MICROBIAL CHARACTERISTICS OF FRESH FISH SOLD AT KEDONGANAN FISH MARKET, BALI, INDONESIA

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1 MICROBIAL CHARACTERISTICS OF FRESH FISH SOLD AT KEDONGANAN FISH MARKET, BALI, INDONESIA Ketut Suriasih Faculty of Animal Husbandry, Udayana University, Denpasar, Indonesia I Nyoman Sucipta Faculty of Agricultural Technology, Udayana University, Denpasar, Indonesia ABSTRACT This study was conducted to find out the bacterial microflora of fresh fish sold at Kedonganan fish market, Bali, Indonesia. Three species of fish (Tuna, Snapper and Milk fish) were bought from the market and aseptically transported to Laboratory of microbiology, Faculty of Agricultural Technology, Udayana University. Section of skin and gills of ten randomly selected fishes from each species were aseptically removed by means of a sterile scalpel and pair of scissors. Five (5) g of each of the section was mix and homogenized in 90 ml of sterile physiological solution (0,85% NaCl). A serial dilution up to 10 8 was carried out, and surface plated on nutrient agar. The average viable bacterial count of each fish species was 2.14 x 10 6 cfu.g -1 in Tuna, 6.86 x 10 6 cfu.g -1 in Milk Fish and 5.27 x 10 6 cfu.g -1 in Snapper. A total of 6 bacterial genus were isolated and identified. Three bacterial genus obtained from Tuna fish were identified as Pseudomonas, Flavobacterium and Staphylococcus. Five genus of bacteria isolated from Snapper fish were : Pseudomonas, Micrococcus, Flavobacterium, and Staphylococcus. Five genus of bacteria isolated from Milk Fish were : Micrococcus, Staphylococcus, Flavobacterium, Vibrio, and Pseudomonas. Frequency of occurrences of the isolated bacteria indicated that Pseudomonas was the most frequently appear in Tuna fish followed by Flavobacterium and Staphylococcus, while in Snapper fish Micrococcus showed the highest frequency followed by Pseudomonas, Flavobacterium, Alcaligeness and Staphylococcus, and in Milk fish again Pseudomonas showed the highest frequency, followed by Micrococcus, Flavobacterium, Staphylococcus and Vibrio. Keywords: bacteria, tuna, snapper, milk fish, Kedonganan fish market Introduction Fresh fish is a healthy source of protein and loved very much by all levels of society including foreign tourists. Fresh fish food commodities which have important role, both in terms of nutrition and socio-economic aspects and culture and is always available on the market throughout the year. The fish in the market are generally supplied from catching by the fishermen and marketed after being caught more than one day, so the possibility of the occurrence of decomposition by microbial contaminants are very strong. Luis and Marth (1979) stated that, to maintain freshness of the fish cooling technique with ice was commonly used. The 1

2 high price of ice used to preserve fresh fish on a fishing boat caused high operational costs, result in high market prices of fresh fish which may not affordable by most consumers, Suriasih (2004). This situation make fishermen to choose material which is not-for-food preservatives (such as formaldehyde) to maintain the freshness of the catched fish. Although the use of formalin to preserve food products have been banned, there are many fishermen still using it, because these chemicals can easily available in the market and it is relatively cheap. Besides, consumer attitude encourage such errors. They prefer fish that brightly colored, durable, compared to product naturally colored. Detection of this case caused a lot of losses for both consumers (dangerous to health) and also for the fisherman itself, because consumers are afraid to eat fish contain toxic materials so that the fish are not bought in the market. This brought harm to consumers, merchants and fishermen themselves (the fish was unsold) because consumers are afraid to buy fish that contain hazardous materials. Indeed, it is very important to know that spoilage of fish was largely due to microorganisms contaminating the fish. Contamination can be of the equipment used in catching, storage and handling of the fish from catching to the market. Knowledge of the kind and number of contaminating microorganisms that caused fis spoilage will contribute to better consideration in choosing safe preservative materials. The aim of this experiment was to conduct microbiological analysis to determine the microbial characteristics of fresh fish (tuna, snapper and milkfish) sold in Kedonganan fish market, Bali. based cell colonies, cell morphology, gram reaction, catalase test, oxidase test, carbohydrate fermentation and gas production from glucose. Benefits of the research, it gives information in term of microbial characteristics of tuna, snapper and milk fish. Academically can be used as a theoretical contribution in the development of science, knowledge and technology Fish is a perishable food commodities because it contains nutrients and high water content. Damage fish microbiologically mostly caused by natural microbial contaminates fish life time. Knowing microbial characteristics (tuna, snapper and milkfish) can practically be used to determine the level of freshness of the fish so that the problem of damage to fish from the fishermen and consumers can be overcome. 2

3 Research methods Materials Fish samples of 60 fish tail consisting of tuna, fish and snapper obtained from fishermen fishing on the beach of Sanur, Denpasar and beach Kedonganan, Badung regency of Bali. taken within a period of 4 months. Fresh fish from boat was transferred into a box containing ice and immediately taken to the laboratory. There is period of 24 hours between the time of arrest at sea and analysis of the fish. Media Media: Nutrient Agar (Oxoid CM3), Nutrient Broth, Mackonkey order, proteose Peotone (Oxoid L-85) lab LEMCO powder (Oxoid L29) glucose, sodium chloride, sodium glycollate (Oxoid L120), order (Oxoxid Ll 1), methylene blue, methyl tetra-p-phenylene diamine hydrochloride, ascorbic acid, tryptone (Oxoid L21), D-glucose, in potassium hydrogen phosphate, L argininc monohydrochlorida, potassium iodide, mercury iodide, potassium hydroksida, Mueller-Hinton order (Oxoid CM337) Chemicals: Standard organic acid, buffer ph 4, ph 7 buffer, phenol red, phenol phtalein (Merck), H 2 SO 4 PA (Merck), Ampicillin, Chloramphenicol, crystal violet, Potassium iodide, safranin, immersion oil, alcohol pa (Merck), NaOH, carbohydrates (glucose, sucrose, lactose, mannitol, trehalose, D-xylose, mannose, glycerol, esculin, ferric citrate, gelatin, mercuric chloride, concentrated hydrochloric acid, distilled water. Equipments: petri dish, erlenmeyer 10 ml erlenmeyer 25 ml, erlemneyer 500 ml, Erle meyer 1000 ml glass beaker 500 ml glass beaker l000m ml, pipette l ml, pipette 10 ml burette ph meter, analytical balance, autoclave, laminar flow, centrifuges, microscopes, incubators, thermometer, knife, caliper / caliper, spatula, tweezers, refrigerator. Isolation and Identification. Isolation and identification of microorganisms of fresh fish were carried out according to Cowan and Steel, (1981); Isolation. 5 grams of fresh fish sample was taken from the skin of the fish that have not been washed, transferred into 45 ml of sterile peptone solution, then homogenized for 10 3

4 minutes. Then a serial dilution to 10-7 was undertaken. After serial dilution. 0.1 ml of the sample filtrate was inoculated on to nutrient agar medium with surface spread method. Further cultures were incubated at 30 C for 48 hours. Colonies that grew were than calculated and observed the shape and color. Six to eight isolates from different colonies were chosen and streaked on to nutrient agar medium to obtain pure culture. Identification of Isolate: Isolates obtained were identified to genus level by gram staining and microscopic observation of the Gram nature, size and shape of bacteria, the size and shape of spores and motility. Furthermore catalase activity test was done: a little colony isolates put up on a glass object,containing a drops of 3% H 2 O 2 solution. Formation of gas bubbles indicates a positive result. Oxidase test is done by applying a slight colony isolates to the filter paper soaked with a solution of 1% Tetramethyl-p-phenylenediamine dihydrochlorida, wait for 10 seconds. The formation of a blue color indicates positive results. Against gram negative then pigmentaton test was done, oxidative/fermentative test in the Hugh & Leifson media, and acid formation with or without gas from glucose using durham tube. Both Fiavobacterium and were not motile but Flavobacterium produce yellow to orange color, while Achromobacter did not produce any color. Positive oxidase test, without acid and gas production from glucose indicate the nature of Pseudomonas. Vibrio and Aeromonas form acid from glucose, but Aeromonas was also produce gas from glucose while vibrio did not. Furthermore isolates were inoculated on to slanted agar, incubated at 37 C for 24 h and stored in the refrigerator as bacterial stock Results and Discussion Microbial analysis showed that the average viable bacterial count of each fish species was 2.14 x 10 6 cfu.g -1 in Tuna, 6.86 x 10 6 cfu.g -1 in Milk Fish and 5.27 x 10 6 cfu.g -1 in Snapper fish. A total of 6 bacterial genus were isolated and identified Three bacterial genus obtained from Tuna fish were identified as Pseudomonas, Flavobacterium and Staphylococcus. Five genus of bacteria isolated from Snapper fish were : Pseudomonas, Micrococcus, Flavobacterium, and Staphylococcus. Five genus of bacteria were isolated from Milk Fish were : Micrococcus, Staphylococcus, Flavobacterium, Vibrio, and Pseudomonas. Frequency of occurrences of the isolated bacteria indicated that Pseudomonas was the most frequently appear in Tuna fish followed by Flavobacterium and Staphylococcus, 4

5 while in Snapper fish Micrococcus showed the highest frequency followed by Pseudomonas, Flavobacterium, Alcaligeness and Staphylococcus, and in Milk fish again Pseudomonas showed the highest frequency, followed by Micrococcus, Flavobacterium, Staphylococcus and Vibrio. Occurrence of Pseudomonas in fresh fish from Kedonganan market is not in line with Shewan and Hobbs (1967) cited by Suriasih (2004) who stated that the microbial population of Conclusion Microbial characteristics of fresh fish sold at kedonganan fish market : Three bacterial genus obtained from Tuna fish were identified as Pseudomonas, Flavobacterium and Staphylococcus. Five genus of bacteria isolated from Snapper fish were : Pseudomonas, Micrococcus, Flavobacterium, and Staphylococcus. Five genus of bacteria were isolated from Milk Fish were : Micrococcus, Staphylococcus, Flavobacterium, Vibrio, and Pseudomonas. Frequency of occurrences of the isolated bacteria indicated that Pseudomonas was the most frequently appear in Tuna fish followed by Flavobacterium and Staphylococcus, while in Snapper fish Micrococcus showed the highest frequency followed by Pseudomonas, Flavobacterium, Alcaligeness and Staphylococcus, and in Milk fish again Pseudomonas showed the highest frequency, followed by Micrococcus, Flavobacterium, Staphylococcus and Vibrio. Reference Cowan, M.M Plants Products as Antimicrobial Agents. Clinical Microbiology Reviews. 12(4): Luis, R.F. dan E.H.Marth Bacterial Flora of Fish from Tropical Sea Water. J. of Food Protection. 42.9: Maibach IH. Raza A Skin microbiology relevance to clinical infection. New York. Hiedelberg, Berlin: Springer~Verlag; hal Purseglove, I.., E.G. Brown, G.L Green and S.R.I. Robbin Spices Longman Inc., New York. Samarasinghe,J Wenk, KFS.T Silva and JM.DM Gunasebera Turmeric (Curcuma Ionja) Root Powder and Manaran Oligosaccharides as Altemativees to antibiotics inbroiler chichen Diets. Asian-Aust.J Ani. Sci 2003 Vol Suriasih, K Microbial Spoilage of Fish and Its Biochemical Changes. Gitayana, 6(1):

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