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A scanning electron microscopic, chemical and microbiological study of two types of chicken skin Sahasrabudhe, Jyoti Madhu 1981

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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<ye/7CJU The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 DE-6 (2/79) / i i ABSTRACT Evaluation of several methods of f i x i n g chicken skin f o r scanning electron microscopy (SEM) indicated standard chemical f i x a t i o n 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 ski n , d i s t i n g u i s h a b l e on the basis of chemical composition and appearance were observed. Type I has a filamentous surface with 55% moisture and 25% f a t , whereas Type II skin has a globular appearance, 52% f a t and 33% moisture. The f a t t y a c i d p r o f i l e s of Types I and II skin are the same. Bacteria have greater a f f i n i t y f o r Type II than Type I skin. Attachment studies indicated that Salmonella typhimurium quickly attach to the skin surface and cannot be removed e a s i l y by washing with water or with water containing a surfactant. / i i i TABLE OF CONTENTS Page ABSTRACT i i .TABLE OF CONTENTS ••• i i i LIST OF TABLES v LIST OF FIGURES v i i ACKNOWLEDGEMENTS x i INTRODUCTION 1 LITERATURE REVIEW 3 A. Scanning Electron Microscopy 3 B. Microbiological Sampling of Chicken Carcasses 5 C. Enumerating Bacteria 9 D. Bacterial Adhesion 10 E. Inoculation of Chicken Skin 14 METHODS AND MATERIALS 16 A. Scanning Electron Microscopy 16 1. Standard Chemical Fixation 16 2. Standard Dehydration 17 Z. Chemical Dehydration 17 4. Thiocarbohydrazide Fixation 20 5. Freeze-Drying 20 B. Proximate Analysis 22 1. Moisture Determination 22 2. Crude Fat Determination 22 3. Protein Determination 25 4. Total Carbohydrate Determination 25 C. Fatty Acid Composition 26 1. Preparation of Methyl Esters 26 2. Gas-Liquid Chromatography 27 D. Bacterial Load of Chicken Carcasses 27 / i v E. Attachment Studies 30 1. Preparation of Inoculum 30 2. Preparation of the Skin Samples 33 3. B a c t e r i o l o g i c a l Analysis 35 4. S t a t i s t i c a l Analysis 38 RESULTS AND DISCUSSION 40 A. Scanning Electron Microscopy 40 B. Proximate Analysis 56 C. Fatty Acid Composition 61 D. Microbiological Sampling 69 E. Bacterial Load 69 F. Attachment Studies 75 1. B a c t e r i o l o g i c a l Analysis 75 2. Scanning Electron Microscopy 89 GENERAL DISCUSSION 95 CONCLUSIONS 102 REFERENCES CITED 104 LIST OF TABLES Table Page I Time and cost estimates for the preparation of ten samples for scanning electron microscopy 51 II 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) 57 III Moisture and fat content of Type I and Type II chicken skin a f t e r immersion c h i l l i n g . T r i a l 1 and 2 62 IV Fatty acid composition of Type I and Type II chicken skin before scalding (Site A) 67 V Fatty acid composition of Type I and Type II chicken skin after immersion c h i l l i n g (Site C) 68 VI Comparison of the drop plate technique to a more conventional method of plating 70 VII Analysis of variance for the attachment study. T r i a l 1 82 VIII Analysis of variance for the attachment study. T r i a l 2 83 IX Analysis of variance for the combined t r i a l s of the attachment study 84 X Estimates of main effect and interaction means for t r i a l and treatment from the analysis of variance 86 Estimates of the main e f f e c t and i n t e r a c t i o n means for time and treatment from the analysis of variance Estimates of main e f f e c t and i n t e r a c t i o n means fo r treatment and skin type from the analysis / v i i LIST OF FIGURES Figure Rage 1 Flow sheet of the standard chemical f i x a t i o n procedure 18 2 Flow sheet of the dehydration procedures 19 3 Flow sheet of the thiocarbohydrazide f i x a t i o n procedure 21 4 Flow sheet of the f r e e z e - d r y i n g procedure 23 5 Aluminum block used i n the f r e e z i n g of chicken s k i n samples 24 6 Sampling s i t e s i n the p o u l t r y processing p l a n t 29 7 Metal template used f o r i n s c r i b i n g c i r c l e s on chicken s k i n (arrow). S k i n i s cut from l o c a t i o n 1 and l o c a t i o n 2 31 8 B r i l l i a n t green agar ( l e f t ) and n u t r i e n t agar ( r i g h t ) p l a t e s . Each p l a t e shows three d i l u t i o n s , each done i n d u p l i c a t e , using the drop p l a t e technique 32 9 Apparatus f o r the attachment s t u d i e s . Chicken legs hanging from r i n g stands i n a s t e r i l e hood (a). Chicken leg s immersed i n attachment medium (b) 34 10 Flow sheet of the attachment procedure 36 11 Metal template used to i n s c r i b e chicken s k i n shown at l o c a t i o n 1. Location 2 i s i n d i c a t e d by / v i i i Figure Page 12 SEM micrographs of chicken breast skin 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 . . . 41 13 Chicken breast skin viewed under the l i g h t microscope 42 14 Bridging reaction between thiocarbohydrazide and osmium tetroxide for the enhancement of contrast in SEM 44 15 SEM micrographs of chicken breast skin prepared (a) by standard chemical f i x a t i o n and (b) by thiocarbohydrazide f i x a t i o n 45 16 Reaction of 2,2-dimethoxypropane with water during dehydration of tissue samples for SEM 47 17 SEM micrographs of chicken breast skin prepared (a) by conventional dehydration and (b) by chemical dehydration using 2,2-dimethoxypropane 48 18 SEM micrographs of chicken breast skin prepared (a) by standard chemical f i x a t i o n and (b) by freeze-drying 50 19 SEM micrographs of (a) Type I and (b) Type II skin obtained from chicken breasts 53 20 SEM micrographs of (a) Type I and (b) Type II skin obtained from chicken breasts 54 21 SEM micrographs of (a) Type I and (b) Type II skin obtained from chicken legs 55 / i x Figure '.Page 22 SEM micrographs of (a) Type I and (b) Type II skin obtained from chicken backs 58 23 SEM micrographs of Type I skin obtained from (a) chicken legs, (b) chicken breasts and (c) chicken backs 59 24 SEM micrographs of Type II skin obtained from (a) chicken legs, (b) chicken breasts and (c) chicken backs 60 25 Fatty acid p r o f i l e of (a) Type I and (b) Type II skin from chicken legs 64 26 Fatty acid p r o f i l e of (a) Type I and (b) Type II skin from chicken breasts 65 27 Fatty acid p r o f i l e of (a) Type I and (b) Type II skin from chicken backs 66 28 Bacterial load on chicken carcasses measured (a) after defeathering and (b) after immersion c h i l l i n g . T r i a l 1 72 29 Bacterial load on chicken carcasses measured (a) after defeathering and (b) after immersion c h i l l i n g . T r i a l 2 74 30 Bacterial population on inoculated, washed and surfactant treated chicken legs. T r i a l 1 78 31 Bacterial population on inoculated, washed and surfactant treated chicken legs. T r i a l 2 80 /x Figure Page 32 SEM micrograph of inoculated Type I chicken skin. Crevices and channels (arrow) are apparent on the skin surface 91 33 SEM micrograph of inoculated Type I chicken skin.... 92 34 SEM micrograph of inoculated Type II chicken skin... 93 /xi ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. B. J. Skura for his time, patience, assistance and constructive criticism throughout the course of this research. I also wish to thank the members of my graduate committee, Dr. R. C. Fitzsimmons, Dr. W. D. Powrie, Dr. J. F. Richards and Dr. M. A. Tung for their valuable suggestions and assistance. Thanks are also extended to Dr. J. V. Zidek for his help in the s t a t i s t i c a l analysis and to Mr. S. Yee and Miss L. Robinson for their invaluable technical advice. Finally, a special thanks to Eric Coyle for his endless time, patience and encouragement during this research and for his work in the preparation of the photographic material. /I INTRODUCTION Poultry meat forms an important part of the diets of many of the world's population, and production i s increasing to s a t i s f y demand. Poultry i s frequently associated with food-borne disease, with Salmonella sp., Staphyloccocus aureus and Clostridium perfrigens being the main e t i o l o g i c a l agents. An increase i n poultry-associated food-borne disease, i n par t i c u l a r salmonellosis, can be related to the increased consumption of poultry meat. This disease i s caused by ingestion of foods contaminated with bacteria of the genus Salmonella. In Canada, 164 of the 1440 cases of food-borne outbreaks reported between 1973 and 1975, were associated with cooked poultry (Todd, 1980). The incidence of Salmonella contamination of poultry caracasses i s of major concern. Various studies (Duitschaever, 1977; Wilder and MacCready, 1966) have reported levels as high as 50% f o r market b r o i l e r s . Processing of b r o i l e r s has been reported (Surkiewicz et al. , 1969; Dougherty, 1974; Campbell, 1979; McBride et al., 1980) to be a major factor i n the contamination of poultry meat. The most probable s i t e s of contamination are thought to be the scalding-defeathering, eviscerating and c h i l l i n g operations (Mulder et al., 1978; McBride et al., 1980). In order to decrease Salmonella contamination of poultry carcasses, the mechanisms of invasion and attachment of bacteria to poultry carcasses must be elucidated. 12 The object of the present study was to develop the methodology f o r the examination o f chicken skin by scanning electron microscopy, since the skin microtopography may y i e l d information on the modes of attachment of Salmonella to poultry carcasses. In addition, the attachment of Salmonella typhimurium was studied i n model systems i n an attempt to provide information that can be applied toward the development of methods f o r decreasing Salmonella-contamination on poultry carcasses at the processing l e v e l . /3 LITERATURE REVIEW A. Scanning Electron Microscopy Scanning electron microscopy has been commercially available since 1965, but has found limited application i n food microbiology. However, i t could well provide information about the ecology of food borne microorganisms and the s p a t i a l relationship between the bacteria and i t s substrate (McMeekin et al., 1979). When t h i s research was commenced, no work had been published on the microstructure of chicken skin. But, recently several authors have reported studies on the topography of chicken skin. McMeekin et al. (1979) examined breast skin from poultry carcasses. Two methods of f i x a t i o n were employed: (i) f i x a t i o n by immersion i n glutaraldehyde (4% w/v i n 0.2M phosphate buffer, pH 7.0) f o r 12 h and ( i i ) f i x a t i o n i n osmium tetroxide vapors (1% w/v). Skin samples were then washed i n d i s t i l l e d water, and post-fixed in glutaraldehyde prior to washing i n d i s t i l l e d water. Dehydration with a graded series of ethanol followed. The SEM results indicated that chicken skin has many "crevices" and "channels" of c a p i l l a r y s i z e , i n which bacteria may become trapped. Microorganisms were c l e a r l y v i s i b l e on the skin a f t e r storage of the carcasses f o r 10 days at 5°C. At high magnifications, f i b r i l s connecting individual b a c t e r i a l c e l l s could be seen. The authors also pointed out the importance of using macerated samples, rather than swabs or rinses for accurately determining viable b a c t e r i a l counts. /4 Suderman and Cunningham (1980) studied the effects of age, method of c h i l l i n g , and scald temperatures on the adhesion of coatings to poultry skin. Samples for scanning electron microscopy were prepared by three methods: (i) glutaraldehyde f i x a t i o n , with ethanol dehydration and c r i t i c a l point drying; ( i i ) f i x a t i o n with glutaraldehyde, post-fixation with osmium tetroxide, dehydration with ethanol followed by c r i t i c a l point drying. In both these methods the samples were coated with carbon and then with gold-platinum. The t h i r d method involved freeze-drying. The samples were fixed i n glutaraldehyde, and frozen i n isopentane p r i o r to immersion i n l i q u i d nitrogen and freeze-drying. Their results show that after the c u t i c l e was removed, "numerous protrusions of the epidermis are evident along with microholes, elevations and recessions"'. Comparisons of the three techniques indicated that freeze-drying after a glutaraldehyde f i x a t i o n yielded micrographs with good c l a r i t y and resolution of d e t a i l . Both of the other previously mentioned methods resulted i n electron charging of the sample, a problem frequently encountered with tissues with high fat content. Thomas and McMeekin (1980) used scanning (SEM) and i transmission electron microscopy (TEM) to examine aspects of contamination of b r o i l e r skin by bacteria during processing. Breast and leg skin samples were excised and fixed overnight at 4°C i n either osmium tetroxide vapour or 5% glutaraldehyde and then /5 dehydrated through a graded series of ethanol, i n f i l t r a t e d with amyl acetate and c r i t i c a l point dried. The skin samples were found to be rough and folded as shown by McMeekin et al. (1979). B. Microbiological Sampling of Chicken Carcasses Numerous studies have been published dealing with the spread of contamination on eviscerated and non-eviscerated carcasses during processing. It has been shown (Surkiewicz et al., 1969; Dougherty, 1974; Campbell, 1979; McBride et al., 1980) that poultry carcasses may become contaminated with Salmonella during processing operations whereupon non-infected birds acquire Salmonella organisms from a contaminated environment (Wilder and MacCready, 1966). Dissemination of Salmonella starts on the farm. Bryan et al. (1967) examined the sources of Salmonella contamination of turkey products. Turkeys and farm environments were evaluated, as well as plant surveys on processing equipment and turkey carcasses. They found that feed, feed ingredients, fecal droppings and trough water were sources of Salmonella. The predominant serotype of Salmonella isolated from the plant environment changed as a new flock was introduced. Defeathering machines were found to be a source of i n i t i a l Salmonella transfer from carcass to carcass. Subsequent spray washing did not remove a l l Salmonella, and therefore processing equipment became contaminated. Carcass contact with contaminated equipment caused cross contamination as the next birds were processed. /6 A study by Zottola et al. (1970) indicated that Salmonella i s present i n the "live bird" area of processing plants. Ziegler et al. (1954) found that the area under the wing and that around the vent to be the most heavily contaminated parts of the skin and the v i s c e r a l cavity, respectively. Different areas of the processing plant have been implicated in the dissemination of Salmonella within the poultry plant. Van Schothorst et al. (1972), using E. coli K12 as an indicator organism, investigated the problem of contamination of chickens during defeathering and subsequent cleaning and c h i l l i n g . They found a d e f i n i t e spread of contamination during feather removal, which could be decreased somewhat i n l a t e r stages of processing. Patrick et al. (1973) compared the effectiveness of water -cooling and steam scalding i n decreasing Salmonella contamination. The number of Salmonella contaminated chickens approximately doubled between scalding and defeathering and between defeathering and c h i l l i n g , i n dicating that scalding and defeathering are important vectors for the spread of Salmonella. They also found that water scalded carcasses had a much higher level of contamination than did steam scalded carcasses. Mulder et al. (1978) also studied cross-contamination of birds during the scalding and plucking operations. They contaminated poultry carcasses, both i n t e r n a l l y and externally with E. coli K12, resistant to n a l i d i x i c acid concentrations up to 200 mg/L. Their results indicated that the number of E. coli K12 on externally contaminated b r o i l e r s decreased 1000-fold during scalding. This decrease was not only due to the high temperatures used i n scalding, but also to the washing effect which occurs during scalding. More cross-contamination during scalding at temperatures between 52-54°C was noted than when a higher scald temperature (60°C) was used. In contrast, cross-contamination during plucking was s l i g h t after internal contamination (Mulder et al., 1978). Wilder and MacCready (1966) found that Salmonella was distributed throughout the poultry plant environment and reached t h e i r highest levels i n areas maintained at low sanitary conditions, and in those areas where the poultry undergoes extensive human handling. Surkiewicz et al. (1969) found that eviscerated chickens 4 2 2 had aerobic plate.counts of 1.5x10 /cm , s i x E. coZ^/cm , and three 2 S. aureus/cm . Salmonella were found to be present on 20.5% of the birds sampled. Passage through the continuous counterflow c h i l l e r diminished the t o t a l b a c t e r i a l load but did not s i g n i f i c a n t l y reduce the incidence of Salmonella contamination. Finlayson (1977), i n a survey of Alberta poultry processing plants, found that salmonellae were common in breeding flocks and in processing plants. The most frequent sources of Salmonella i n the plant were the defeathering area and the drains. The c h i l l i n g operation i s also very important i n the cross-contamination of poultry. Numerous studies have focused on t h i s stage of poultry processing. Peric et al. (1971) showed that spin /8 c h i l l i n g i n i t i a l l y caused a decline i n the surface b a c t e r i a l counts of b r o i l e r s early i n the day, but as the day progressed, an increase in b acterial counts was noticed. The time that the increase occurred was found to be dependent upon the ba c t e r i a l load of the carcasses before spin c h i l l i n g , the number of carcasses processed and the amount of water used i n the c h i l l e r . They also found that spray cooling was more effec t i v e than spin c h i l l i n g . Mead and Thomas (1973a) found that chlorine i n the c h i l l water destroyed v i r t u a l l y a l l the bacteria present i n the c h i l l water and therefore aided i n the prevention of cross-contamination during the cooling operation. The majority of a l l bacteria were destroyed by the use of 45 to 50 ppm t o t a l chlorine i n the c h i l l water at a volume rate of 5 L of water per carcass. I f the water rate was increased to 8 L of water per carcass, 25 to 30 ppm residual chlorine was s u f f i c i e n t . In a companion paper, Mead and Thomas (1973b) studied the effects of a three-stage c h i l l e r . Total viable counts at 20 and 37°C, as well as the levels of Coli-aerogenes bacteria were decreased by one log cycle. They concluded that the main effect of the chlorine in the water was to inactivate the organisms that were washed from the carcasses, thereby avoiding recontamination. Notermans et al. (1973), using a n a l i d i x i c resistant s t r a i n of E. coli K12, found that cross-contamination can occur either i n the spin c h i l l e r or i n spray cooling. However, the p o s s i b i l i t y of /9 cross-contamination was higher i n the spin c h i l l e r , than i n the spray cooler. McBride et al. (1980) studied the incidence of Salmonella at three stages of processing: (i) before scalding, ( i i ) after evisceration and ( i i i ) after c h i l l i n g . The average incidence of Salmonella was found to be between 1.2 and 74.4%, and flocks with a high incidence of Salmonella before scalding were s t i l l contaminated after c h i l l i n g . They concluded that i t i s possible to predict the incidence of Salmonella at one s i t e from the incidence at another s i t e i n the processing operation. C. Enumerating Bacteria on Poultry Skin There are several methods for determining the numbers of bacteria on poultry meat and carcasses. Walker and Ayres (1956) used 2 cotton swabs to sample 2 cm of both internal and external surfaces. The area was delineated by s t e r i l e metal guides. Fromm (1959) quantitatively compared four methods: direct contact p l a t i n g , swab sampling, rinse sampling and skin tissue removal. His results indicated that skin tissue removal was the most accurate. The major disadvantage of t h i s technique i s that i t lowers the carcass grade and therefore may not be suitable for routine quality control analysis. It may be better in such cases to employ swab techniques. Clark (1965) used a non-destructive method, i n which a known area of skin was sprayed with a f l u i d under constant pressure. The bacteria were recovered from a measured volume of the f l u i d . no Avens and M i l l e r (1970) compared a skin "blending" technique to the cotton swab sampling method on turkey carcass skin. The blending technique involved the use of a laboratory blender to release the bacteria from skin samples suspended i n 0.1% peptone broth or physiological saline as a diluent. The skin blending method permitted enumeration of s i g n i f i c a n t l y more bacteria than did the swab method. The swab method was found to y i e l d incomplete and inconsistent r e s u l t s . Thomson et al. (1976) compared the use of a l u c i t e template anchored to the poultry skin by means of stainless steel pins,to a fiberboard template. The l u c i t e template prevented skin slippage but the bacte r i a l counts did not d i f f e r s i g n i f i c a n t l y from those obtained using the fiberboard template. D. Bacterial Adhesion Notermans and Kampelmacher (1974), concluded that attachment of bacteria to poultry skin was largely dependent on the presence of f l a g e l l a on the bacteria. Studies using a non-flagellated E. coli K12 97+ mutant indicated minimal attachment compared to that of the f l a g e l l a t e d E. coli K12. They also noted that pH and temperature were important parameters. By lowering the pH of the attachment medium, the attachment rate was decreased due to the decreased m o t i l i t y of the organisms. The optimal temperature for the attachment of the various bac t e r i a l strains studied was 21°C. Increasing / I I the temperature from 0° to 21°C was found to increase the attachment rate. This seemed to be due to the increased metabolic a c t i v i t y of the organism and the f l a g e l l a (Notermans and Kampelmacher, 1974). Notermans and Kampelmacher (1975a) also found that a proportion of the b a c t e r i a l f l o r a of skin was present i n the surrounding water f i l m and could be removed by adequate r i n s i n g . The remainder of the f l o r a are very d i f f i c u l t to remove, even with mechanical cleaning, such as spin c h i l l i n g . The bacteria present i n the water f i l m played a key role i n the attachment of bacteria to the skin. The attachment was time dependent and proportional to the number of bacteria present (Notermans and Kampelmacher, 1975a). In contrast, McMeekin et al. (1978) found that m o t i l i t y had a n e g l i g i b l e effect on the number of organisms retained on the skin. They observed that there was no preferential accumulation of motile bacteria on the skin surfaces. The time of immersion was also of minor importance when compared to the effect of population densities on retention. Bacterial attachment seems to take place i n two separate stages. Marshall et al. (1971), i n t h e i r work with marine bacteria, separated the attachment process into an instantaneous phase and a time-dependent i r r e v e r s i b l e phase. In the f i r s t phase, the bacteria were attracted to a surface and then weakly held by a balancing of London - van der Waals a t t r a c t i v e forces and the e l e c t r i c a l repulsive charges of two surfaces, or a gain i n entropy. In the second phase, /12 the bacteria became i r r e v e r s i b l y attached by the formation of polymeric bridges between the b a c t e r i a and the surface. This "polymeria bridging" was v i s u a l i z e d by Fletcher and Floodgate (1973). Transmission electron microscopy, a f t e r the samples were stained with ruthenium red and a l c i a n blue, demonstrated the presence of an e x t r a c e l l u l a r polysaccharide layer which was involved i n the adhesion o f marine b a c t e r i a to surfaces. This adhesive substance was found to be present before attachment, but a secondary fibrous a c i d i c polysaccharide was produced once natural attachment had occurred. McCowan et al. (1978) used both SEM and TEM to investigate the mechanisms of attachment of b a c t e r i a to the reticulo-rumen of c a t t l e . Bacteria could avoid being washed out of the rumen by adhering to the mucosa. TEM of the ruthenium red stained samples showed that the attachment was mediated by glycocalyx and carbohydrate coat of the b a c t e r i a . The glycocalyx aided i n the attachment of b a c t e r i a to epithelium, food p a r t i c l e s and to other b a c t e r i a . Intermittent c o l o n i z a t i o n of the e p i t h e l i a was observed, as well as microcolonies formed by groups of b a c t e r i a . Costerton et al. (1978) attested that b a c t e r i a l adhesion was mediated by the formation of glycocalyx. It i s t h i s "mass of tangled fibers of polysaccharide" which help the b a c t e r i a survive in a competitive environment. The glycocalyx seems to serve many purposes. It not only p o s i t i o n s the b a c t e r i a , but may conserve and /13 concentrate the digestive enzymes released by the bacteria against the host c e l l . I t may function as a food reservoir and protect the organism against predatory bacteria and b a c t e r i a l viruses. This leads to the concept that i f adhesion of bacteria can be prevented, perhaps bact e r i a l invasion of poultry skin could also be prevented. Costerton et al. (1978) suggested three methods of achieving t h i s ; f i r s t l y , by disrupting glycocalyx synthesis; secondly, by preventing attachment of glycocalyx to the bacteria or t h i r d l y , by preventing attachment of glycocalyx to the host. Notermans et al. (1979) examined the attachment of bacteria to cows' teats. They studied the attachment at different storage temperatures and found results s i m i l a r to t h e i r e a r l i e r work on poultry skin (Notermans and Kampelmacher, 1974). After the i n i t i a l attachment, the strength of adhesion increased. This increase was faster at higher storage temperatures, perhaps due to an increase i n b a c t e r i a l metabolism and a subsequent increase i n the formation of glycocalyx. After longer periods, the strength of adhesion decreased, due to the formation of colonies. As the bact e r i a l numbers increase, more are attached to each other and not to the surface, and therefore can be e a s i l y removed. This was further demonstrated by Firstenberg-Eden et al. (1979). Scanning electron miscroscopy showed that during storage, polymers i n the form of thi n f i b e r s , were produced. These fibe r s thickened to form slime. This work supported the observations of Notermans et al. (1979) and showed the existence of microcolonies of bacteria. /14 Butler et al. (1979) examined the attachment of micro-organisms to pork skin and to the surfaces of beef and lamb carcasses. The authors developed a model system in which the samples were embedded in solidified wax with the skin surfaces exposed. The wax cubes were then dipped into attachment media. They reported that different organisms have different attachment rates. Motile gram-negative organisms such as E. coli, P. putrefaciens, E. herbicola exhibited greater attachment than did non-motile gram-positive organisms such as Lactobacillus and S. aureus. There is also a direct relationship between the bacterial counts on the skin and the concentration of the bacteria in the attachment medium which is in agreement with the work of Notermans and Kampelmacher (1974) and Notermans et al. (1975b). The effects of temperature and pH were not significant. These discrepancies could be due to the fact that Butler et al. (1979) used pork skin whereas Notermans and Kampelmacher (1974) and Notermans et al. (1975) used chicken skin. E. Inoculation of Chicken Skin Several authors have studied model systems for the intro-duction of bacteria to food myosystems. Clark (1965) developed a method of using a spray gun to uniformly inoculate nutrient surfaces with a bacterial suspension. The uniformity of inoculation was shown to be affected by the ionic strength of the bacterial suspension, the distance between the spray nozzle and the sample surface, the rate of air flow, and the exposure /15 time. It was reported that the use of ionic strengths less than 0.25 resulted i n uneven inoculation and a 25-50% decrease of bacterial numbers when compared to ionic strengths between 0.25 and 1.5. A minimum exposure time of 30 sec was reported to be required for obtaining a consistent, uniform inoculation on a l l s i x plates. The density of the inoculum could be s a t i s f a c t o r i l y controlled by the exposure time and the concentration of the inoculating suspension. Notermans and Kampelmacher (1974) employed the use of a physiological solution (NaCl, 8.7 g/L) containing phosphate buffer (pH'7.2) and EDTA. They immersed whole chicken carcasses into 25 L baths of attachment medium inoculated with a known number of bacteria. Butler et al. (1979) embedded t h e i r samples i n l i q u i d wax with a piece of s t e r i l e s t r i n g . This sample was then immersed i n 180 mL of s t e r i l e attachment medium simi l a r to that used by Notermans and Kampelmacher (1974). Barrow et al. (1980) used a quite different approach. E p i t h e l i a l c e l l s were obtained from the gut of a 4 week old pig and suspended i n phosphate buffered saline. Equal volumes of bacter i a l and e p i t h e l i a l c e l l suspensions were mixed and incubated on a rotating platform at 37°C for 30 min. The number of attached •microorganisms per 20 e p i t h e l i a l c e l l s was counted by phase contrast microscopy. /16 MATERIALS AND METHODS Chicken carcasses (42 to 45 day old broi l e r s ) were obtained from a l o c a l poultry processing plant, placed i n s t e r i l e poly-ethylene bags (1 Mrad y i r r a d i a t i o n , Gammacell 200 (Atomic Energy of Canada Ltd.)) a n a transported on ice to the laboratory. A. Scanning Electron Microscopy Three methods of preparation of chicken skin for scanning electron microscopy (SEM) were employed. They were ( i ) standard chemical f i x a t i o n ; ( i i ) thiocarbohydrazide f i x a t i o n (TCH) and ( i i i ) freeze-drying. In addition, two methods of dehydration were compared: standard ethanol dehydration with amyl acetate i n f i l t r a t i o n and chemical dehydration using 2,2-dimethoxypropane. In a l l the f i x a t i o n procedures, the chicken skin was f i r s t excised into 6 cm x 6 cm pieces. Large pieces were cut since curling occurred during f i x a t i o n . The skin samples contracted i n a l l directions and had to be kept f l a t with forceps u n t i l immersed i n f i x a t i v e . 1. Standard chemical fixation The samples were fixed i n 6.3% (v/v) electron microscopic grade glutaraldehyde (CAN-EM Chemicals, Guelph, Ont; Marivac, Halifax, N.S.) i n Millonig's phosphate buffer at 4°C overnight (Dawes, 1971). /17 After f i x a t i o n i n glutaraldehyde the tissue was washed three times i n Millonig's phosphate buffer p r i o r to post-fixation i n 1% (w/v) osmium tetroxide (CAN-EM Chemicals, Guelph, Ont; Marivac, Halifax, N.-X.) i n Millonig's buffer for 1 h. Samples were then rinsed three times i n Millonig's buffer p r i o r to dehydration and c r i t i c a l point drying (Figure 1). 2. Standard dehydration The samples were dehydrated through an ascending series of ethanol: 50%, 70%, and 80% (v/v) ethanol for 5 min each, two changes of 90% ethanol for 10 min each and f i n a l l y three changes of 100% ethanol for 20 min each. A l l ethanol dilutions were made with d i s t i l l e d deionized water. This was followed by amyl acetate (Fisher S c i e n t i f i c Co., Fairlawn, NJ) i n f i l t r a t i o n . A graded series of amyl acetate solutions i n 100% ethanol was used: one change of 10 min duration each i n 25%, 50% and 75% amyl acetate and f i n a l l y 100% amyl acetate for 1 h (Figure 2). S. Chemical Dehydration Samples were dehydrated using a c i d i f i e d 2,2-dimethoxy-propane (2,2 DMP) following the method of Maser and Trimble (1976). 2,2 DMP reacts with water to form methanol and acetone. Two changes of 15 min duration of 2,2 DMP were used (Figure 2). Samples were c r i t i c a l point dried i n a Parr bomb (Parr Instruments Co., Moline, IL) and mounted on aluminum stubs with /18 S A M P L E F I X I N G L U T A R A L D E H Y D E I W A S H I N B U F F E R \ F I X I N O s O , • W A S H I N B U F F E R I D E H Y D R A T E C R I T I C A L P O I N T D R Y I G O L D C O A T E X A M I N E FIGURE 1: Flow sheet o f the standard chemical f i x a t i o n procedure. /19 S A M P L E F I X A T I O N D E H Y D R A T I O N I N A L C O H O L j D E H Y D R A T I O N I N | 2,2 - D M P ( a c i d i f i e d ) A M Y L A C E T A T E I N F I L T R A T I O N C R I T I C A L P O I N T D R Y I N G FIGURE 2: Flow sheet of the dehydration procedures. /20 epoxy cement and s i l v e r paste (Structure Probe Inc., West Chester, PA). Samples were then coated with gold i n a sputter coater (Technics Inc., Alexandria, VA) and examined with a Hitachi S-500 Scanning Electron Microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 20 kV. Images were recorded on I l f o r d Pan F 135 fine grain black and white f i l m ( I l f o r d Ltd., Essex, England). 4. Thiocharbohydrazide fixation Sample preparation was performed according to the method of Malik and Wilson (1975). The chicken skin was fixed i n 6.3% (v/v) glutaraldehyde i n Millonig's phosphate buffer (as described previously) and washed three times i n the phosphate buffer. Following post-fixation i n 1% oxmium tetroxide for 3 h, samples were rinsed s i x times with d i s t i l l e d deionized water. The samples were then placed i n 1% aqueous solution of thiocarbohydrazide (TCH, Eastman Chemicals, Rochester, NY) for 30 min, after which they were again rinsed s i x times with d i s t i l l e d deionized water. Another f i x a t i o n with 1% osmium tetroxide for 2 h followed. This step was then repeated, giving a t o t a l of three fixat i o n s i n osmium tetroxide and two treatments with TCH (Figure 3). 5. Freeze-drying I n i t i a l l y , samples of chicken skin were dipped into l i q u i d N^ and then freeze-dried. This method was then modified such that the samples were dipped i n isopentane (Fisher S c i e n t i f i c , S A M P L E I F I X I N G L U T A R A L D E H Y D E W A S H I N B U F F E R • F I X I N O s 0 4 W A S H I N B U F F E R • T R E A T W I T H T C H - 1 I W A S H I N B U F F E R ^ r e p e a t F I X I N O s 0 4 \ W A S H I N B U F F E R » • D E H Y D R A T E I C R I T I C A L P O I N T D R Y \ E X A M I N E FIGURE 3: Flow sheet of the thiocarbohydrazide fixation procedure. 122 Co., Fairlawn, NJ) which was cooled i n a l i q u i d bath. This method did not y i e l d satisfactory results either, so another modification was necessary. The samples were placed i n wells of an aluminum block (Figure 4). The block was cooled i n l i q u i d N 2 > 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 • 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°C 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°C 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°C, 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°C for 4 h. The solution was stored under N^, at 4°C, 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°C and ending at 200°C, increasing by 4°/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°C. 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°C. 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°C 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„HP0., 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°C and the nutrient agar plates were incubated for 48 h at 35°C. 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 . [•' 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 '•I 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 • 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 ± + 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° 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* ± 6. 54** a 27.13 ± 6 .54 a 1 II 35.32 ± 2. 28 b 53.06 ± 3 .91 b I 52.91 ± 10. 32 a 22.79 ± 5 . 2 l a 2 II 31.86 ± 5. 58 b 59.67 ± 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 • • <A 8 10 0 4 6 TIME (min) (b) 8 10 FIGURE 25: F a t t y a c i d p r o f i l e o f (a) Type I and (b) Type I I s k i n from chicken l e g s . FIGURE 26: F a t t y a c i d p r o f i l e of (a) Type I and (b) Type II s k i n from chicken b r e a s t s . 16 0 T IME (min) (a) 18 I i6o n 0 2 4 6 8 1 0 TIME (min) (b) FIGURE 27: Fatty ac i d p r o f i l e of (a) Type I and (b) Type II skin from chicken backs. TABLE IV: Fatty acid composition of Type I and Type II chicken skin * before scalding (Site A). Leg Fatty acid composition, % Location Skin Type C < 1 4 CU.Q C 1 4 ; 1 C16_Q C 1 6 : 1 C l g. 0 C l g : 1 C l g : 2 C l g. 3 I 0.173**a 0.706b 0.304a 22.09C 10.14a 4.54a 45.09C 14.14° 2.00d II 0.087 0.710 0.272 23.16 9.52 4.56 45.23 14.46 1.98 Breast I 0.302 0.831 0.314 23.14 10.86 4.00 44.19 14.06 1.85 II 0.429 0.770 0.303 22.55 11.00 4.07 45.05 14.32 2.01 Back I 0.177 0.727 0.262 23.70 9.59 4.85 44.86 13.97 1.91 II 0.091 0.738 0.245 22.95 10.43 4.68 45.51 14.06 1.86 means of quintuplicate samples done i n duplicate, row totals may not add up to exactly 100%, since the values are means. ** c o e f f i c i e n t s of var i a t i o n for means in the same column range from a) 0.049-0.081; b)0.016-0.054; c) 0.009- 0.029 and d) 0.010-0.058 TABLE V: Fatty acid composition of Type I and Type II chicken ski after immersion c h i l l i n g (Site C). Fatty acid composition, % Location Skin Type Leg Breast Back II II II J<14 '14:0 14:1 16:0 16:1 0.282**a 0.7353 0.275a 22.33[ 9.79 C18:0 C 1 8 : l C18:2 C18:3 4.52a 46.60d 13.68C 2.88 0.040 0.297 0.109 0.159 0.105 0.609 0.264 22.91 10.26 4.39 46.80 12.86 1.84 0.707 0.624 0.897 0.637 0.269 0.265 0.276 0.267 22.78 22.43 23.10 23.03 10.99 11.27 10.38 10.24 4.22 4.17 4.39 4.48 45.76 46.34 45.77 46.35 13.19 12.99 13.03 13.05 1.80 1.81 1.86 1.78 ** means of quintuplicate samples done i n duplicate, row tot a l s may not add up to exactly 100% since the values are means coefficients of va r i a t i o n for means i n the same column range from a) 0.022-0.083; b) 0.018-0.072; c) 0.063-0.101 and d) 0.0019- 0.031 /69 have a higher proportion of short chain f a t t y acid and unsaturated fat t y acids. This was not indicated i n the fatt y acid p r o f i l e (Tables IV and V). Other considerations such as glyceride composition and skin permeability may be determinant factors. D. Microbiological Sampling Due to the large number of di l u t i o n s and samples required in some of the l a t e r experiments, the f e a s i b i l i t y of using the drop plate technique (ICMSF, 1978) was evaluated. I t saves time i n both preparation of plates and i n the actual plating. Six plates can be replaced by one plate having three sectors (Figure 8). T r i a l s were conducted i n order to determine whether the modified drop plate technique was reproducible, since 0.1 ml pipettes calibrated to 0.01 ml were used rather than special c a p i l l a r y tubes calibrated to 0.02 ml. The drop plate technique was found to give reproducible results which compare favorably with those obtained by a more conventional method of plating (Table VI). E. Bacterial Load The bacte r i a l load present on chicken legs at the processing plant was evaluated. Chicken legs were taken after defeathering (Site B) and after immersion c h i l l i n g (Site C). They were examined for viable bacteria present on both Type I and Type II skin (Figures 28, 29). TABLE VI: Comparison of the drop plate technique to a more conventional method of p l a t i n g . concentration o^ S. typhimuvivm culture (cfu/cm ) T r i a l Standard method Drop plate technique 1 2.6 A ± 0.2 x 1 0 8 B Q 2.6 ± 0.2 x 10 2 4.7 ± 0.1 x 1 0 5 4.7 ± 0.1 x 1 0 5 3 7.5 ± 0.1 x 1 0 3 7.5 ± 0.1 x 1 0 3 mean of t r i p l i c a t e samples plated i n duplicate standard deviation FIGURE 28: B a c t e r i a l load on chicken carcasses measured (a) a f t e r defeathering and (b) a f t e r immersion c h i l l i n g . T r i a l 1. Aerobic ( wmm ) and psychrotrophic ( i / v x i ) b a c t e r i a were measured i n duplicate. Ill 10< 1 I I j II j II 0 0 J J ll_ SKIN TYPE _A B _ LEG 6 CHICKEN Ill FIGURE 29: B a c t e r i a l load on chicken carcasses measured((a) a f t e r defeathering and (b) a f t e r immersion c h i l l i n g . T r i a l 2. Aerobic ( mm ) and psychrotrophic ( E E S ) b a c t e r i a were measured i n duplicate. /74 TO J 310'' u -O I s s s i \ \ S \ I ioJ S K I N TYPE L E G C H I C K E N 10 2 io3J u 10' TO S §R s $R S $R 8 $R $ $ R S l U J •! I s ip S R K S Is IS J H J M J H I H * B_ J_ B_ 6 7 (b) SR j u_ i A 8 S K I N TYPE L E G C H I C K E N /75 There i s considerable variation i n the b a c t e r i a l population between individual chickens as well as between legs from the same chicken. There are also differences i n b a c t e r i a l counts on Type I and Type II chicken skin from the same leg. In general, Type II skin has a higher b a c t e r i a l load than does Type I skin. The b a c t e r i a l population seems to decrease ten f o l d between defeathering and the c h i l l tank. The washing action of the c h i l l e r s presumably reduced the number of coliforms, E. coin., S. aureus, and Salmonella and lowered the aerobic plate count (Surkiewicz et al., 1969). Psychrotrophic bacteria represent a greater proportion of the t o t a l b a c t e r i a l population after c h i l l i n g (Site C) than after defeathering (Site B). This i s probably due to cross-contamination at the c h i l l tank (Surkiewicz et al., 1969). Previous studies (Barnes, 1960; Mead and Impey, 1970; Mead and Thomas, 1973b; van Schothorst et al., 1972; Notermans et al., 1973 and McBride et al., 1980) have demonstrated that the numbers of bacteria on carcass surfaces vary considerably at different stages of processing and both increases and decreases i n numbers of bacteria may occur. F. Attachment Studies 1. Bacteriological analysis Inoculation of chicken legs was carried out by immersion g i n an attachment medium containing approximately 10 Salmonella /76 typhimurium/mL. After 15 min immersion i n the attachment medium, the legs were removed and allowed to hang for the desired length of time. At each sampling period, two legs were removed for each test procedure.. The control samples, which were dipped i n s t e r i l e attachment buffer, showed ba c t e r i a l counts between 0 and 600 c e l l 2 forming units (cfu)/cm . This indicates the extreme v a r i a b i l i t y of the autochthonous population on chicken leg skin. This, i n i t s e l f , leads to problems i n interpreting the data. Enumeration of the inoculated samples at time 0 4 5 (Figures 30 and 31) shows attachment i n the range of 10 to 10 2 5 6 2 cfu/cm for Type I skin and 10 to 10 cfu/cm for Type II skin. As expected, this i s lower than the concentration of Salmonella typhimurium i n the attachment medium. Similar results have been reported by Notermans and Kampelmacher (1974) and McMeekin and Thomas (1978). These bacteria are firmly attached almost immediately and cannot be ea s i l y removed by washing. Notermans and Kampelmacher (1975a) proposed the existence of a "water film" on the surface of chicken skin. Bacteria are present i n t h i s water f i l m before physical attachment occurs. These bacteria were thought to be ea s i l y removable by replacing the water f i l m with fresh uncontaminated water. Their results showed that a three 3 minute wash removed approximately 2x10 organisms per gram, regard-less of the number of bacteria present i n or on the skin. The Ill FIGURE 30; B a c t e r i a l population on inoculated, washed and surfactant treated chicken legs. T r i a l 1. Inoculated ( » , 0 ), washed (•, • ), and surfactant treated ( A , A ). Closed symbols indic a t e geometric means of two samples, each done i n duplicate. Open symbols indi c a t e range. Control samples had values between 0 and 965 cfu/cm^. /78 /79 FIGURE 31: Bacterial population on inoculated, washed and surfactant treated chicken legs. T r i a l 2. Inoculated (•, o ), washed ( •, • ) and surfactant treated ( A , A ). Closed symbols indicate geometric means of two samples, each done i n duplicate. Open symbols indicate range. Control samples had values between 0 and 579 cfu/cm2. j " " / n o /81 remainder of the bacteria were considered "attached". The population of attached bacteria could be decreased by a maximum of one log unit with either immersion c h i l l i n g or spray cooling. Notermans and Kampelmacher (1975a) believe that the b a c t e r i a l f l o r a i n the water f i l m i s of great importance with regard to the number of bacteria i n or on the skin, since the water f i l m appears to play a key role as far as the "attachment" of bacteria to the skin i s concerned. I f Notermans and Kampelmacher's hypothesis was to hold true, one would expect a dramatic decrease i n the bacterial counts on the samples washed i n buffer containing surfactant. The surfactant would act to reduce the surface tension, and the bacteria present i n the water layer should be e a s i l y washed away The data presented i n Figures 30 and 31, however, does not support t h i s theory. S t a t i s t i c a l analysis (Tables VII, V I I I , IX) indicates that the two t r i a l s are not exact replicates. This i s probably due to the different chickens used i n each t r i a l , d i f f e r i n g c e l l densities i n each of the two t r i a l s , and a host of other factors. E a r l i e r data showed that the autochthonous population on chickens varies greatly. T r i a l 1 (Table VII) indicates that treatment, time, sk type, and t h e i r interactions are s i g n i f i c a n t (p < 0.01). The reason f o r the large F-value for treatments i s the large difference between the counts for the control and the other treatments. /82 TABLE VII: Analysis of variance f o r the attachment study. T r i a l 1. Source DF Sum Sq F-Value Prob Treatment Time Skin type Treatment x Time Treatment x Skin type Time x Skin type Treatment x Time x Skin type Error 3 3 1 9 3 3 9 96 427.65 1.86 11.43 5.76 2.78 2.36 3.51 11.45 1222.8 5.20 95.81 5.36 7.76 6.61 3.27 0.694E-16 0.227E-02 0.805E-15 0.623E-05 0.108E-03 0.417E-03 0.166E-02 TOTAL 127 476.81 /83 TABLE VIII: Analysis of variance for the attachment study. T r i a l 2. Source DF Sum Sq F-Value Prob Treatment 3 173.52 166.39 0.139E-15 Time 3 0.90 0.86 0.462 Skin type 1 6.07 17.42 0.641E-04 Treatment x Time 9 8.11 2.59 0.101E-01 Treatment x Skin type 3 1.90 1.83 0.148 Time x Skin type 3 1.22 1.17 0.326 Treatment x Time x Skin type 9 3.84 1.23 0.287 Error 96 33.37 TOTAL 127 228.94 /84 TABLE IX: Analysis of variance for the combined t r i a l s of the attachment study. Source DF Sum Sq F-Value Prob T r i a l Treatment Time Skin type Tr i a l x Treatment Trial x Time Tri a l x Skin type Treatment x Time Treatment x Skin type Time x Skin type Tr i a l x Treatment x Time Trial x Treatment x Skin type Trial x Time x Skin type Treatment x Time x Skin type Error 1 3 3 1 3 3 1 9 3 3 9 201 2.95 556.66 4.76 15.52 24.95 11.12 0.05 9.64 2.11 0.74 9.33 0.42 0.88 1.41 62.16 9.539 600.03 5.131 50.20 26.892 1.206 0.171 3.463 2.277 0.794 3.351 0.545 0.951 0.506 0.229E-02 0.422E-70 0.194E-02 0.252E-10 0.157E-13 0.309 0.680 0.548E-03 0.809E-01 0.498 0.773E-03 0.715 0.417 0.870 TOTAL 255 692.69 /85 T r i a l 2 (Table VIII) indicates that only treatment and skin type are s i g n i f i c a n t (p < 0.01). Using Neuman-Keuls1 multiple range t e s t , the treatments can be divided into three groups. The f i r s t includes the control samples; the second, inoculated samples; and the t h i r d , both washed and surfactant treated samples. Skin types are 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), with Type II having more b a c t e r i a attached. When the data from both t r i a l s are combined (Table IX), i t i s evident that the t r i a l s are s i g n i f i c a n t l y d i f f e r e n t , as are treatment, time, and skin type. Several in t e r a c t i o n s are also important. These are the trial-treatment i n t e r a c t i o n , treatment-time i n t e r a c t i o n , and the combined i n t e r a c t i o n between t r i a l -treatment -time. Since the sample s i z e i s small, and the b a c t e r i a l counts may not represent a normally-distributed population, the n u l l hypothesis that the parameters studied are the same, may be in c o r r e c t . Therefore these r e s u l t s cannot be taken as conclusive. However, i n both of the t r i a l s , treatment with wash water, with and without the i n c l u s i o n of a surfactant show no s i g n i f i c a n t d i f f e r e n c e . In a d d i t i o n , Type II skin had s i g n i f i c a n t l y higher b a c t e r i a l counts than d i d Type I skin. In order to examine the data and separate out the i n t e r a c t i o n e f f e c t s , i t i s possible to estimate the main e f f e c t s . In Tables X, XI, XII, the i n t e r a c t i o n s are examined, and the /86 TABLE X: Estimates of main e f f e c t and i n t e r a c t i o n means for t r i a l and treatment from the analysis of variance. Interaction means (cfu/cm^") T r i a l Control Inoculated Washed Surfactant 1 1.42X101 4.36xl0 5 2.05xl0 5 1.73xl0 5 2 5.12xl0 2 3.23xl0 5 1.63xl0 5 1.40xl0 5 T r i a l means 2.17x10 4.14xl0 4 Treatment , r c 5 , ^ 4* means 8.52X101 3.75xl0 5 1.72x10^ 1.55x10 3.09x10 grand mean /87 TABLE XI: Estimates of main e f f e c t and i n t e r a c t i o n means f o r time and treatment from the analysis of variance. Interaction means (cfu/cm ) Time means Time Control Inoculated Washed Surfactant 0 3. .08X101 2. 78xl0 5 2.39x10 5 1 .13xlO S 2. 19xl0 4 5 1. ,64xl0 2 5. ,82xl0 5 1.68xl0 5 1 .69xlO S 4. 05xl0 4 10 2. ,27xl0 2 3. 05xl0 5 1.37xl0 5 1 .37xlO S 3. 34x10 4 15 4. .60X101 4. 03xl0 5 2.10xl0 5 2 .14xlO S 2. 98xl0 4 Treatment means 8. .52X101 3. ,75xl0 5 1.84xl0 5 1 .55xlO S 3. 09x10 4 * grand mean /88 TABLE XII: Estimates of main e f f e c t and i n t e r a c t i o n means for treatment and skin type from the analysis of variance. 2 Interaction means (cfu/cm ) Skin type Skin type Control Inoculated Washed Surfactant means I 2.47X101 2.39xl0 5 1.21xl0 5 1.06xl0 5 1.66xl0 4 II 2.93xl0 2 5.90xl0 5 2.79xl0 5 2.28xl0 5 5.76xl0 4 Treatment ^ means 8.52x10 3.75xl0 5 1.84xl0 5 1.55xl0 5 4* 3.09x10 * grand mean /89 estimated means due to each f a c t o r are presented. The control samples have the greatest deviation from the o v e r a l l mean. Inoculation r a i s e s the b a c t e r i a l counts to 3.72x10'' from the control l e v e l of 8.5x10^. Washing with water decreases the b a c t e r i a l numbers to 1.72x10^ and the i n c l u s i o n of Tween 80 further reduces the b a c t e r i a l counts to 1.55x10^. There i s no s i g n i f i c a n t d i f f e r e n c e (p > 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°C, 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. American Type Culture Collection, Rockville, Maryland. Association of O f f i c i a l Analytical Chemists. 1975. Methods of Analysis. Twelfth edition. Horowitz, W., Senzel, A., Reynolds, H. and Park, D.L. eds. p. 927. Association of O f f i c i a l A nalytical Chemists, Washington, D.C. Avens, J.S. and M i l l e r , B.F. 1970. Quantifying bacteria on poultry carcass skin. Poultry S c i . 49: 1309. Babayan, V.K. 1974. Fats and Oils in Encyclopedia of Food Technology. Johnson, A.A. and Peterson, M.S. eds. AVI Publishing, Westport, Connecticut, Barnes, E.M. 1960. Bacteriological problems i n b r o i l e r preparation and storage. Roy. Soc. Health. 3: 145. Barrow, P.A., Brooker, B.E., F u l l e r , R. and Newport, M.J. 1980. The attachment of bacteria to the gastric epithelium of the pig and i t s importance i n the microecology of the intestine. J. Appl. B a c t e r i o l . 48: 147. Bryan, F.L., Ayers, J.C. and Kraft, A.A. 1967. Contributory sources of Salmonellae on turkey products. Amer. J . Epidemiol. 87: 578. Butler, J.L., Steward, J.C.,Vanderzant, C , Carpenter, Z.L. and Smith, G.C. 1979. Attachment of microorganisms to /105 pork skin and surfaces of beef and lamb carcasses. J. Food Prot. 42: 401. Campbell, K.A. 1979. The incidence of Salmonella i n chicken flocks undergoing processing at a commercial poultry processing plant. B.Sc. (Agr.) thesis i n Food Science, University of B r i t i s h Columbia. Clark, D.S. 1965. Improvement of the spray gun method of estimating bact e r i a l populations on surfaces. Can. J . Microbiol. 11: 1021. Coleman. CH. 1968. Calculations Used In Food Analysis. Defense Subsistence Testing Laboratory, Chicago, I l l i n o i s . Concon, J.M. and Soltess, D. 1973. Rapid micro Kjeldahl digestion of cereal grains and other b i o l o g i c a l materials. Anal. Biochem. 53: 35. Costerton, J.W., Geesy, G.C. and Cheng, K.-J. 1978. How bacteria s t i c k . S c i . Amer. 238: 86. Dawes, C.J. 1971. Biological Techniques in Electron Microscopy. Barnes and Noble Inc., New York. Dubois, M., G i l l e s , K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem 28: 350. Duitschaever, C.L. 1977. Incidence of Salmonella i n r e t a i l e d raw cut-up chicken. J . Food Prot. 40: 191. Dougherty, T.J. 1974. Salmonella contamination i n a commercial poultry (broiler) processing operation. Poultry S c i . 53: 814. /106 Edwards, H.M., Denman, F., Abou-Ashour, A. and Nigara. 1973. Carcass composition studies 1. Influence of age, sex and type of dietary fat supplementation on total carcass and fatty acid composition. Poultry Sci. 52: 934. Emswiler, B.S., Pierson, C.J. and Kotula, A.W. 1977. Stomaching vs blending. Food Technol. 31: 40. Finlayson, M. 1977. Salmonella in Alberta poultry products and their significance in human infections. In Proceedings of the International Symposium on Salmonella and prospects for control. Barnum, D.A. ed. p. 156. University of Guelph. Firstenberg-Eden, R., Notermans, S., Theil, F., Henstra, S. and Kampelmacher, E.H. 19791 Scanning electron microscopic investigations into attachment of bacteria to teats of cows. J. Food. Prot. 42: 305. Fletcher, M. and Floodgate, G.D. 1973. An electron-microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74: 325. Fletcher, D.L. and Thomason, D.M. 1980. The influence of environ-mental and processing conditions on the physical carcass quality factors associated with the oily bird syndrome. Poultry Sci. 59: 731. Fromm, D. 1959. An evaluation of techniques commonly used to quantitatively determine the bacterial population of /107 chicken carcasses. Poultry S c i . 38: 887. Garrett, R.L. 1975. The " o i l y b i r d " syndrome, pp 38 - 47. In Proceedings of the Maryland Nutritional Conference. University of Maryland. Hayat, M.A. 1978. Introduction to Biological Scanning Electron Microscopy. University Park Press, Baltimore, Maryland. Horvat, R.J. 1978. Oily b i r d skin l i p i d s . Poultry S c i . 57: 1187. ICMSF. 1978. Microorganisms in Food I. Their significance and methods of enumeration. University of Toronto Press, Toronto, Ontario. Jensen, L.S., Bartov, I., Beirne, M.J., Veltman, J.R. J r . , and Fletcher, D.L. 1980. Reproduction of o i l y bird syndrome i n b r o i l e r s . Poultry S c i . 59: 2256. Kahn, L.E., Frommes, S.P. and Ca n c i l l a , P.A. 1977. Comparison of ethanol and chemical dehydration methods for the study of c e l l s i n culture by scanning and transmission electron microscopy. In Proceedings of Workshop on Biological Specimen Preparation Techniques. IITRI. p. 501. Kelley, R.O., Dekker, R.A.F. and Bluemink J.G. 1973. Ligand mediated osmium binding: i t s application i n coating b i o l o g i c a l specimens for scanning electron microscopy. J . Ultrastruct. Res. 45: 254. Kotula, A.W., Banwart, G.J. and Kinner, J.A. 1967. Effect of p o s t c h i l l washing on ba c t e r i a l counts on b r o i l e r chickens. Poultry S c i . 46: 1210. /108 Le, CD. 1980a. UBC MFAV Analysis of Variance/Covarianoe. Computing Center, University of Bri t i s h Columbia, Vancouver, B.C. Le, CD. 1980b. UBC BMD 02V Analysis of Variance for F a c t o r i a l Designs. Computing Center, University of British Columbia, Vancouver, B.C. Lindgren, B.W. and McElrath, G.W. 1969. Introduction to Probability and S t a t i s t i c s . Third edition. MacMillan Co., New York. McBride, G.B., Skura, B.J., Yada, R.Y. and Bowmer, E.J. 1980. Relationship between incidence of Salmonella contamination among pre-scalded, eviscerated and post-chilled chickens in a poultry processing plant. J. Food Prot. 43: 538. McCowan, R.P., Cheng, K.-J., Bailey, C.B.M. and Costerton, J.W. 1978. Adhesion of bacteria to epithelial surfaces within the reticulo-rumen of cattle. Appl. Environ. Microbiol. 35: 149. McMeekin, T.A and Thomas, C J . 1978. Retention of bacteria on chicken skin after immersion in bacterial suspensions. J. Appl. Bacteriol. 45: 383. McMeekin, T.A., Thomas, C J . and McCall, D. 1979. Scanning electron microscopy of microorganisms on chicken skin. J. Appl. Bacteriol. 46: 195. Malik, L.E. and Wilson, R.B. 1975. Modified thiocarbohydrazide procedure for SEM: routine use for normal, pathological or experimental tissue. Stain Technol. 50: 265. /109 Marshall, K.C., Stout, R.,and Mitchell, R. 1971. Mechanism of the i n i t i a l events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68: 337. Maser, M.D. and Trimble, J.J. 1976. Rapid chemical dehydration of biological samples for scanning electron microscopy using 2,2-dimethoxypropane. J. Histochem. Cytochem. 25: 247. Mead, G.C. and Impey, C.S. 1970. The distribution of C l o s t r i d i a in poultry processing plants. Br. Poult. Sci. 11: 407. Mead, G.C. and Thomas, N.L. 1973a. Factors affecting the use of chlorine in the spin-chilling of eviscerated poultry. Br. Poult. Sci. 14: 99. Mead, G.C. and Thomas, N.L. 1973b. The bacteriological condition of eviscerated chickens processed under controlled conditions in a spin-chilling system and sampled by two different methods. Br. Poult. Sci. 14: 413. Mendenhall, W. 1968. Introduction to Linear Models and the design and Analysis of Experiments. Duxbury Press, Belmont, California. Mulder, R.W.A.W. and Veerkamp, CH. 1974. Improvements in poultry slaughterhouse hygiene as a result of cleaning before scalding. Poultry Sci. 53: 1690. Mulder, R.W.A.W., Dorresteijn, L.W.J, and van der Broek, J. 1978. Cross-contamination during the scalding and plucking of broilers. Br. Poult. Sci. 19: 61. / n o Notermans, S. and Kampelmacher, E.H. 1974. Attachment of some bacter i a l strains to the skin of b r o i l e r chickens. Br. Poult. S c i . 15: 573. Notermans, S. and Kampelmacher, E.H. 1975a. Further studies on the attachment of bacteria to skin. Br. Poult. S c i . 16: 487. Notermans, S. and Kampelmacher, E.H. 1975b. Heat destruction of some bac t e r i a l strains attached to b r o i l e r skin. Br. Poult. S c i . 16: 351. Notermans, S., Jejunink, J . , van Schothorst, M. and Kampelmacher, E.H. 1973. Vergleichende untersuchungen uber die mbglichkeit von kreuzkonminationen im s p i n c h i l l e r unde bei der spruhkiihlung. Fleischwirtschaft 53: 573. Notermans, S., Kampelmacher,E.H. and van Schothorst, M. 1975. Studies on sampling methods used i n the control of hygiene i n poultry processing. J . Appl. B a c t e r i o l . 39: 55. Notermans, S., Firstenberg-Eden, R. and van Schothorst, M. 1979. Attachment of bacteria to teats of cows. J . Food Prot. 42: 228. Patrick, T.E., C o l l i n s , J.A. and Goodwin, T.L. 1973. Isolation of Salmonella from carcasses of steam and water scalded poultry. J . Milk Food Technol. 36: 34. Patterson, J.T. 1971. M i c r i b i o l o g i c a l assessment of surfaces. J . Food Technol. 6: 63. Pereira, A.S., Evans, R.W. and Stadelman, W.J. 1976. The effects / I l l of processing on some cha r a c t e r i s t i c s , including f a t t y acid composition of chicken f a t . Poultry S c i . 55: 510. Peric, M., Rossmanith, E. and Leistner, L. 1971. Untersuchungen liber die beeinsflussiing des oberflachkeimgehaltes von schlachthahnchen durch die spinchiller-kuhlung. Fleischwirtschaft 51: 216. Sahasrabudhe, M.R. 1979. L i p i d composition of oats (Avena sativa L.) J . Am. O i l Chemists Soc. 56: 80. Sanders, D.H. and Blackshear, CD. 1971. Effect of chlorination i n the f i n a l wash water on bact e r i a l counts of b r o i l e r chickens. Poultry S c i . 50: 215. Suderman, D.R. and Cunningham, F.E. 1980. Factors affecting the adhesion of coatings to poultry skin: effect of age, method of c h i l l i n g and scald temperature on poultry skin u l t r a -structure. J. Food S c i . 45: 444. Surkiewicz, B.F., Johnston, R.W., Moran, A.B. and Krumm, G.W. 1969. A bacteriological survey of chicken eviscerating plants. Food Technol. 23: 1066. Sweeny, L.R. and Shapiro, B.L. 1977. Rapid preparation of uncoated b i o l o g i c a l specimens for scanning electron microscopy. Stain Technol. 52: 221. Thomas, C.J. and McMeekin, T.A. 1980. Contamination of b r o i l e r carcass skin during commercial processing procedures: An electron microscope study. Appl. Environ. Microbiol. 40: 133. /112 Thomas, J.E., Cox, N.A., Bailey, J.S., Holliday, J.H. and Richardson, R.L. 1976. Bacteriological sampling of poultry carcasses by a template swab method. Poultry S c i . 55: 459. Todd, E.D.C. 1980. Poultry associated foodborne disease - i t s occurrence, cost, sources and prevention. J. Food Prot. 43: 129. van Schothorst, M., Notermans, S. and Kampelmacher, E.H. 1972. Einige hygeinische aspekte der geflgtielschachtung. Fleischwirtschaft :'.S2: 749. Walker, H.W. and Ayers, J.C. 1956. Incidence and kind of micro-organisms associated with commercially dressed poultry. Appl. Microbiol. 4: 345. Wilder, A.N. and MacCready, R.A. 1966. Isolation of Salmonella from poultry, poultry products and poultry processing plants i n Massachusetts. New Eng. J . Med. 274: 1453. Ziegler, F., Spencer, J.V. and Stadlman, W.J. 1974. A rapid method for determining spoilage i n fresh poultry meat. Poultry S c i . 33: 1253. Zottola, E.A., Schmeltz, D.L. and Jezeski, J . J . 1)970. Isolation of salmonellae and other air-borne microorganisms i n turkey processing plants. J. Milk Food Technol. 33: 395. 

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