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Selective Media for In Vitro Activity Evaluation of Bacterial Biocontrol Against Pathogenic Vibrio ISNANSETYO, ALIM; MUHTADI, .; ISTIQOMAH, INDAH; NITIMULYO, KAMISO HANDOYO; TRIYANTO, .
HAYATI Journal of Biosciences Vol 18, No 3 (2011): September 2011
Publisher : Bogor Agricultural University, Indonesia

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Abstract

In vitro activity test is a critical evaluation to screen the potential biocontrol agent. We developed a selective medium for quantitative in vitro activity evaluation of bacterial biocontrol agents against pathogenic Vibrio in aquaculture. Sensitivity test of bacterial biocontrol and Vibrio spp. to nine antibiotics showed that oxytetracycline inhibited the growth of Vibrio spp., but did not inhibit the growth of the bacterial biocontrol. This selective inhibition activity of oxytetracycline suggested this antibiotic might be supplemented to establish a selective medium. The MIC of oxytetracycline against V. harveyi, V. parahaemolyticus, and V. algosus were 120, 250, and 120 mg/ml, respectively. These concentration did not inhibit the growth of bacterial biocontrols. Therefore, oxytetracycline was supplemented at 250 µg/ml in Zobell agar medium. This medium was used as a selective medium to enumerate the density of bacterial biocontrol. In vitro activity test of bacterial biocontrol (RLP1) against V. parahaemolyticus showed that strain RLP1 at density of 104 and 106 cells/ml was able to kill V. parahaemolyticus during 6 h incubation. At lower density, 102 cells/ml, this bacterial biocontrol agent was able to kill the pathogenic Vibrio during 12 h incubation. This study discovered a selective medium for the bacterial biocontrol and Vibrio spp. and provided the results of its application in the evaluation of in vitro activity of a bacterial biocontrol agent against V. parahaemolyticus. The results also revealed that strain RLP1 is a potential bacterial biocontrol against vibriosis in marine aquaculture.
Independent Effects of Temperature, Salinity, Ammonium Concentration and pH on Nitrification Rate of the Ariake Seawater Above Mud Sediment ISNANSETYO, ALIM; GETSU, SAI; SEGUCHI, MASAHIRO; KORIYAMA, MASUMI
HAYATI Journal of Biosciences Vol 21, No 1 (2014): March 2014
Publisher : Bogor Agricultural University, Indonesia

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Abstract

The Ariake Sea located in the west parts of Kyushu Island is a semi-closed and macro-tidal shallow sea, and has the largest tidal flat in Japan. A large mud tidal flat with a productive ecosystem found along the western shoreline of the sea makes this area ideal as a major production site of nori (Porphyra yezoensis) in Japan. We determined the independent effect of temperature, salinity, ammonium concentration and pH on nitrification rates (NR) in the Ariake seawater above the mud sediment. The NR was determined by measuring accumulation of NO2-N production after adding sodium chlorate, an inhibitor of NO2-N to NO3-N oxidation. NRs were relatively high at 20-35 oC (optimum at 29.5 oC), but the rates were very low at 5, 10, and 40 oC. NRs increased sharply when increasing the salinity from 13 to 20 ppt, but it decreased drastically at salinity levels more than 35 ppt (optimum at 19 ppt). The relationship between ammonium concentration and NR showed a typical kinetic curve of enzymatic reaction with the maximum NR (Vmax) of 0.029 mM N.h-1 at 200 mM NH4-N (the half saturation constant (Ks) = 35 mM NH4-N). High NRs were determined at pH 7.5-8.0 (optimum pH 7.8). This is the first report on the independent effects of temperature, pH, salinity and NH4-N concentration on the NR of seawater, specifically the Ariake seawater.
Distribution of Ammonium-Oxidizing Bacteria in Sediment with Relation to Water Quality at the Musi River, Indonesia Melki, Melki; Isnansetyo, Alim; Widada, Jaka; Murwantoko, Murwantoko
HAYATI Journal of Biosciences Vol 25, No 4 (2018): October 2018
Publisher : Bogor Agricultural University, Indonesia

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Abstract

The Musi River is located in the southern Sumatra, Indonesia. Most of activities, i.e. agricultural, industrial, and urban activities are considered as being major sources of chemicals and nutrients with their waste products effluent into the river. Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, occurs in a wide variety of environments and naturally remove anthropogenic N pollution. The purpose of this research was to determine of distribution of ammonium-oxidizing bacteria (AOB) in sediment with relation to water quality at the Musi river area. This study was conducted on rainy and dry season 2016 at five sampling sites from the freshwater to seawater at high and low tide conditions, the sampling sites are station St1 (Gandus), station St2 (Palembang city), station St3 (Upang), station St4 (Sungsang), and station St5 (Sea). Sediment samples were collected from the surface layer by using an Ekman grab. Some water quality such as salinity, temperature, pH, and dissolved oxygen (DO) were directly analyzed in the field, while other water quality such as NH4-N, NO2-N, and NO3-N were analyzed in the laboratory. The Density of AOB was determined by the most probable number of (MPN) method. The PCA was used to correlate variations of the AOB with physicochemical properties using software Xlstat. The results showed that the physicochemical properties had a range of salinity of 0 to 20 ppt, temperature of 29.21 to 31.82oC, pH of 4.88 to 7.93, DO of 3.44 to 11.33 mg/l, NH4-N in sediment of 0.04 to 0.87 mg/l, NO2-N in sediment of 0.01 to 1.77 mg/l, NO3-N in sediment of 0.09 to 2.08 mg/l. The density of AOB ranged from 7.2 x 102 to 6.1 x 103 cells/g sediment. Principal component analyses showed that temperature, pH, DO, and concentrations of nutrient contributed to density of AOB.
IMMUNOSTIMULATORY EFFECT OF FATTY ACID FROM STAR FISH (ACANTHASTER PLANCI) ON LYMPHOCYTE PROLIFERATION IN-VITRO Achmad, M. Janib; Isnansetyo, Alim; Kasanah, Noer; Ustadi, Ustadi; Kamiso, Kamiso
Squalen, Buletin Pascapanen dan Bioteknologi Kelautan dan Perikanan Vol 9, No 3 (2014): December 2014
Publisher : Research and Development Center for Marine and Fisheries Product Processing and Biotechnol

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Abstract

The aims of this study were to investigate lymphocyte proliferation activity and to identify chemical constituents of active fractions of star fish Acanthaster planci. A. planci was collected from Ternate Island, North Moluccas, extracted with distilled methanol and water, partitioned with gradient chloroform-hexane-methanol-water and fractionated in column chromatography using silica gel and hexane-ethyl acetate-methanol. The active compound had been purified by Thin Layer Chromatography (TLC) and identified by Gas Chromatography-Mass Spectrometry (GC-MS). The lymphocyte proliferation activity was measured based on % Stimulation Index (SI) from sample absorbency and control absorbency. The result showed that the 3 fractions of hexane fraction exhibited lymphocyte proliferation activity. Fraction 1 was able to increase lymphocyte proliferation at 48 hours and 72 hours by 55% (80 µl/ml) and 88% (160 µl/ml) increase, and fraction 2 had 77% (160 µl/ml) and 86% (640 µl/ml) increase. Meanwhile, fraction 3 had 75% (640 µl/ml) and 89% (640 µl/ml) increase. Metabolite analysis of active fraction using GC-MS yielded a number of chemical constituents that was dominated by fatty acid. The study concluded that star fish A. planci from Ternate Island has a potential source of immunostimulator.
Antibacterial Compounds from Red Seaweeds (Rhodophyta) Kasanah, Noer; Triyanto, Triyanto; Seto, Drajad Sarwo; Amelia, Windi; Isnansetyo, Alim
Indonesian Journal of Chemistry Vol 15, No 2 (2015)
Publisher : Universitas Gadjah Mada

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Abstract

Seaweeds produce great variety of metabolites benefit for human. Red seaweeds (Rhodophyta) are well known as producer of phycocolloids such agar, agarose, carragenan and great variety of secondary metabolites. This review discusses the red algal secondary metabolites with antibacterial activity. The chemical constituents of red algae are steroid, terpenoid, acetogenin and dominated by halogenated compounds mainly brominated compounds. Novel compounds with intriguing skeleton are also reported such as bromophycolides and neurymenolides. In summary, red seaweeds are potential sources for antibacterial agents and can serve as lead in synthesis of new natural medicines.
EFEKTIVITAS VAKSIN POLIVALEN UNTUK PENGENDALIAN VIBRIOSIS PADA KERAPU TIKUS (Cromileptes altivelis) Nitimulyo, Kamiso Handoyo; Isnansetyo, Alim; Triyanto, Triyanto; Murdjani, Muhammad; Sholichah, Lili
Jurnal Perikanan Universitas Gadjah Mada Vol 7, No 1 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

The objective of this research was to know the effectiveness of polyvalen  Vibrio vaccine to control vibriosis in humpback grouper (Cromileptes altivelis). The effectiveness of vaccine was evaluated by the survival rate (SR), relative percent survival (RPS), mean time to death (MTD) as well as growth rate of vaccinated fish. This research consisted of 4 treatments (control, injection, immersion, and oral vaccinations) in quadruplicates. Injection  vaccination was conducted by intraperitoneal injection of polyvalen vaccine at 107 cells/fish. Immersion vaccination was done by immersing the fishes at 107 cells/ml for 30 minutes. Oral administration of vaccine was also carried out  at 107 cells/fish. One week after the first vaccination, second vaccination (booster) was carried out at the same dosage and by the same administration. One week after the second vaccination, fishes were challenged with 3.16x104 cells/fish of Vibrio ordalii 3J by intraperitoneal injection, and reared for 20 days post infection. Results indicated that polyvalen Vibrio vaccine increased SR (P<0.01) up to 100%. Vaccination was also able to delay MTD of fishes. However, the vaccination was not influence the growth rate of fish.
EFEKTIVITAS EKSTRAK BAWANG PUTIH (Allium sativum) UNTUK MENCEGAH DAN MENGOBATI MOTILE AEROMONAS SEPTICEMIA (MAS) PADA IKAN MAS (Cyprinus carpio) Lukistyowati, Iesje; Windarti, Windarti; Morina, Morina; Isnansetyo, Alim; Kurniasih, Kurniasih
Jurnal Perikanan Universitas Gadjah Mada Vol 10, No 1 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

The aim of study was to know the effectiveness of garlic (Allium sativum) extract in preventing and curing Motile Aeromonas Septicemia (MAS) in common carp (Cyprinus carpio). Garlic extract was obtained by juicing fresh garlic and filtering by using filter paper. Common carp (8-12 cm in total lenght) were used. Infection was done by injecting intraperitonially 0.1 ml 0f 24 h Aeromonas hydrophila culture at bacterial density of 108 cells/ml. Garlic extract was given by immersing the fish at various concentrations. Results indicated that the minimum inhibitory concentration (MIC) of the garlic extract againtst A. hydrophila ranged from 61.300-125.000 ppm, while the LD50 of this extract was 3.438 ppm. The use of garlic extract to prevent MAS was effective as survival rate of the treated fish with the extract at 1000, 2000, and 3000 ppm ranged from 83 to 100% while the survival rate of untreated fish was 60.71%. However, the use of garlic extract to cure MAS was not effective as the survival rate of the treated fish ranged 50-80% which were the same or even lower than that of the control (76.67%).
PENAMBAHAN VITAMIN C PADA IKAN LELE DUMBO (Clarias gariepinus) UNTUK MENINGKATKAN TANGGAP KEBAL TERHADAP VAKSIN Aeromonas hydrophilia Isnansetyo, Alim
Jurnal Perikanan Universitas Gadjah Mada Vol 1, No 1 (1996)
Publisher : Universitas Gadjah Mada

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Abstract

Motile Aeromonas Septicimia is a dangerous disease on fresh water fish especially walking catfish and carp. Vaccination against the disease has been developed in Indonesia. However, there were high result variations of the vaccination. Immune response is influenced by level of vitamin C dietery. Objective of this experiment was to know effect of vitamin c levels on walking catfish’s specific immune respose. The levels of vitamin (0, 50, 500, and 1000 mg ascorbic acid (AA)/kg feed) were given to the fish. Immersion vaccination for 30 minutes (108 sel/ml) was carried out 15 days after feeding trial. Challenge test was conducted by injection of 0,2 ml bacteria suspension (LD80 concentration) was carried out 15 days after vaccination. Growth rate of the fish was observed monthly. Result of this experiment indicated that mortality in the challenge test due to A. hydrophila infection considerably decreased for treatment combination among vaccination; and 500 and 1000 mg AA/kg feed. These AA levels without vaccination could not reduce mortality.
Patogenisitas Vibrio Fluvialis 24SK terhadap Kerapu Tikus (Cromileptes altivelis) Istiqomah, Indah; Isnansetyo, Alim; Triyanto, Triyanto; Nitimulyo, Kamiso Handoyo; Murdjani, Muhammad
Jurnal Perikanan Universitas Gadjah Mada Vol 8, No 1 (2006)
Publisher : Universitas Gadjah Mada

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Abstract

This research was aimed to investigate the pathogenicity of Vibrio fluvialis 24SK in humpback grouper (Cromileptes altivelis) based on its Lethal Dosage 50 (LD50). V. fluvialis 24SK was isolated from ren of humpback grouper cultured in floating net cage at Brackishwater Aquaculture Development Center (BADC) Situbondo, with vibriosis signs. The bacterium was cultured in Tryptone Soy Broth (TSB) medium dissolved in trisalt solution (KCl, 0.75 g/l; MgSO4.7H2O; 14.2 g/l; NaCl, 18.4 g/l), incubated at 300C for 24 h. Infection was carried out by interperitoneal injection to humpback grouper (8-9 cm of total length) at 102, 104, 106, and 108 cfu/fish. Control fishes were injected with 0.2 ml trisalt solution. Disease sign and mortality of fishes were observed every eight hour for 40 days. LD50 was calculated based on Dragstedt Behrens method (Hubert, 1980). Result indicated that infection of the bacteria at 106 and 108 cfu/fish caused sub-acute disease signs, such as haemorhagic on operculum, base of fins (pinnae pectorales, pinnae abdominales, pinna analis), and also head and abdomen, while infection at 102 and 104 cfu/fish caused chronic disease signs, such as haemorhagic on fins base which was followed by necrotic on fins and skin tissue in prolonged time. Histopathologically, infection of the bacteria caused atrophy on the gills, infiltrations of lymphocyte, heterofel and plasma cell on the gills and fins base, vacuolar degeneration on the liver, and also present the bacteria colony on the fins base and intestine tissues. V. fluvialis 24SK has LD50 at (1,1±0,5)x107 cfu/fish.
Protein Aeromonas Hydrophila Sebagai Vaksin untuk Pengendalian Mas (Motile Aeromonas Septicemia) pada Jambal Siam (Pangasius hypophthalamus) Olga, Olga; Rini, Ririen Kartika; Akbar, Junius; Isnansetyo, Alim; Sembiring, Langkah
Jurnal Perikanan Universitas Gadjah Mada Vol 9, No 1 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Objectives of this study were to find out the protein of Aeromonas hydrophila for vaccine, and to evaluate the efficacy of the protein to control Motile Aeromonas Septicemia (MAS) in catfish (Pangasius hypophthalamus). A. hydrophila was isolated from the kidney of diseased catfish. The isolate was cultured, sonicated and sentrifuged into cell-free extract and debris. The protein in the cell-free extract was precipitated with ammonium sulphate, dialized, then fractionated by gel filtration column chromatography on Sephadex G150.The concentration of protein were estimated by spectrophotometer at 280 nm. Fractions obtained from the gel filtration with high concentration of protein were analized by sodium dodocyl sulphate-polyachrylamide gel electrophoresis (SDS-PAGE). The protein bands obtained from SDS-PAGE with molecular weight more than 10 kDa were tested for immunogenicity to mice. Futhermore, the most immunogenic protein was used for fish vaccination. The vaccinated and unvaccinated fishes were chalenged with A. hydrophila.The results indicated that vaccination with the protein significantly increased (P<0.01) the antibody titer either in mice or catfish. Relative Percent Survival of catfish vaccinated with the protein at 5; 7.5; and 10 µg/fish were 61.54%, 80.77% and 76.92%, respectively. The optimum dose of vaccine was 8.33 µg/fish with maximum RPS of 82.05%.