Nutrient Broth Medium Research Articles

Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric

Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

The effect of pH and incubation time on rude protease enzymes activity of Bacillus mycoides from anchovy isolates (Stolephorus sp.)

Proteases are several enzymes that are in important positions in the enzyme industry. eyebrow hydrolysis of peptide bonds on protein. Bacillus mycoides bacteria can grow in Nutrient Broth medium, these bacteria are included in facultative anaerobic bacteria. Bacillus mycoides are resistant to penicillin, positive to form Beta-hemolysis, positively catalyze hydrogen without oxidase, positively reduce nitrates and reduce methylene This study aims to determine the effect of pH and time on the protease enzyme activity of Bacillus mycoides from salted anchovy isolates. This research was conducted using a descriptive method with the treatment of pH concentration which has 5 levels, namely pH 5, 6, 7, 8 and 9 which are grouped based on the incubation time which has 4 levels, namely 6 hours, 12 hours, 18 hours and 24 hours and each was repeated 2 times. The results showed that pH and time affected the protease enzyme activity of Bacillus mycoides from salted anchovy isolate. Different pH treatments gave significantly different effects (F count> F5%) and the optimum activity was obtained at pH 5 of 5.243 U/ml. The different incubation time treatments had a significantly different effect (F count> F5%) and the optimum activity at 24 hours was 5.154 U/ml.

Bacterial Degradation of Synthetic Dye by Pseudomonas sp. Obtained from Dyeing Mill Effluent

Environmental pollution is one of the major concerns of today’s world. Due to rapid industrialization and urbanization, a large number of wastes are generated and discharged into the environment and causing major pollution problems. For obtaining reactive dye decolorizing bacterial isolates, effluent samples were collected from a dyeing mill. From bacterial pure culture, 10 isolates were selected for screening. Screening of these isolates for the capability to decolorize and degrade reactive dye was performed in a nutrient broth medium containing reactive dye. 6 isolates among these bacterial isolates showed dye decolorizing ability within 120 hours of incubation. These isolates were further identified based on cultural, morphological, and biochemical characteristics. These characteristics indicated that these six bacterial isolates were distributed to the bacterial genus of Bacillus (2 isolates), Pseudomonas (2 isolates), Aeromonas (1 isolate), and Alcaligenes (1 isolate). For the study of dye decolorization and degradation, Reactive Dark Blue dye used in jute and textile industries was chosen. Pseudomonas, a prominent dye decolorizing isolate during screening, was taken for the optimization of different physicochemical parameters. This bacterium decolorized and grew well up to 500 mgL-1 of Reactive Blue dye. Pseudomonas sp. showed noteworthy decolorization of approximately 84% at 200 mgL-1 of dye concentration after 96 h of incubation. The optimum temperature for dye degradation was at 37 °C. The maximum level of decolorization for Pseudomonas sp. was observed at pH 8.0. This isolate showed better decolorization extent under static conditions rather than shaking conditions. This result indicated that the dye had been utilized by this bacterial isolate. It can be concluded that Pseudomonas is a prospective candidate in the biodegradation of Reactive Blue dye and might be useful in bioprocess technology used for the bioremediation of dyeing mill effluents.

Development of a Sustainable Alternative for the Ammoniacal Cyanidation of Copper–Gold Ores Through a Biological Approach

Due to the problems arising from the presence of copper in the gold cyanidation process, including high cyanide consumption and occupation of the activated carbon surface, biological ammoniacal cyanidation was implemented as a resolution. Bacillus megaterium was implemented to produce cyanide and ammonia in nutrient broth and vinasse media. Maximum cyanide concentrations of 29.8 and 47.5 mg/L were produced using 3 g/L glycine in the mentioned media, respectively. Afterward, the Box-Behnken experiment design method was implemented to optimize ammonia biogenesis in vinasse culture, where 1974 mg/L ammonia was synthesized at 13.56 g/L calcium chloride, 7.2 g/L urea, and pH = 7.9. Three different strategies, namely: (1) bio-cyanidation, (2) bio-ammoniacal cyanidation, and (3) bio-ammonia pretreatment/bio-cyanidation, were employed to recover gold and copper from a 1% solid pulp. Afterward, using the nutrient broth medium, 49% gold was recovered by the first approach; the second approach extracted 9.2% gold and 33.5% copper, and 77% copper removal and 76% gold recovery were achieved through the third technique. Nutrient broth substitution with vinasse increased the gold recovery to 97% in the third approach. Hence, ammoniacal pretreatment, in combination with bio-cyanidation, is suggested to recover gold from copper–gold ores. In conclusion, the originality of the present work lies first in developing a bacterial culture procedure in which nutrient broth was replaced with vinasse as an industrial byproduct to make the process more eco-friendly. And secondly, in using the biogenic ammonia to leach out copper from the copper–gold ore and prepare it for gold biocyanidation.

Characterization of Multi-Drug Resistant Gram-Negative Bacteria Present in Fresh Leafy & Salad Vegetables in Dhaka, Bangladesh

This study was conducted to determine the microbiological quality of fresh raw and unwashed leafy and salad vegetables in Dhaka, Bangladesh. Six different types of samples (red amaranth, spinach, carrot, radish, tomato, and cucumber) were collected in pre-sterilized zip-lock bags from various local markets in Dhaka. All samples were transported to the Centre of Excellence for Global Health Research Laboratory of Primeasia University at earliest convenience. Samples were enriched in Nutrient Broth media and were then cultured on selective media for isolation purpose. Serial dilution was performed for the total viable count. Biochemical and carbohydrate profiling was conducted for the presumptive identification of the isolates. Commercial antibiotic discs were used for antibiogram by Kirby-Bauer disc diffusion method on Mueller-Hinton agar medium. The total microbial load ranged from 8x107 to 1.70x108. Total 36 isolates were identified, having 7 differentorganisms. The most predominant organism was Vibrio sp. (23%) followed by Klebsiella sp.(20%), Acinetobacter sp. (19%), Pseudomonas sp. (19%), Salmonella sp. (8%), Moraxella sp.(8%) and Escherichia coli (3%). 11% of the Vibrio sp. isolates were V. cholerae, found from 4 samples. No presence of V. cholerae was observed in the tomatosamples. E. coli was observed only in Carrot sample. Antibiotics from 7 different groups were tested against the organisms among which Imipenem showed the highest sensitivity (86%). Following Ceftriaxone (100%), Nitrofurantoin (94%), Erythromycin (89%) and Amoxicillin (83%) had the highest resistance against the isolated organisms. Moreover, most of the isolates showed a multi-drug resistance pattern where they were resistant to at least four drugs. Prevalence of pathogenic bacteria in raw unwashed vegetables can cause potential adverse health effects and therefore the consumers need to be conscious about the matter.

Characterization of Multi-Drug Resistant Gram-Negative Bacteria Present in Fresh Leafy & Salad Vegetables in Dhaka, Bangladesh

This study was conducted to determine the microbiological quality of fresh raw and unwashed leafy and salad vegetables in Dhaka, Bangladesh. Six different types of samples (red amaranth, spinach, carrot, radish, tomato, and cucumber) were collected in pre-sterilized zip-lock bags from various local markets in Dhaka. All samples were transported to the Centre of Excellence for Global Health Research Laboratory of Primeasia University at earliest convenience. Samples were enriched in Nutrient Broth media and were then cultured on selective media for isolation purpose. Serial dilution was performed for the total viable count. Biochemical and carbohydrate profiling was conducted for the presumptive identification of the isolates. Commercial antibiotic discs were used for antibiogram by Kirby-Bauer disc diffusion method on Mueller-Hinton agar medium. The total microbial load ranged from 8x107 to 1.70x108. Total 36 isolates were identified, having 7 differentorganisms. The most predominant organism was Vibrio sp. (23%) followed by Klebsiella sp.(20%), Acinetobacter sp. (19%), Pseudomonas sp. (19%), Salmonella sp. (8%), Moraxella sp.(8%) and Escherichia coli (3%). 11% of the Vibrio sp. isolates were V. cholerae, found from 4 samples. No presence of V. cholerae was observed in the tomatosamples. E. coli was observed only in Carrot sample. Antibiotics from 7 different groups were tested against the organisms among which Imipenem showed the highest sensitivity (86%). Following Ceftriaxone (100%), Nitrofurantoin (94%), Erythromycin (89%) and Amoxicillin (83%) had the highest resistance against the isolated organisms. Moreover, most of the isolates showed a multi-drug resistance pattern where they were resistant to at least four drugs. Prevalence of pathogenic bacteria in raw unwashed vegetables can cause potential adverse health effects and therefore the consumers need to be conscious about the matter.

The Antibacterial Effect of Soursop Leaf Extract on <i>Staphylococcus aureus</i> ATCC<sup>®</sup> 25923<sup>tm</sup> (<i>In Vitro</i>)

Purpose: This study was to determine the Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) soursop leaf extract antibacterial activity against bacteria Staphylococcus aureus ATCC® 25923TM at a concentration of 100, 90, 80, 70, 60 and 50%. Method: This study is an experimental laboratory with design Post Test Only Control Group Design by using soursop leaf extract at a concentration of 100, 90, 80, 70, 60 and 50% respectively. Each test is repeated four times to determine the average Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC). Soursop leaf extract is made by the dilution method using a medium Nutrient Broth (NB) and sub-cultured on Mannitol Salt Agar (MSA). The number of colonies of Staphylococcus aureus ATCC® 25923TM are counted using a manual calculation in the media Mueller Hinton Agar (MHA). Test Kruskkal Wallis there are differences in the antibacterial effect that was significant (p <0.05), soursop leaf extract against Staphylococcus aureus ATCC® 25923TM from each treatment group. Results: The minimum inhibitory concentration was 90% and the minimum bactericidal concentration is 100% committed. The number of colonies of Staphylococcus aureus bacteria ATCC® 25923TM at 100% concentration 0 CFU/mL 90% 234.50 CFU/ml and at concentrations of 80, 70, 60 and 50% could not count the number of colonies due to > 300 CFU/mL. As for the diameter of inhibitory zone at 100% concentration 10,625 mm, 90% 8,875 mm, 6,750 mm 80% and 70%, 60%, 50% reporting no inhibition zone diameter. Conclusion: The p-value of Kruskkal Wallis is p<0,05 which shows that there is a significant difference between the inhibition of soursop leaf extract with a concentration of 100, 90, 80, 70, 60 and 50% on growth of Staphylococcus aureus ATCC® 25923TM. Thus, soursop leaf extract has antibacterial effects against the growth of Staphylococcus aureus ATCC® 25923TM.

Evaluation of Biological Control Agents and Conventional Products for Post-Harvest Application on Potato (Solanum tuberosum L.) to Manage Leak

Biological control agents applied post-harvest may provide an effective way to manage leak of potatoes by competing with Pythium ultimum. The objective of this paper was to test the efficacy of various biological control agents and conventional post-harvest fungicides to manage leak. Two studies were performed with a Pseudomonas fluorescens triculture including desiccation tolerant variants of strains S11P12, P22Y05, and S22T04 in three formulations (a “fresh” triculture, dried on Kenite 700, or dried on Attapulgite clay) applied at a rate of 3.5 mL kg−1. A third study contained treatments of the P. fluorescens “fresh” triculture, triculture dried on Kenite, triculture dried on Kenite blended with a fungicide containing a three-way mixture of azoxystrobin, fludioxonil, and difenoconazole (Azo+Flu+Dfz, 0.033 mL kg−1), and triculture dried on Kenite rehydrated with nutrient broth Medium 1 blended with the same fungicide. Other treatments included Pseudomonas syringae (3.5 mL kg−1) in Studies 1 and 2, while hydrogen peroxide and peroxyacetic acid (0.042 mL kg−1), phosphorous acid (4.2 mL kg−1), and Azo+Flu+Dfz (0.033 mL kg−1) were used in all three studies. The three studies were each repeated twice using unwashed tubers (cv. ‘Russet Burbank’) that were wounded and inoculated with P. ultimum. After inoculation, tubers were treated, then stored at 21 C for four days prior to disease evaluation. In Study 1, none of the formulations of P. fluorescens triculture significantly controlled leak compared to the inoculated control; however, the “fresh” triculture formulation significantly decreased leak incidence by 24% in Study 2. In all three studies the three-way fungicide mixture Azo+Flu+Dfz resulted in lower leak incidence (1, 7, and 24%, in studies 1, 2, and 3 respectively) compared with the inoculated control (12, 37, and 50% leak incidence, in studies 1, 2, and 3 respectively). Study 3 showed the formulations of P. fluorescens strains were not effective in leak control but when combined with Azo+Flu+Dfz leak incidence significantly decreased compared to the inoculated control. The lowest leak incidence was seen when the triculture component of the fungicide mix was rehydrated with Medium 1 in Study 3. The P. fluorescens triculture formulations mixed with Azo+Flu+Dfz and rehydrated with Medium 1 had slightly higher bacterial counts than the “fresh” triculture and the triculture dried on Kenite, potentially indicating a greater activity level afforded by cell revival in dilute culture medium. P. syringae, phosphorous acid and hydrogen peroxide-peroxyacetic acid were ineffective in controlling leak in all three studies. The efficacy of the Azo+Flu+Dfz mixture appears promising for the management of leak in storage. The efficacy of P. fluorescens tricultures used alone was inconsistent in limiting leak incidence (only significant in 1 of 3 studies), and additional research and development may be warranted to realize its potential as biological control agent.

Precise quantification of the antibacterial activity of chitosan by NB medium neutralizer

In the present research, nutrient broth (NB) medium was identified to be able to neutralize the antibacterial activity of chitosan and its derivatives. Therefore, an improved test method independent of NB medium was proposed to precisely quantify the antibacterial effectiveness and efficiency of chitosan. The minimum bactericidal concentration (MBC) of chitosan was 60 μg mL−1 against S. aureus and E. coli, and 0.01 % (w/v) chitosan could kill 100 % of bacteria within 3 min. From another point of view, the neutralizing efficiency of NB could be tripled by adding 25 g L−1 of sodium chloride. Then the neutralizing mechanism of NB medium was ascribed to flocculation between chitosan and protein. Adding extra sodium chloride could significantly reduce the size of floccules, and smaller floccules would lose the ability of binding with bacteria directly, showing higher neutralizing rate on the macro scale.

A smart and active film with tunable drug release and color change abilities for detection and inhibition of bacterial growth.

Antimicrobial resistance has become a global issue and thus the development of natural products/biomedical materials composites with antibacterial activities is urgently needed. When acute wounds develop into chronic wounds, the wound environments become alkaline. As long as infections occur, the wound pH further increases, making the wounds difficult to heal. Besides, bacterial growth in poultry, meat, fish and seafood products is usually reflected in a marked increase of pH values. Herein, smart, stimuli responsive self-assembled multilayer and complex film were constructed through the formation of hydrogen bonds and hydrophobic interactions between hydroxypropyl methylcellulose (HPMC) and epigallocatechin-3-gallate (EGCG), thereby greatly reducing the hydrophilicity of HPMC and offering enhanced mechanical strength, superior free radical scavenging capability, and improved water vapor and light barrier properties. The EGCG/HPMC complex film was able to control EGCG release by tuning pH or temperature of the release medium. Furthermore, incorporation of CuS nanoparticles into the film allowed it to triggers EGCG release in an on-demand fashion under near-infrared (NIR) exposure. Bacterial growth in glucose-free nutrient broth medium caused pH to rise (near pH8.0), leading to transformation of EGCG from phenol type to phenolate ion and then quinone, allowing for spontaneous generation of H2O2 to kill bacteria. The complex films changed their color in response to bacterial growth because EGCG transformed from phenol type to quinone type under alkaline condition. The green synthesized EGCG/HPMC complex films can be used as a colorimetric pH indicator and an antibacterial material for wound dressing and food packaging applications.

Crude extract from a hardcoral-associated bacterium Virgibacillus salarius PHC-44-04 inhibiting growth of Multidrug-Resistant Enterobacter aerogenes human pathogen

Abstract. Ayuningrum D, Muchlissin SI, Trianto A, Radjasa OK, Sabdono A. 2018. Crude extract from a hardcoral-associated bacterium Virgibacillus salarius PHC-44-04 inhibiting growth of Multidrug-Resistant Enterobacter aerogenes human pathogen. Biofarmasi J Nat Prod Biochem 18: 78-83. Hardcoral-associated bacteria are potential sources of natural product compounds with wide range of activities, i.e. antibacterial activity, antiviral, anticancer, antifungal, etc. Virgibacillus salarius PHC-44-04 is a gram-positive bacterium that was isolated in prior research from hard coral Pavona sp. collected from Panjang Island, Jepara, Indonesia. This bacterium at the screening phase was showing high antibacterial activity against Multidrug-Resistant Enterobacter aerogenes (MDR-EA). Therefore, this paper has aim to deliver the result of crude extract antibacterial test from V. salarius PHC-44-04 after being cultivated in liquid medium, to know the efficiency of using supernatant and pellet extract and to determine in which concentration was the minimum for antibacterial activity. The cultivation of bacterial isolates was using liquid medium Nutrient Broth, and the production of crude extract was using liquid-liquid extraction method. The liquid medium containing bacterial cell was separated using centrifuge with 8000 rpm became supernatant and pellet. The supernatant was extracted using ethyl acetate, while the pellet was extracted using methanol. The antibacterial test from both crude extract from supernatant and pellet was using disk diffusion method with several concentration as follows: 15 µg/mL, 30 µg/mL, 60 µg/mL, 90 µg/mL, 180 µg/mL, 250 µg/mL, 350 µg/mL, and 500 µg/mL. Each concentration was repeated in three replicates. The crude extract produced from 700 mL supernatant was 0.0667 g and from 50mL pellet was 0.0320. The pellet crude extract has higher mass but not having antibacterial activity against MDR-EA. The result showed only supernatant crude extract of V. salarius showed antibacterial activity against MDR-EA bacterium. The minimal crude extract concentration to inhibit the growth of MDR-EA was 60 µg/mL, meanwhile, the best concentration for exhibiting antibacterial activity was at 500 µg/mL with zone of inhibition (ZOI) diameter of 11.77 ± 0.8730 mm. Thus, supernatant should be the main source of crude extract production rather than the pellet to get high antibacterial activity.

Cadmium (Cd) Resistance of Isolate Bacteria from Poboya Gold Mining in Palu, Central Sulawesi

The mining industry in Indonesia is growing rapidly, giving an environmental impact from the waste produced because it contains dangerous heavy metals, one of which is cadmium metal (Cd). The purpose to know the ability of cadmium resistance in bacterial isolates. Bacterial isolates were obtained from soil samples from the Poboya Gold Mine field, Palu. Bacterial isolation and selection were carried out by the plate count method, resistance testing using nutrient broth media containing heavy metals Cd, analysis of Cd concentrations using atomic absorption spectrophotometers. From the results of the study, four types of Cd resistant isolates were obtained based on macroscopic and microscopic characterization results, namely isolates EK1, EK2, EK3 and EK4. The two best isolates of Cd resistance, EK2 and EK4 isolates were tested on media containing 20 ppm Cd, showing that both of these isolates were able to significantly reduce Cd concentrations in the supernatant and pellets.

First Report of Rhizome Rot on Ginger (Zingiber officinale) Caused by Enterobacter cloacae in Shandong Province, China.

Ginger (Zingiber officinale Roscoe) is an economically important crop that is planted on 60,000 to 70,000 ha every year in Shandong Province, China. During June to July 2019, a bacterial disease was observed on ginger in Shandong Province. The disease incidence was about 15% in each of three fields in Rushan (36°41'-37°08'N, 121°11'-121°51'E). Symptoms included leaves chlorosis, basal root and stem rot, rhizome rot, and wilting of foliage. The symptoms were similar to those associated with bacterial wilt of ginger, caused by Ralstonia solanacearum (Smith) Smith, which is a devastating and destructive disease of ginger in China (Ren and Fang 1981). Bacterial colonies isolated from infected ginger rhizomes surface-disinfested with 1% NaClO were purified on nutrient agar (NA) medium. Eighteen isolates were obtained from 20 diseased tissue samples. Three virulent isolates (SDGR1, SDGR2, and SDGR3) that differed morphologically from strains of R. solanacearum were selected for characterization based on morphological, biochemical, and molecular analyses. The colonies were light yellow, circular, smooth, and convex on NA. Individual cells were rod-shaped with peritrichous flagella (Dong and Cai 2001). The isolates were Gram negative, facultatively anaerobic, negative for urease, oxidase, and pectate degradation, as well as positive for pectate degradation, catalase, lactose and asparagine utilization (Dong and Cai 2001). These characteristics were consistent with Enterobacter spp., a member of the Enterobacteriaceae. For further identification, bacterial DNA of each of the three isolates was extracted from pure culture grown in liquid nutrient broth (NB) medium on a shaker at 28°C for 16 h. The sequence of the 16S rRNA gene was amplified using primers 27F/1492R (Lane 1991). The three isolates had identical 16S rRNA sequences and the consensus sequence (GenBank Accession No. MT233300) was analyzed by NCBI BLASTn, revealing 99.8% identity with the sequence of the 16S rRNA gene from E. cloacae strain R8-2 (GQ406570) (Wang et al. 2008). Construction of a phylogenetic tree of 16S rRNA sequences using a maximum likelihood analysis showed that the three ginger isolates clustered most closely with E. cloacae. The pathogenicity of three isolates was tested on ginger plantlets and immature rhizomes (Nishijima et al. 2004). Fifteen plantlets for each of the three isolates were each injected with 0.3 ml of a bacterial suspension (107 to 108 CFU/ml) at the stem base. The same number of plantlets were injected with sterile water as a control treatment. Inoculated plants were placed in a chamber at 90% relative humidity (RH) and 28°C with a photoperiod of 16 h. Rhizomes were surface-disinfected in 1% NaClO for 3 min, rinsed in distilled water, then cut into segments. Fifteen segments for each of the three isolates were injected with 0.2 ml of bacterial suspension (107 to 108 CFU/ml). Control rhizomes were treated similarly with sterile water. All rhizomes were placed in a chamber at 90% RH and incubated at 30°C in the dark. The pathogenicity tests were conducted three times independently. Brown discoloration, chlorosis, and necrosis of leaves developed 2 to 3 weeks after inoculation of plantlets for all three isolates. Water-soaked, brown and rotted rhizome tissue developed about 1 week after inoculation of rhizomes for all three isolates. No obvious symptoms were observed on the control plantlets and rhizome segments. The bacterial isolates that caused symptoms on the inoculated plants were re-isolated and identified as E. cloacae based on the same morphological, biochemical, and molecular analyses. The pathogen was not isolated from the control plantlets and rhizomes. E. cloacae has been reported as the cause of ginger rhizome rot in the United States and mulberry wilt in China (Nishijima et al. 2004; Wang et al. 2008). To our knowledge, this is the first report of ginger rhizome rot caused by E. cloacae in China. Confirming the existence of this pathogen in this area will be useful to adopt effective field management measures to control this disease on ginger.

DETEKSI KRISTAL PROTEIN PADA ISOLAT BACILLUS SP. DENGAN PEWARNAAN COOMASSIE BRILLIANT BLUE

Bacillus is one of the genera of bacteria that has the potential to be developed in the field of biotechnology. Bacillus is able to produce protein crystals during the stationary phase in its life cycle. This protein crystal is a protoxin that is active will be pathogenic in insects. In this study, protein crystal staining was carried out using coomassie brilliant blue dye on the isolate of Bacillus sp. with isolate code B1, B2, and B3. Before being stained, the three isolates were bred on Nutrient Broth (NB) medium at pH 5, pH 7, and pH 9, then incubated for 72 hours. Based on research results, the three isolates were detected to produce protein crystals. Crystal protein of Bacillus sp. on media with a pH of 5 and pH 7 located at the end of the spore, while the protein crystal of Bacillus sp. isolate on media with a pH of 9 located on the outside of the spores.

First report about the screening, characterization, and fosmid library construction of Xanthomonas oryzae pv. oryzae strain with resistance to Fubianezuofeng

Bacterial blight (BB), which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is a common bacterial disease that seriously harms rice production in major rice-growing areas worldwide. Fubianezuofeng (FBEZF), a sulfone bactericide that contains an oxadiazole moiety, exerts good control effect on BB. In this study, FBEZF-resistant strains of Xoo were screened for the first time in the laboratory to evaluate the risk of Xoo developing resistance to FBEZF. Three strains with moderate resistance to FBEZF, were obtained and named as F1, F2, and F3, which have resistance factors (RF) of 14.69, 15.72, and 11.12, respectively. FBEZF lacked positive cross-resistance to bismerthiazol, thiodiazole copper, zhongshengmycin and phenazino-1-carboxylic acid. The growth rates of the resistant strains F1 and F2 were similar to those of the wild-type strain in nutrient broth medium, but differed in nutrient agar medium. The extracellular polysaccharide production and pathogenicity of F1, F2, and F3 were reduced relative to those of the wild-type strain. A fosmid library containing 2304 transformants was constructed based on the genome of F2, and transformants 2193 and 2202 exhibited FBEZF resistance. The results are helpful for further study on the molecular mechanism of resistance to FBEZF in Xoo.

Identification, Determination and Quantification of Indole-3-Acetic Acid Produced by Pseudomonas aeruginosa UPMP3 and Its Effect on The Growth of Oil Palm (Elaeis guineensis Jacq)

Pseudomonas species have founded as greatest and potentially most promising group of plant growth promoting rhizobacteria (PGPR). Pseudomonas aeruginosa UPMP3 is an important PGPR isolated from oil palm rhizosphere. This rhizobacteria is likely to synthesize and release phytohormone indole-3 acetic acid (IAA). Production of IAA is one of the main reasons to promote plant growth and yield. The aim of this study was to detect, identify and quantify the IAA production by P. aeruginosa UPMP3 in vitro and its influence on oil palm seedling growth. Nutrient broth medium supplemented with 1-5 mg/ml L-tryptophan and without L- tryptophan were used for bacterial culture. The pH levels of culture media were optimized under shaken and static conditions and incubated at 28±2°C in different incubation periods. The production of IAA by P. aeruginosa UPMP3 was extracted, purified, detected and quantified by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) analyses. Production of IAA was quantified by HPLC in liquid culture and achieved 12.08µg/ml with a retention time of 13.711 min. On the other hand, the maximum 52 µg/ml IAA was recorded in the medium supplemented with 4 mg/ml L- tryptophan in compare to control. The optimum pH level of the culture medium was recorded as 7 under shaken conditions at 150 rpm with 5 days incubation. The influence of IAA produced by the UPMP3 on oil palm seedling growth was carried out in the pot experiment. The germinated oil palm seedlings were treated with the extract of bacterial strain and observed a positive effect on seedling growth in respect to average root and leaf number, root, shoot, and leaf length compare to the synthetic IAA and the control.

The compound paraquat dichloride hydrate significantly affects the in vitro growth rate of a Chromobacterium violaceum wild type strain

The present study aimed to assess the <em>in vitro</em> growth rate of the <em>C. violaceum</em> wild type strain ATCC12472 when exposed to the compound paraquat dichloride hydrate, one of the most herbicides largely employed in agriculture around the world. The bacterium was monitored using spectrophotometry to estimate the growth rate of bacterial inocula incubated in a nutrient broth medium containing the compound paraquat at different concentrations. Moreover, the number of viable <a title="Bacteria" href="https://en.wikipedia.org/wiki/Bacteria">bacteria</a> cells in the samples was also estimated based on colony-forming units on the nutrient broth agar. Results showed that the compound paraquat promoted significant decreases in the growth rate of the <em>C. violaceum </em>bacterium in response to incubation time and dose tested. Significantly lower values of bacterial growth were also corroborated by counting the total number of colony-forming units at final concentrations of 100 (<10<sup>4 </sup>CFU) and 1,000 µg mL<sup>-1 </sup>(<10<sup>2 </sup>CFU) in comparison to control and 0 µg mL<sup>-1 </sup>(>10<sup>8 </sup>CFU) on the nutrient broth agar. Thus, the findings described herein report the effects of the compound paraquat dichloride hydrate on the <em>in vitro </em>growth rate of a <em>C. violaceum</em> wild type strain.

Potential effect of Allium sativum bulb for the treatment of biofilm forming clinical pathogens recovered from periodontal and dental caries.

Biofilm producing clinical bacterial isolates were isolated from periodontal and dental caries samples and identified as, Lactobacillus acidophilus, Streptococcus sanguis, S. salivarius, S. mutansand Staphylococcus aureus. Among the identified bacterial species, S. aureus and S. mutansshowed strong biofilm producing capacity. The other isolated bacteria, Streptococcus sanguis, S. salivarius showed moderate biofilm formation. These pathogens were subjected for the production of extracellular polysaccharides (EPS) in nutrient broth medium and the strain S. aureus synthesized more amounts of EPS (610 ± 11.2 µg/ml) than S. sanguis (480 ± 5.8 µg/ml).EPS production was found to be less in S. salivarius (52 ± 3.8 µg/ml).The solvent extract of A. sativum bulb showed the phytochemicals such as, carbohydrate, total protein, alkaloids, saponins, flavonoids, tannins and sterioids. The solvent extract of A. sativum bulb showed wide ranges of activity against the selected dental pathogens. The difference in antibacterial activity of the solvent extract revealed differences in solubility of phytochemicals in organic solvents. Ethanol extract was highly active againstS. aureus (25 ± 2 mm). The Minimum Inhibitory Concentration (MIC) of crude garlic bulb varied widely and this clearly showed that bacteria exhibits different level of susceptibility to secondary metabolites. MIC value ranged between 20 ± 2 mg/ml and 120 ± 6 mg/ml and Minimum Bactericidal Concentration (MBC) value ranged from 60 ± 5 mg/l to 215 ± 7 mg/ml. To conclude, A. sativum bulb can be effectively used to treat periodontal and dental caries infections.

Antibacterial activity tests of isolate endophytic bacteria from the tea plant (Camellia sinensis) againts Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus is one of the most common types of bacteria in Asia that causes local infectious diseases of the skin, nose, urethra, vagina, digestive tract, pneumonia, endocarditis, septic arthritis, and septicemia. Staphylococcus aureus and Staphylococcus epidermidis are the most common types of Staphylococcus in Asia. Tea plants contain bioactive compounds and endophytic bacteria which are widely used as antimicrobial agents. Endophytic bacteria are bacteria that exist in plant tissues, not pathogenic, and have the ability as the host plant. The purpose of this study was to determine the antibacterial activity of endophytic bacterial isolates of tea plants (Camellia sinensis) against the growth of Staphylococcus aureus and Staphylococcus epidermidis bacteria. The antibacterial activity test of endophytic bacteria of tea plants includes a series of processes such as sample selection, surface sterilization of samples, isolation of endophytic bacteria in agar medium, screening, suspension of endophytic bacteria in 0.9 % NaCl and standardized with 0.5 McFarland, making endophytic bacterial culture in nutrient broth medium, making endophytic bacterial supernatant and antibacterial activity test with paper disc diffusion method. The result is that there is an antibacterial activity from the endophytic bacterial supernatant isolates B14, B23, and A2 to the growth of Staphylococcus aureus and Staphylococcus epidermidis. The best antibacterial activity was found in endophytic bacterial B14 isolates with inhibition zones of 7.75 mm and 12.5 mm followed by B23 isolates with 7.5 mm and 8.25 mm inhibition zones and A2 isolates with large inhibition zones of 7.42 mm and 8.16 mm. Endophytic bacteria of tea plants showed antibacterial activity against the growth of Staphylococcus aureus and Staphylococcus epidermidis.

Immobilization of Bacterial Cells and Chitinolytic Activity ofStreptomycessp. (PB 2)

This study was aimed to immobilize the chitinolytic bacteriaStreptomycessp. PB 2 using sodium alginate as immobilization agent.Streptomycessp. PB 2 was reported have a good chitinolytic activity and immobilization is known to increase the stability of bacteria during repeated used. Sodium alginate has been used on several methods of immobilization and sodium alginate are reported as a good agent. Cell immobilization was done by growing the cell on nutrient broth (NB), mixed with 1% sodium alginate to form the beads, then the mixture were put to a syringe and droped into CaCl2 solution. The beads were washed with 0.85% NaCl solution and filtered. To test the bacterial activity, beads were applied into chitin liquid medium and the chitinolytic activity was observed every 24 hours for 5 days. To test its stability, after the 5thday, the beads was filtered and put in to chitin liquid medium and tested the chitinolytic activity. This experiment was repeated twice. The highest chitinolytic activity ofStreptomycessp. PB2 was observed on the 4thday with the value of 0.00014 U/ml and the concentration of NAG of 5.42087 µg/ml with the treatment of immobilized NB medium without the addition of chitin.