutaneous components than the l e g or the breast s k i n . Back s k i n i s more f i r m l y h e l d to the bone, whereas leg and breast s k i n overlays muscle t i s s u e and are e a s i l y removeable. B. Proximate Analysis Once i t was e s t a b l i s h e d that there were at l e a s t two types of chicken s k i n , i t was of i n t e r e s t to see what d i f f e r e n c e s , other than appearance, e x i s t e d between Type I and Type I I chicken s k i n . Chickens were obtained before the s c a l d i n g ( s i t e A) and a f t e r immersion c h i l l i n g operations ( s i t e C) of the processing p l a n t . Proximate a n a l y s i s (Table I I ) i n d i c a t e s that Type I chicken s k i n has 46% moisture before s c a l d i n g and t h i s increases to TABLE I I : Proximate analysis of Type I and Type II chicken skin before scalding (Site A) and after immersion c h i l l i n g (Site C). Site Skin Type Moisture, % Fat, % Protein, % Carbohydrate, % A I II 46. 33. * ,21 ,98 + + 1. 2. ,92 a ,63b 22. 35, .70 .00 + + 5.26a 4.09b 16.80 13.08 \u00C2\u00B1 + 4, 4 .80a .19a 0.308 0.254 + + 0.059a 0.098a I 55. .04 + 5. ,06C 25, .16 + 5.68a 10.75 + 4 .47a 0.155 + 0.046a C II 33. ,64 + 0. ,65b 52. .14 + 3.12\u00C2\u00B0 8.63 + 2 .60a 0.148 + 0.028a mean of quintuplicate samples, means bearing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p < 0.01) standard deviation (a) (b) FIGURE 22: SHM micrographs of I'ype I and (b) Type 11 s k i n obtained from chicken backs. /59 (a) lb) FIGURE 24 : SEM micrographs of Type II skin obtained from (a) chicken Legs, lb) chicken breasts and (c) chicken backs. o /61 55% after c h i l l i n g . This high moisture content may be due to water uptake during processing (Mulder and Veerkamp, 1974; Notermans and Kampelmacher, 1975a). Type II skin absorbs very l i t t l e water during processing. The moisture content of Type II i s s i g n i f i c a n t l y (p < 0.01) lower than that of Type I skin. There are no s i g n i f i c a n t differences (p > 0.01) i n protein or carbohydrate content between Type I and Type II skin. There i s considerable difference (p < 0.01) in fat content between the two skin types. In Type I skin, there i s l i t t l e change i n fat content during processing. Type II skin has approximately double the fat content of Type I skin after the c h i l l i n g operation. Before scalding, Type II skin contains 35% f a t , but after c h i l l i n g , the fat content i s 52% (Table I I ) . Since Type II skin i s located around the feather t r a c t s , subcutaneous fat may be mobilized through the skin v i a pores and feather f o l l i c l e s during scalding, producing the higher fat content observed after processing. Further moisture and fat analysis were carried out on two groups of f i v e chickens obtained from S i t e C. The r e s u l t s , shown in Table I I I , display the same trend as that seen i n Table I I . The fat content of Type II chicken skin i s approximately twice that of Type I chicken skin. C. Fatty Acid Composition The f a t t y acid composition of both types of chicken skin was determined to ascertain whether the differences i n fat content were TABLE I I I : Moisture and f a t cantent of Type I and Type chicken skin a f t e r immersion c h i l l i n g . T r i a l 1 and 2. T r i a l Skin Type Moisture, % Fat, % I 58.39* \u00C2\u00B1 6. 54** a 27.13 \u00C2\u00B1 6 .54 a 1 II 35.32 \u00C2\u00B1 2. 28 b 53.06 \u00C2\u00B1 3 .91 b I 52.91 \u00C2\u00B1 10. 32 a 22.79 \u00C2\u00B1 5 . 2 l a 2 II 31.86 \u00C2\u00B1 5. 58 b 59.67 \u00C2\u00B1 1 .20 b mean of quintuplicate samples, means bearing the same superscript are not s i g n i f i c a n t l y d i f f e r e n t (p < 0.01). standard deviation /63 just a difference i n t o t a l amount of f a t , or i f there was a difference i n the l i p i d s themselves. Chickens were obtained from Site A and Site C. Samples of Types I and II skin were taken from the leg, breast and back. The f a t t y acid p r o f i l e s (Figures 25, 26 and 27) show that Type I and Type II skin display e s s e n t i a l l y no difference i n f a t t y acid composition. The fatt y acid compositions of leg, breast and back skin are also e s s e n t i a l l y the same. The fa t t y acid composition (Tables IV and V) indicate that the major component i s C^g.-^ (oleate) at 46%, followed by C J ^ . Q (palmitate) at 28%. The amounts of short chain f a t t y acids (C <^^) are approximately equal, and are therefore combined into one value. These results are i n agreement with the work by Pereira et al. (1976) on chicken tissue f a t . They reported 38% of the fat was o l e i c acid and 28% was palmitic acid. They found that processing effects on the f a t t y acid composition of chicken fats was low, but dietary factors were found to be s i g n i f i c a n t . Since t h i s research involved chicken skin and not chicken tissue, the discrepancies are to be expected. Since proximate analysis (Table II) suggested the mobilization of fat through Type II skin during processing, i t was anticipated that l i p i d s of Type II skin would have lower melting temperatures. Since short chain f a t t y acids and unsaturated f a t t y acids have lower melting temperatures (Babayan, 1974) than long chain saturated f a t t y acids, i t was thought that Type II skin would 160 18 I Z o o. CO LU cr o o 4 TIME 6 (min) Ca) - J O \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 0.05) between the water and surfactant treatments, however. There i s also a d i f f e r e n c e between T r i a l 1 and T r i a l 2. 4 The average counts i n T r i a l 2 are higher (4.14x10 ) than those i n T r i a l 1 (2.17xl0 4). 4 The b a c t e r i a l counts on Type I skin are 1.65x10 , whereas on Type II skin they are s i g n i f i c a n t l y higher (p < 0.01) at 5.75xl0 4. From these r e s u l t s i t i s evident that the attachment procedure i s very complex and i s further complicated by the inherent v a r i a b i l i t y of b i o l o g i c a l systems. 2. Scanning electron microscopy Samples of Type I and Type II skin from each of the treatments were prepared f o r SEM. Micrographs of skin, from a l l the treatments, were prepared. Since the micrographs of the controls were s i m i l a r to those presented e a r l i e r , they are not repeated here. Examination of the micrographs of washed and surfactant treated samples f a i l e d to reveal the presence of any b a c t e r i a , /90 even though enumeration on B r i l l i a n t green agar yielded b a c t e r i a l 4 5 2 numbers i n the range of 10 to 10 cfu/cm (Figures 30, 31). 5 2 2 On a microscopic l e v e l , 10 cfu/cm translates to 0.7 cfu/700 ym 2 assuming that the bacteria are evenly distributed. 700 um corresponds roughly to the skin surface area represented i n Figure 32. McMeekin et al. (1979), using SEM to study the attachment of microorganisms to chicken skin, found a ten-fold discrepancy between the counts obtained on nutrient agar and those calculated from the density of microorganisms on a micrograph. This could be due to the formation of a scum during f i x a t i o n i n glutaraldehyde. This scum i s l i k e l y to have been unfixed l i p i d material or material washed from the surface of the skin by the f i x a t i v e which presumably contains many organisms. Micrographs from the inoculated skin samples (Figures 32, 33, 34) show the presence of bacteria on the surface of Type I and Type II chicken skin. The crevices and channels (arrow, Figure 32) i n the surface of chicken skin, are i n most cases, larger than the bacteria, thus favoring physical entrapment. -Once trapped, the bacteria could secrete the exopolysaccarides necessary for attachment. These observations may explain i n part, the d i f f i c u l t i e s i n decreasing b a c t e r i a l populations on chicken carcasses at the processing plant (McMeekin et ail., 1979). I t may also explain FIGURE 32: SEM micrograph of i n o c u l a t e d Type I c h i c k e C r e v i c e s and channels (arrow) are apparent s k i n s u r f a c e . fit FIGURE 35: SEM micrograph of i n o c u l a t e d Type I chicken s k i n . FIGURE 34: Sem m i c r o g r a p h o f i n o c u l a t e d T y p e II c h i c k e n s k i n . /94 the observation that viable counts from macerated samples of skin are always greater than those obtained by swabbing or rinsing the skin (Avens and Miller, 1970; Patterson, 1971; Notermans et al., 1975a). /95 GENERAL DISCUSSION Several methods of f i x i n g chicken skin f o r examination i n the SEM were examined. The standard chemical method using f i x a t i o n with 6.3% buffered glutaraldehyde at 4\u00C2\u00B0C, and post f i x a t i o n with osmium tetroxide was compared to the thiocarbohydrazide method, and to freeze-drying. The thiocarbohydrazide method involves f i x a t i o n of the sample with glutaraldehyde, three p o s t - f i x a t i o n s with osmium tetroxide interspersed with two treatments with thiocarbohydrazide (TCH). This method does not require the use of gold coating f o r conduction of electrons, since the OsO^-TCH-OsO^ bridging renders the tissues conductive. Micrographs of chicken skin f i x e d with TCH show poor d e t a i l and electron charging e f f e c t s i n d i c a t i n g that the TCH method i s not su i t a b l e f o r the examination of chicken skin, although McCowan et al. (1978) recommended i t s use i n the study of the rumen of c a t t l e . The t h i r d method involves freezing the chicken skin i n isopentane cooled i n l i q u i d nitrogen. L i t t l e s t r u c t u r a l d e t a i l could be observed i n the micrographs. The sample surface was obscured by a layer of what may be melted f a t . This problem may be overcome i f the isopentane was eliminated from the procedure and the sample frozen i n l i q u i d nitrogen. However, when skin samples are frozen i n l i q u i d nitrogen alone, the skin c u r l s and cracks severely. Other studies i n the laboratory showed that i n freeze-dried samples of beef, the bac t e r i a were e a s i l y l o s t onto other surfaces, such as tweezers, sides of /96 beakers, etc., due to el e c t r o s t a t i c effects (R. Yada, personal communication). These results are i n contrast to those of Suderman and Cunningham (1980) who found freeze-drying to be the method of choice. However, they fixed the chicken skin samples with glutaraldehyde p r i o r to freezing. The standard chemical f i x a t i o n yielded micrographs with good c l a r i t y and resolution. Some samples, however, s t i l l showed some charging effects. This problem i s quite common when examining samples with high l i p i d content (Suderman and Cunningham, 1980). In addition, chemical dehydration using 2,2-dimethoxy-propane was evaluated against the standard ethanol dehydration and amyl acetate i n f i l t r a t i o n . The rapid chemical dehydration was found to y i e l d good qua l i t y micrographs with an appreciable time saving. SEM indicated that chicken skin does not have a smooth surface, but i s filamentous with many cracks and crevices. Other authors have reported si m i l a r results using different methods of f i x a t i o n . McMeekin et al. (1979) used f i x a t i o n above OSO4 vapors to prepare chicken skin for SEM. Suderman and Cunningham (1980) used freeze-drying as well as several other methods i n t h e i r study. Light microscopy also yields s i m i l a r structures. Scanning electron microscopy revealed the existence of at least two types of skin on the chicken carcass. The f i r s t type (Type I) has a filamentous surface structure, whereas the second type (Type II) has a more globular appearance. These two types of /97 chicken skin can be found at several locations on the chicken carcass. Leg and breast skin are very si m i l a r i n appearance, but back skin i s somewhat less d i f f e r e n t i a t e d . This could be due to the d i f f e r i n g subcutaneous structures.. Breast and leg skin overlay muscle tissue, whereas l i t t l e muscle tissue i s found under back skin. Back skin i s more firmly held to the underlying fat and bone. Type II skin seems to follow the feather tracts on the chicken carcass. The two skin types d i f f e r i n chemical composition. Type I chicken skin has approximately 55% moisture and 25% f a t , whereas Type II skin has 52% fat and 33% moisture. Processing of chicken carcasses led to a s i g n i f i c a n t increase (p < 0.01) i n the fat content of Type II chicken skin. There i s no difference i n f a t t y acid composition between the two types of chicken skin. Therefore, the difference between the two skin types may reside i n the more complex l i p i d s or lipoproteins. The i d e n t i f i c a t i o n of the two types of chicken skin may help i n the examination of the \"oily bird syndrome\" (OBS). Oily b i r d syndrome has recently received much attention. It i s characterized by \"oily or greasy birds, water pockets under loose skin and broken skin\" (Garrett, 1975). OBS i s thought to be caused by several factors such as environmental temperatures, fat deposition due to diet or processing plant stress. OBS appears most frequently i n warmer months and almost completely disappears with the onset of cooler weather. Edwards et al. (1973) suggested that the degree of /98 saturation i n carcass fat can be influenced by the type of fat i n the diet. Energy level alone i s not a causative factor i n producing OBS since nutrient density could not be related to greasy appearance of a carcass. Female birds exhibit a greater tendency to be o i l y than males (Garrett, 1975). Females have a higher level of body fat than males of the same age (Edwards et al., 1973). Garrett (1975) and Horvat (1978) were unable to detect s i g n i f i c a n t differences i n the fat t y acid p r o f i l e between birds classed as o i l y or non-oily. Similar observations were made between the fat t y acid p r o f i l e s of Type I and Type II skin. Fletcher and Thomason (1980) and Jenson et al. (1980) investigated the effects of processing conditions on the incidence of OBS. Their r e s u l t s indicated that an increase i n scald temperatures increases o i l y skin scores. This could be due to the mobilization of fat through the skin, onto the surface of the chicken carcass. In addition, Jensen et al. (1980) found that an increase i n plucking stress increased water sorption and o i l y , loose and broken skin scores. This could be due to the mechanical action of the plucker, spreading the melted fat over the entire carcass. It would be of interest to examine chickens exhibiting OBS and determine whether they had a greater prevalence of Type II skin or just an elevated fat content. The b a c t e r i a l load on Type I and Type II skin was determined after defeathering (Site B) and after c h i l l i n g (Site C). Type I /99 skin generally had a lower bac t e r i a l load than did Type II skin. However, there was considerable v a r i a b i l i t y i n bacterial.counts between chickens from the same flock and between legs from each chicken. The bac t e r i a l load decreases approximately ten-fold from Site B to C. Psychrotrophic bacteria form a greater proportion of the bact e r i a l f l o r a at S i t e C. These results are i n agreement with Surkiewicz et at. (1969), who found that the aerobic count was lowered by the washing action of the c h i l l e r s , whereas the psychrotrophic count increased due to cross-contamination i n the c h i l l tank. Attachment studies also showed s i g n i f i c a n t l y higher (p < 0.01) bacter i a l counts on Type II skin, compared to Type I skin. The control samples, which were dipped into s t e r i l e attachment medium showed 2 b a c t e r i a l counts between 1 and 600 cfu/cm , indicating a wide v a r i a b i l i t y i n the autochthonous population on chicken leg skin. S t a t i s t i c a l analysis using Neuman-Keul's multiple range test indicated a wide v a r i a b i l i t y i n the autochthonous population on chicken leg skin. S t a t i s t i c a l analyses using Neuman-Keul1s multiple range test indicated that there was a s i g n i f i c a n t difference (p < 0.01) between inoculated and washed samples; inoculated and surfactant treated samples, but no s i g n i f i c a n t difference (p > 0.05) between washed and surfactant treated samples. These results tend to refute Notermans and Kampelmacher*s (1974) theory that bacteria are i n i t i a l l y present /100 in a l i q u i d f i l m , since one would expect a dramatic decrease i n bacterial counts when surfactant i s incorporated i n the wash water. Therefore, i t would seem that Notermans and Kampelmacher's (1974) suggestion that replacement of the l i q u i d f i l m would reduce bact e r i a l attachment may not adequately describe the sit u a t i o n . Physical attachment of Salmonella typhimurium to chicken skin takes place more rapidly than o r i g i n a l l y suggested by Notermans and Kampelmacher (1974). These results indicate that simple washing techniques w i l l not be effe c t i v e i n decreasing the Salmonella load on SalmoneZZa-contamined chicken skin. McBride et al. (1980) and Campbell (1979) showed that i n most cases, the incidence of Salmonella-contaminated carcasses did not decrease markedly after the spin-c h i l l i n g operation. Incorporation of surface active agents i n the c h i l l water would probably not be effective in decreasing the bacterial load on the chicken skin since t h i s study shows that the addition of Tween 80 to rinse water did not produce a s i g n i f i c a n t decrease in the population of S. typhmurium on inoculated chicken skin. The addition of chlorine to the c h i l l - t a n k acts to k i l l the bacteria i n the c h i l l water (Sanders and Blackshear, 1971, Mead and Thomas, 1973b; Notermans et al., 1973; Mulder and Veerkamp, 1974), and thus prevent cross-contamination. Those bacteria already attached to the skin surface, however, w i l l not be affected. Kotula et al. (1967) found no p r a c t i c a l advantage in spray-washing with 50 ppm chlorine immediately after c h i l l i n g . Results of the attachment process. The and seemed to f i t i n the surface. SEM study yielded l i t t l e bacteria were evident on crevices and channels on /101 information on the the skin surface, the chicken skin /102 CONCLUSIONS The present study revealed that the standard chemical method of f i x a t i o n using 6.3% glutaraldehyde and 1% osmium tetroxide, followed by chemical dehydration with 2,2-dimethoxypropane i s the method of choice f o r preparation of chicken skin f o r scanning electron microscopy (SEM). SEM revealed that the surface of chicken skin i s not smooth. Rather, i t i s convoluted with many crevices and channels. Two types of chicken skin were discerned. Type I has a filamentous surface, whereas Type II chicken skin has a globular appearance. Skin samples from the leg and breast are s i m i l a r i n appearance but back skin i s somewhat coarser. Proximate analysis showed that the major chemical d i f f e r e n c e between Type I and Type II skin to be the moisture and f a t contents. Type I skin had s i g n i f i c a n t l y higher (p < 0.01) moisture content and approximately h a l f the f a t content of Type II chicken skin. The f a t t y a c i d p r o f i l e s of Type I and II skin were very s i m i l a r . M i c r o b i o l o g i c a l sampling of chicken carcasses showed that the b a c t e r i a l load decreased approximately t e n - f o l d between the scalding and the c h i l l i n g operations. Psychrotrophic b a c t e r i a form a greater proportion of the m i c r o f l o r a on chicken skin at the c h i l l tank. There i s considerable v a r i a t i o n i n the autochthonous b a c t e r i a l population between i n d i v i d u a l chickens. /103 Attachment studies revealed that the physical attachment of b a c t e r i a occurs r a p i d l y and these b a c t e r i a cannot be e a s i l y removed by washing with water, or with water containing a surfactant. Thus, i t i s important to decrease Salmonella contamination i n poultry f l o c k s since, once attachment occurs, i t i s d i f f i c u l t to remove the Salmonella from the poultry carcasses during processing. Further chemical c h a r a c t e r i z a t i o n of the chicken ski n , p a r t i c u l a r l y the complex l i p i d s and glycoproteins, may be of i n t e r e s t to determine why b a c t e r i a have a greater a f f i n i t y f o r Type II chicken skin than f o r Type I skin. /104 REFERENCES CITED American Type Culture Collection Catalogue of Strains I. Twelfth Edition. 1976. Gherna, R.L. and Hatt, H.D. eds. p. VII. 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"For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "A scanning electron microscopic, chemical and microbiological study of two types of chicken skin"@en . "Text"@en . "http://hdl.handle.net/2429/22588"@en .