Biofilm inhibiting activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

AIMS: To investigate the biofilm inhibitory activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. METHODS AND RESULTS: The pulp of red pitahaya and the leaves of red spinach were extracted using methanol followed by subfractionation to obtain betacyanin fraction. The anti-biofilm activity was examined using broth microdilution assay on polystyrene surfaces and expressed as minimum biofilm inhibitory concentration (MBIC). The betacyanin fraction from red spinach showed better anti-biofilm activity (MBIC: 0·313-1·25 mg ml-1 ) against five Staph. aureus strains while the betacyanin fraction from red pitahaya showed better anti-biofilm activity (MBIC: 0·313-0·625 mg ml-1 ) against four P. aeruginosa strains. Both betacyanin fraction significantly reduced hydrophobicity of Staph. aureus and P. aeruginosa strains. Numbers of Staph. aureus and P. aeruginosa attached to polystyrene were also reduced without affecting their cell viability. CONCLUSION: Betacyanins can act as anti-biofilm agents against the initial step of biofilm formation, particularly on a hydrophobic surface like polystyrene. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to investigate the use of betacyanin as a biofilm inhibitory agent. Betacyanin could potentially be used to reduce the risk of biofilm-associated infections.

CsgA production by Escherichia coli O157:H7 alters attachment to abiotic surfaces in some growth environments.

The role of curli expression in attachment of Escherichia coli O157:H7 to glass, Teflon, and stainless steel (SS) was investigated through the creation of csgA knockout mutants in two isolates of E. coli O157:H7. Attachment assays using epifluorescence microscopy and measurements of the force of adhesion of bacterial cells to the substrates using atomic force microscopy (AFM) force mapping were used to determine differences in attachment between wild-type (wt) and csgA-negative (ΔcsgA) strains following growth in four different media. The hydrophobicity of the cells was determined using contact angle measurements (CAM) and bacterial adhesion to hydrocarbons (BATH). The attachment assay results indicated that ΔcsgA strains attached to glass, Teflon, and SS surfaces in significantly different numbers than their wt counterparts in a growth medium-dependent fashion (P < 0.05). However, no clear correlation was seen between attachment numbers, surface type, or growth medium. No correlation was seen between BATH and CAM results (R(2) < 0.70). Hydrophobicity differed between the wt and ΔcsgA in some cases in a growth medium- and method-dependent fashion (P < 0.05). AFM force mapping revealed no significant difference in the forces of adhesion to glass and SS surfaces between wt and ΔcsgA strains (P > 0.05) but a significantly greater force of adhesion to Teflon for one of the two wt strains than for its ΔcsgA counterpart (P < 0.05). This study shows that CsgA production by E. coli O157:H7 may alter attachment behavior in some environments; however, further investigation is required in order to determine the exact relationship between CsgA production and attachment to abiotic surfaces.

Significance of the rdar and bdar morphotypes in the hydrophobicity and attachment to abiotic surfaces of Salmonella Sofia and other poultry-associated Salmonella serovars.

AIMS: To investigate the relative role of the red dry and rough (rdar) and brown dry and rough (bdar) morphotypes on hydrophobicity and ability to attach to abiotic surfaces of poultry-associated Salmonella strains with a focus on S. Sofia. METHODS AND RESULTS: Cellulose synthase gene null mutants were constructed in five Salmonella strains converting them from rdar to bdar morphotypes. One S. Sofia null mutant displayed reduced hydrophobicity and attachment to Teflon® relative to its parent strain. The S. Virchow and S. Infantis null mutants attached less well to glass relative to their parent strains. CONCLUSIONS: The rdar or bdar morphotype may influence S. Sofia persistence but did not explain why bdar strains predominate in this serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides some insight into why some Salmonella strains survive in poultry environments and may ultimately contribute to their control.

Relative prevalence of Salmonella Sofia on broiler chickens pre- and postprocessing in Australia.

A survey was conducted to determine the relative prevalence of Salmonella serovars on whole chicken carcasses before and after processing in 3 Australian poultry abattoirs. Ninety and 180 whole chicken carcasses were tested for Salmonella serovars before and after processing, respectively. Each carcass was subjected to a buffered peptone water rinse according to Australian Standard methodologies and Salmonella prevalence was determined using Australian Standard methodologies. After isolation, Salmonella isolates were serotyped and results were analyzed to determine the relative percentage of each serovar at both processing points. Salmonella Sofia was shown to significantly increase its relative prevalence (P < or = 0.05) after processing and proved to be the dominant serovar accounting for 45/89 (51%) isolations before processing and 51/69 (74%) isolations after processing. The reasons for the increased relative prevalence of Salmonella Sofia are currently unknown and require further investigation but may involve factors related to prevalence and numbers on chickens and the ability of Salmonella Sofia to respond to environmental stressors and attach to surfaces.

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524.

AIMS: To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced. METHODS AND RESULTS: Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga. xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3.83 and 3.75 g l(-1) respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2.5 g l(-1). Solid state (13)C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles. CONCLUSIONS: The production rate of bacterial cellulose by Ga. xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features. SIGNIFICANCE AND IMPACT OF THE STUDY: Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga. xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.

Efficacy of sericea lespedeza hay as a natural dewormer in goats: dose titration study.

Gastrointestinal nematodes (GIN) parasitism is the greatest threat to economic sheep and goat production in the southern USA, and there is widespread prevalence of GIN resistance to broad-spectrum anthelmintics in this region. A natural alternative for controlling GIN in small ruminants is feeding hay of sericea lespedeza [SL, Lespedeza cuneata (Dum.-Cours., G. Don)], a perennial warm-season legume high in condensed tannins. To determine the level of SL needed to reduce GIN infection, a confinement study was completed with 32 Spanish/Boer/Kiko cross yearling bucks offered one of four diets with 75% hay and 25% concentrate (n=8, 2 pens/treatment, 4 goats/pen). The hay portion of each diet consisted of a combination of ground SL (0%, 25%, 50%, and 75% of the diet) and bermudagrass [BG, Cynodon dactylon (L.) Pers.; 75%, 50%, 25%, and 0% of the diet]. The bucks were allowed to acquire a natural GIN infection on pasture prior to moving to the pens. After a 3-week adjustment period in the pens, the goats were stratified by fecal egg count (FEC) and packed cell volume (PCV), randomly assigned to treatments and pens, and then fed the treatment diets for six weeks. During the experimental period, fecal and blood samples were collected from individual animals weekly to determine FEC and PCV, respectively. Adult worms from abomasum and small intestines were collected for counting and identification of species at slaughter. Goats fed SL hay at 25%, 50%, and 75% of the diet had 45.3% (P=0.2048), 66.3% (P=0.0134), and 74.5% (P=0.0077) lower FEC than control animals (75% BG hay) after 21 days. The 50% and 75% SL goats had 84.6% (P=0.0625) and 91.9% (P=0.0340) lower FEC than controls by day 42. The 75% SL-fed goats tended to have higher (P=0.0624) PCV and had fewer (P=0.035) abomasal worms than control animals, while PCV and adult worm numbers of the 50% and 25% SL goats were not different from controls. The optimum level of SL hay in the diet for reducing worm numbers of small ruminants appears to be 75%, whereas 50-75% SL reduces FEC, which could lead to reduced larval infection on pasture.

Issues in determining factors influencing bacterial attachment: a review using the attachment of Escherichia coli to abiotic surfaces as an example.

An understanding of the mechanisms which facilitate the attachment of Escherichia coli and other bacterial species to abiotic surfaces is desired by numerous industries including the food and medical industries. Numerous studies have attempted to explain bacterial attachment as a function of bacterial properties such as cellular surface charge, hydrophobicity and outer membrane proteins amongst others. Conflicting evidence in the literature both for and against a positive relationship may arise from the nature of the test methods used to measure them. A handful of recent studies utilizing technologies such as atomic force microscopy have begun to look at bacterial attachment at a single cell and molecular level. These studies may provide the information required to fully understand the underlying factors which influence bacterial cell attachment to abiotic surfaces. A number of issues in determining the influential factors of bacterial attachment have been identified from the literature: a lack of standardization and sensitivity of methods, as well as the value of measuring bulk properties of a number of cells rather than the behaviour of single cells which may overlook key interactions at a molecular level. These issues will need to be addressed in future studies in this area.

Attachment of different Salmonella serovars to materials commonly used in a poultry processing plant.

Salmonella can adhere to poultry and food contact surfaces and persist to cause diseases. Adhesion of Salmonella Sofia (n = 14), S. Typhimurium (n = 6), S. Infantis (n = 3) and S. Virchow (n = 2) to Teflon, stainless steel, glass, rubber and polyurethane were assayed using epifluorescence microscopy. Surface free energies of bacteria and materials were calculated using contact angle values and interfacial free energy between isolates and materials determined. Surface roughness of the materials was analysed using atomic force microscopy. S. Sofia isolates adhered in higher numbers (P < 0.05) to all materials compared to other serovars. The mean number of cells of S. Sofia isolates attaching to Teflon were significantly higher (P < 0.05) compared to all materials except stainless steel (P > 0.05). Mean roughness values ranged from 82.26 nm (Teflon) to 1.34 nm (glass). Correlations between the apolar component of the surface free energy of materials (gamma(S)(LW)) and bacterial adhesion (R(2) = 0.80), and between gamma(S)(LW) and the surface roughness of the materials (R(2) = 0.71) were found. Materials more positive in interfacial free energies had the highest number of adhering bacteria. Generalised surface property measurements were found to be useful in characterising Salmonella attachment but the degree of variability in results suggests that other factors, such as flagella or membrane proteins, could also contribute.

Salmonella Sofia differs from other poultry-associated Salmonella serovars with respect to cell surface hydrophobicity.

Salmonella enterica is one of the most important foodborne pathogens. Salmonella enterica subsp. II 4,12:b:- (Salmonella Sofia) is commonly found in Australian poultry. It has been suggested that physicochemical properties such as surface charge and hydrophobicity may affect bacterial attachment to surfaces and their ability to persist in food systems. A possible link between hydrophobicity cell surface charge and persistence of Salmonella from the poultry system was examined. Hydrophobicity of Salmonella Sofia (n = 14), Salmonella Typhimurium (n = 6), Salmonella Infantis (n = 3), and Salmonella Virchow (n = 2) was assayed using hydrophobic interaction chromatography, bacterial adherence to hydrocarbons (BATH), using xylene or hexadecane, and the contact angle method (CAM). Cellular surface charge (CSC) of the isolates was determined using zeta potential measurements. The majority (12 of 14) of Salmonella Sofia isolates were found to be hydrophobic when assayed using BATH with xylene, except isolates S1635 and S1636, and the other serovars were found to be hydrophilic. Salmonella Sofia isolates were not significantly different (P > 0.05) from isolates of other serovars as measured by hydrophobic interaction, BATH with hexadecane, or the CAM. No significant differences (P > 0.05) in zeta potential measurements were observed between isolates. Principal component analysis using results from all four measures of hydrophobicity allowed clear differentiation between isolates of the serovar Salmonella Sofia (except S1635 and S1636) and those of other Salmonella serovars. Differences in physicochemical properties may be a contributing factor to the Salmonella Sofia serovar's ability to attach to surfaces and persist in a food system.

Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats.

A bacterially produced cellulose film containing nisin was developed and used in a proof-of-concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of vacuum-packaged frankfurters. Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus K3 in Corn Steep Liquor-Mannitol Medium and were subsequently purified before nisin was incorporated into them. Investigations into the effect of nisin concentrations and contact times on incorporation of nisin into cellulose films showed that the lowest nisin concentration and shortest time needed for production of an effective antimicrobial cellulose film were 625IUml(-1) and 6h, respectively. The active cellulose films produced under these conditions did not, however, significantly reduce L. monocytogenes populations on frankfurters (P>0.05) during refrigerated storage for 14 days as compared to the controls. Films produced using a higher concentration of nisin (2500IUml(-1)) with the same exposure time (6h) resulted in a significant (P<0.05) decrease in L. monocytogenes counts on frankfurters of approximately 2logCFUg(-1) after 14 days of storage as compared to the control. Both the above-mentioned films showed a similar effectiveness in reducing total aerobic bacterial populations as measured by total aerobic plate counts on frankfurters. For both films, total aerobic bacterial levels were significantly (P>0.05) reduced by approximately 3.3logCFUg(-1) after 14 days of storage as compared to control samples. Bacterial cellulose films were demonstrated in this study to have potential applicability as antimicrobial packaging films or inserts for processed meat products.

Selection and fitness in bacteriocin-producing bacteria.

Bacteriocins are proteinaceous anticompetitor molecules produced by bacteria against closely related species. A number of theoretical models have been used to explain experimental data that indicate high polymorphisms among bacteriocins and a frequency-dependent nature of selection for bacteriocin-producing strains. The majority of these experimental data were, however, obtained from investigations into the colicin group of bacteriocins produced by Gram-negative bacteria. The conclusions drawn from these models have been extrapolated to other bacteriocins and allelopathic compounds in general. Examination of more recent experimental investigations into the bacteriocins of Gram-positive bacteria indicate a lower degree of polymorphism and a less frequency dependent mode of selection among these strains them among the colicin-producing strains. Here we examine these contradictions in the light of the assumptions and conclusions of the theoretical models and reported data. We show that fitness costs as indicated by decreased relative maximum growth rate associated with bacteriocin production may be much lower in many cases than is assumed in the present models. A lower fitness cost associated with bacteriocin production adequately explains the newer data from Gram-positive bacteria cited here, and indicates that extrapolation of existing models to all bacteriocins and other allelopathic compounds is not appropriate.

Combined antimicrobial effect of nisin and a listeriophage against Listeria monocytogenes in broth but not in buffer or on raw beef.

The effect of nisin and listeriophage LH7, alone and in combination, on the growth and survival of two strains of Listeria monocytogenes in broth and two model food systems, with appropriate controls, was determined. Growth curves for both bacterial strains in tryptic soy broth incubated at 7 or 30 degrees C, and with the addition of nisin and/or listeriophage at lag, mid-exponential or early stationary phase, were obtained by measuring absorbance at 550 nm. Numbers of mixed populations of both L. monocytogenes strains in phosphate buffered saline (pH 5.5) and on vacuum-packaged fresh beef, both stored for 4 weeks at 4 degrees C, and with the addition of nisin and/or listeriophage, were determined. This was achieved by plating appropriately diluted samples on both Tryptic Soy Agar and Modified Oxford Agar to determine both L. monocytogenes numbers and the presence of sub-lethal injury. In broth nisin alone, reduced levels or prevented growth of the two strains under the conditions studied, but regrowth to levels equivalent to those of untreated cells, occurred. Listeriophage LH7 alone, on the other hand, had no effect in broth under the conditions studied. Notably, however, a mixture of nisin and listeriophage displayed a combined effect in broth and reduced levels of cells substantially without regrowth under the conditions studied. In both model food systems only nisin appeared to be active, in a manner consistent with existing literature, and no combined action was apparent. The use of nisin and listeriophage has potential to control L. monocytogenes in foods but a further understanding of the interactions in this complex system needs to be achieved before it could be applied practically.

Survival of osmotic and acid stress by Listeria monocytogenes strains of clinical or meat origin.

The ability of 30 Listeria monocytogenes strains, 15 of meat origin and 15 of clinical origin, to use carnitine as an osmoprotectant and to resist acid stress was determined. All strains examined were able to use carnitine as an osmoprotectant, indicating the importance of this characteristic to the survival of L. monocytogenes in natural environments. Clinical and meat strains, however, differed with respect to this characteristic. Specifically, 73% of meat strains reached a lower maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. Only 33% of clinical strains displayed the same feature whereas the remaining clinical strains reached a higher maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. The physiological reasons and advantage of this difference are unclear. When exposed to conditions of severe acid stress (pH 2.5) for 2 h, only two L. monocytogenes strains (L66 and L78), both of meat origin, displayed significant reductions (P < 0.05) in number (3.51 and 2.79 log cfu, respectively). Acid-sensitive strains were not found among the clinical isolates examined, highlighting the importance of acid stress resistance in the infection process.

Planktonic or biofilm growth affects survival, hydrophobicity and protein expression patterns of a pathogenic Campylobacter jejuni strain.

The effect of planktonic or biofilm modes of growth on survival, hydrophobicity and cellular protein expression patterns of a pathogenic Campylobacter jejuni strain were determined. This was achieved by growing the strain in brain heart infusion broth (with 1% yeast extract), or attached to glass beads in the same medium, at 37 degrees C for 48 h under microaerophilic conditions. Cells from the broth or the bead surfaces were stored at different temperatures (4, 10, 25 and 37 degrees C) for 28 days in phosphate buffered saline (PBS) and monitored at appropriate time intervals for culturable numbers and hydrophobicity by standard methods. In addition, cells were inoculated onto the surface of two processed meat products (a bologna and a summer sausage) vacuum packaged and stored at 4 degrees C for 28 days. Numbers of culturable cells were monitored at appropriate time intervals by standard methods. Cells from the broth or the bead surfaces were also examined for protein expression using two-dimensional protein electrophoresis. Results indicated that numbers of culturable cells in phosphate buffered saline decreased from approximately 6 log colony forming units (cfu) g(-1) to undetectable levels within 14-day storage in a temperature dependent manner. Hydrophobicity of broth grown cells decreased from 15% to 0% adherence to xylene over the same time in a temperature independent manner. Cells grown in a biofilm mode initially displayed a <0.3% adherence to xylene which was maintained during storage. Furthermore, cells grown in the biofilm mode decreased in number more rapidly on storage in buffer than their counterparts grown in broth. Numbers of culturable cells on meat decreased from approximately 5 log cfu g(-1) to undetectable levels within 14-day storage in a product dependent manner, with the most rapid decrease observed for the more acidic summer sausage. Cells grown in a biofilm mode decreased in number more rapidly on storage than broth grown cells. The protein expression patterns differed between planktonic and biofilm cells with seven unique and 12 up-regulated protein spots expressed in a growth mode specific manner. A number of the differentially expressed spots were tentatively identified, by comparison to existing literature, as surface- and stress-associated proteins. Despite the elicitation of some putative stress proteins, this study importantly indicates that biofilm cells of C. jejuni are less resistant to stress than their planktonic counterparts and may lack a sophisticated adaptive stress-resistance response. These findings have implication in determining the risks of infection associated with C. jejuni contamination on food.

Quantification of microbial populations associated with the manufacture of vacuum-packaged, smoked vienna sausages.

Sources of contamination of vacuum-packaged vienna sausages by spoilage microorganisms were examined in a meat-processing plant. Microbial numbers present in the environment, on working surfaces and workers' hands and aprons were quantified by plate counting on selective media. Product line samples were taken at critical control points in the manufacturing process and analysed before and after preliminary incubation of vacuum-packaged samples at 25 degrees C for 24 h. In all samples the numbers of aerobic bacteria, Enterobacteriaceae, lactic acid bacteria and yeasts were determined by standard procedures. Contamination of sausage surfaces by lactic acid bacteria occurred as a result of the manufacturing and handling processes. The environment and specifically packers' hands, as well as working surfaces contributed to microbiological contamination of various types after removal of the peel from individual sausages. The preliminary incubation procedure allowed detection of low numbers of spoilage microorganisms.

Yeasts associated with Vienna sausage packaging.

A total of 123 representative yeast isolates from a previous study of a Vienna sausage processing plant were identified according to conventional methods and long-chain fatty acid analyses. The most prevalent isolates belonged to the genera Candida and Debaryomyces. Other genera encountered were Rhodotorula, Yarrowia, Pichia, Galactomyces, Cryptococcus, Trichosporon and Torulaspora.

Taxonomy of lactic acid bacteria associated with vacuum-packaged processed meat spoilage by multivariate analysis of cellular fatty acids.

The taxonomy of lactic acid bacteria from vacuum-packaged processed meats is problematic, and atypical members of the leuconostocs and the Lactobacillus sake / curvatus group are often encountered. In order to resolve this problem the cellular fatty acid (CFA) content of 61 isolates from vacuum-packaged vienna sausages and 18 reference strains was determined by gas chromatography. The relationship between strains was derived by principal component analysis of data. The CFA profiles were highly reproducible. Although no relationships could be derived using only one or two differentiating CFAs, plots of the first two principal components based on only the six most variable CFAs allowed grouping of strains. The two genera (Leuconostoc and Lactobacillus) could not be clearly separated when analysed together, but differentiation of species within each of the genera was achieved when they were analysed independently. Examination of plots for the reference strains confirmed previously established relationships between these strains. From the plot of the Lactobacillus sake / Lactobacillus curvatus component of the study it was found that most atypical Lactobacillus sake / curvatus strains were closely related to the typical Lactobacillus sake isolates and reference strain, while the Lactobacillus curvatus strains formed an independent grouping. A small cluster of atypical strains, however, indicated that this relationship may not be true for all these strains. Among the leuconostocs only isolates of Leuconostoc mesenteroides could be clearly differentiated.

Survival of Escherichia coli O157:H7 and Salmonella on chill-stored vacuum or carbon dioxide packaged primal beef cuts.

The ability of two Escherichia coli O157:H7 strains (E27, a cattle isolate, and B6-914 gfp-91, a fluorescent marker strain) and two Salmonella serotypes (S. typhimurium and S. brandenberg) to survive on chilled preservatively packaged primal beef cuts was examined. Each of the strains was inoculated separately at two dilution levels (10(3) and 10(5) cfu g(-1)) onto 500 g beef steaks, packaged under vacuum or 100% carbon dioxide, and stored, with uninoculated controls, for 6 weeks at - 1.5 degrees C, then for 2 weeks at 4 degrees C. Bacterial numbers were determined by dilution and incubation at 37 degrees C for 24 h on either Sorbitol McConkey Agar or Xylose Lysine Desoxycholate Agar for E. coli O157:H7 and Salmonella samples, respectively. Counts were corrected for background growth and their accuracy checked using immunological tests. Fluorescent E. coli O157:H7 B6-914 gfp-91 was also counted under ultra-violet light. No significant changes in numbers of the E. coli O157:H7 or Salmonella strains occurred during storage at either - 1.5 or 4 degrees C packaged under either vacuum or carbon dioxide. The ability of these pathogens to survive standard preservative packaging conditions is different from that reported from their generic counterparts and therefore a cause for public health concern.

Stochastic modelling of the growth of a microbial population under changing temperature regimes.

The application of models of microbial growth to the design of food safety systems requires consideration of the effect of arbitrary changes in external variables on growth of bacteria. In particular, the effect of changes in external variables, such as temperature, on the probability that the microbial population size will not exceed acceptable levels at a given time needs to be predicted. This paper presents a method of calculating the time-dependent probability distribution of the microbial population size under arbitrary changes of temperature through time. To illustrate this method, the effect of a sudden temporary increase in temperature on the evolution of the probability distribution of Lactobacillus plantarum population size is presented. The effect of this change in temperature on the time taken for the population to reach a critical size, with a given probability, is also calculated and the application of this calculation to the design of HACCP protocols is discussed.

Determination of quarantine period in African catfish (Clarias gariepinus) fed with pig (Sus sp.) offal to assure compliance with halal standards.

Pig (Sus sp.) and pig by-products are considered as najasa (impurities) in Islam and forbidden in Muslim consumer products. Animals fed on najasa are categorised as al-jallalah (contaminated animals) which are allowed to be consumed as long as they have been quarantined for a certain period of time. During this quarantine period the animals will have undergone a natural purification process or istihalah. African catfish (Clarias gariepinus) are commonly consumed in Malaysia and may be fed on najasa. This study was carried out to estimate the istihalah period for catfish after feeding with pig offal, based on the absence of pig DNA in catfish gut and to suggest the quarantine period in catfish fed with pig offal. The results indicated that the maximum istihalah period could reach 36h in the stomach, 6h in the midgut and less than 2h in the hindgut although in many cases shorter periods were observed. Based on these results it is estimated that the minimum quarantine period for catfish fed with pig offal is 1.5days.