الفهرس 
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Abstract
The aim of the work was to determine Listeria species in imported frozen fish by phenotypic methods and characterization of the recovered isolates by genotypic methods. To obtain this aims , a total of 189 imported frozen fish samples ( 104 mackerel , 52 horse mackerel , 25 catfish and 8 herring ) were used in this study and the following tests were done. Firstly Bacteriological examination was done by traditional method acc.to ISO 11290-1 1996- Amendment 2004 as follow: Preparation of test sample by adding 25 g of tissue to 225ml of the selective primary enrichment medium , to obtain a ratio of test portion to selective primary enrichment medium of 1/10 which is the initial suspention.then The initial suspension, was incubated at 30 °C for 24 h ± 2 h. A black coloration was develop during the incubation.After incubation of the initial suspension (primary enrichment) for 24 h ± 2 h , 0,1 ml of the culture was transferred to a tube containing 10 ml of secondary enrichment medium (Full Fraser broth) , then incubated at 35 °C or 37 °C for 48 h ± 2 h. from the primary enrichment culture incubated for 24 h ± 3 h at 30 °C , a portion of the culture was inoculated on the surface of the first selective plating medium, ALOA. The seeded plates were incubated to obtain well separated colonies. The same procedures were repeated with the second selective plating-out medium (OXFORD or PALCAM). from the secondary enrichment medium incubated for 48 h ± 2 h at 35 °C or 37 °C , the procedure described before was repeated with the two selective plating-out media. The dishes was inverted and placed in an incubator set at 37 °C for 24 h ± 3 h (and for an additional 24 h ± 3 h if the growth is weak or if no colony was observed after 24 h incubation) .The dishes were examined for the presence of colonies presumed to be Listeria spp.” Then Identification of Listeria spp.by Purification on TSYEA then Catalase reaction was performed as follow : an isolated colony was Taken and suspended in a DROP of hydrogen peroxide solution on a slide. gas bubbles was formed immediately which indicates a positive reaction. Then Gram staining was done as follow : the gram stain was performed on a colony. gram-positive slim and short rods were seen under light microscope. Then Motility test was done as follow :By using hanging DROP technique , an isolated colony was suspended in a tube containing TSYEB then Incubated at 25 °C for 8 h to 24 h. A DROP of the above culture was deposited using a loop onto a clean glass microscope. ThenBiochemical identification of L.monocytogeneswas donw as follow: firstly haemolysis test by inoculation of horse blood agar plates to determine the haemolytic reaction. incubated at 35 °C or 37 °C for 24 h ± 2 h. L. monocytogenes gave narrow, clear, light zones ( β- haemolysis) L. ivanovii usually show wide, clearly delineated zones of β- haemolysis. Then Carbohydrate utilization was done by inoculation of The carbohydrate utilization broths with a culture from TSYEB. Incubated at 35 °C or 37 °C for up to 5 days. Positive reactions (acid formation) were indicated by a yellow colour within 24 h to 48 h. then, Serological characterization of L. monocytogenes as follow :Listeria O antiserum types 1 and 4 (Difco Laboratories) were used for serological identification of L. monocytogenes by using the slide agglutination test according to the manufacturer’s instructionsin two steps firstly Preparation of the test organism by culturing of The test organism in Tryptic Soya Yeast extract Broth then the growth was suspended in FA Buffer. The Organism Suspension was Heated at 80 -100 oc for one hour in a water bath. The suspension was centrifuged and then the bulk of the supernatant fluid was removed. The organism was resuspended in the remaining portion of liquid. Then rapid test procedures were done as follow : One DROP of the heated organism suspension was added to one DROP of the diluted antiserum (1:20 in saline solution) on a glass slide. Similarly, one DROP of the organism suspension was added to one DROP of the negative control buffer solution as well as positive control. the test was read for agglutination with in 1-2 minutes the result was seven L. monocytogenes isolates were grouped into 3 different serotypes ( Type1 , Type4 and not typeable). serotype 1 : 3 isolates,serotype 4 : 3 Isolates and Un typeable : 1 isolate. Then,Antimicrobial Susceptibility testingof L. monocytogenes was done for six most commonly used antibiotics in veterinary and human therapy, using the standard disk diffusion method on Mueller Hinton Agar (MHA), following the procedures recommended by the Clinical and Laboratory Standards Institute.All L. monocytogenes isolates were resistant to ampicillin and tetracycline. Two isolates were resistant to erythromycin. However, they were susceptible to rifampicin,vancomycin,and streptomycin.then Multiplex pcr for detection of genus gene and genes responsible for pathogenicity was doneand results were All the isolates were positive for prs gene confirming the. AllL. monocytogenes isolates only were positive for prfA, hlyA, actA and inlA genes. All in all, Listeriamicroorganismwas isolated from certain types of imported frozen fish specially catfish. from the results of this study, it is advisable to use molecular methods beside biochemical tests and serological characterization to confirm presence of Listeria. Economically, multiplex PCR confirmation for L.monocytogenes is more beneficial for detection of Listeria in foods.