Blood Agar Test Research Articles

VARIATIONS OF COLIFORM BACTERIA ISOLATED FROM BALI CATTLE CALVES FECES, MARKETED AT BERINGKIT ANIMAL MARKET

Beringkit Animal Market is one of the largest animal markets in Bali. Which is a place for livestock transactions, especially bali cattle calves, which are usually used as breeds. Bali cattle calves need digestive tract microbes such as bacteria to carry out the digestives process. Coliform bacteria are one of the bacteria that play an important role in the digestive tract of bali cattle calves. Coliform bacteria are one of the bacteria that plays an important role in the digestive tract of bali cattle calves. Coliform bacteria which are included in the Enterobacteriaceae group consist of Escherichia coli, Enterobacter sp., Klebsiella sp., and Citrobacter sp. This research aims to determine what variations of Coliform bacteria are found in the feces of bali cattle calves marketed at the Beringkit Animal Market. This research was observational with the number of samples used were 50 fecal samples from 50 bali cattle calves. The samples were isolated on Eosin Methylene Blue Agar (EMBA) media and then tested and identified using the Indole Methyl Voges Proskauer Citrate (IMViC) principles and Blood agar test. The result showed that 82% of samples were positive Coliform. Based on the result, it can be concluded that in the feces of bali cattle calves marketed at the Pasar Hewan Beringkit contain Coliform bacteria consisting of Escherichia coli, Enterobacter sp., Klebsiella sp., and Citrobacter sp. with the highest percentage was Escherichia coli at 58%, followed by Citrobacter sp. at 12% and Enterobacter sp. and klebsiella sp. respectively 6%. It is necessary to count the total number of each bacteria to determine the pathogenicity of the bacteria.

Screening, biochemical characterization and antibiotics resistance/susceptibility of bacteria isolated from native soil and water samples.

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.

Antimicrobial activity of Limosilactobacillus fermentum strains isolated from the human oral cavity against Streptococcus mutans

Oral probiotics have been recently gaining much attention owing to their potential to inhibit the progression of dental caries by controlling the cariogenic effects of Streptococcus mutans. We isolated and genotypically identified 77 lactic acid bacteria including 12 Limosilactobacillus fermentum probiotic candidates from the oral cavity of healthy volunteers. Among the 12 L. fermentum isolates, nine isolates effectively inhibited the growth of S. mutans via hydrogen peroxide (H2O2) production. The others neither suppressed the growth of S. mutans nor produced H2O2. Eight out of the nine H2O2-producing L. fermentum isolates exhibited strong adherence to oral epithelial KB cells while inhibiting the adherence of S. mutans to KB cells. The eight H2O2-producing isolates were neither haemolytic based on a blood-agar test, cytotoxic according to lactate dehydrogenase assay, nor resistant to eight antibiotics represented by the European Food Safety Authority guideline, indicating that the isolates have potential to suppress the cariogenesis driven by S. mutans while providing general probiotic benefits.

Penapisan dan Karakterisasi Amilase dari Bakteri Asal Ekoenzim

Eco-enzymes are one of the potential sources for obtaining amylolytic bacterial isolates. The study aims to screen amylolytic bacteria from eco-enzymes, characterize semi-purification amylase, and identify amylolytic bacteria molecularly using the 16S rRNA gene. Screening for amylolytic bacteria was carried out by measuring the amylolytic index on a Nutrient agar medium containing 1% tapioca starch. The amylolytic isolate which had the highest index and was non-pathogenic was selected for the amylase characterization process. Testing of amylase activity was carried out using the Bernfeld method while the protein enzymes were measured using the Bradford method. The extracellular extract was concentrated using ammonium sulfate precipitation. PKL2 is gram-positive bacteria that was derived from eco-enzymes with the highest amylolytic indexes of 1.77, which were not pathogenic on the blood agar test. Optimum amylase was produced by PKL2 at the stationary phase at 21 h. The optimum pH and temperature of the amylase activity were observed to be 7.0 and 50°C, respectively. The amylase enzyme from PKL2 increased its purity 1.82-fold upon precipitation of ammonium sulfate at a concentration of 60%. Identification of bacteria based on molecular identification showed that PKL2 obtained was putatively identified as Bacillus amyloliquefaciens.
 
 Keywords: Amylase activity, Bacillus amyloliquefaciens, eco-enzyme, optimum pH

Application of 233\xa0nm far-UVC LEDs for eradication of MRSA and MSSA and risk assessment on skin models

A newly developed UVC LED source with an emission wavelength of 233 nm was proved on bactericidal efficacy and skin tolerability. The bactericidal efficacy was qualitatively analysed using blood agar test. Subsequently, quantitative analyses were performed on germ carrier tests using the MRSA strain DSM11822, the MSSA strain DSM799, S. epidermidis DSM1798 with various soil loads. Additionally, the compatibility of the germicidal radiation doses on excised human skin and reconstructed human epidermis was proved. Cell viability, DNA damage and production of radicals were assessed in comparison to typical UVC radiation from discharge lamps (222 nm, 254 nm) and UVB (280–380 nm) radiation for clinical assessment. At a dose of 40 mJ/cm2, the 233 nm light source reduced the viable microorganisms by a log10 reduction (LR) of 5 log10 levels if no soil load was present. Mucin and protein containing soil loads diminished the effect to an LR of 1.5–3.3. A salt solution representing artificial sweat (pH 8.4) had only minor effects on the reduction. The viability of the skin models was not reduced and the DNA damage was far below the damage evoked by 0.1 UVB minimal erythema dose, which can be regarded as safe. Furthermore, the induced damage vanished after 24 h. Irradiation on four consecutive days also did not evoke DNA damage. The radical formation was far lower than 20 min outdoor visible light would cause, which is classified as low radical load and can be compensated by the antioxidant defence system.

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

The potential of endophytic bacteria isolated from Tagetes sp. to control Meloidogyne spp. infection on tomato plants

Abstract. Munif A, Nursalim M, Pradana AP. 2021. The potential of endophytic bacteria isolated from Tagetes sp. to control Meloidogyne spp. infection on tomato plants. Biodiversitas 22: 3229-3236. The root-knot nematode (Meloidogyne spp.) is one of the plant-parasitic nematodes that cause economical loss in the vegetable plantations in the world. The endophytic bacteria may act as a potential biological agent to control the plant-parasitic nematodes. The aim of this study was to explore the endophytic bacteria from Tagetes sp., which had a potential to control Meloidogyne spp. and act as growth promoter for tomato plants. The endophytic bacteria were isolated from the roots and stems of Tagetes sp. and tissues were cultured on the tryptic soy agar (TSA) media. The endophytic bacterial isolates were selected using hypersensitive and blood agar tests to identify their biological safety. The selected endophytic bacteria were also assessed for growth promoter test in the tomato plants. The selected endophytic bacteria were assessed in vitro against the Meloidogyne spp. juvenile 2 and their effectiveness in suppressing the root-knot nematodes on tomato plants in greenhouse. The results showed that total of 184 endophytic bacterial isolates were successfully isolated from the roots and stem of Tagetes sp. In hypersensitive and hemolytic tests, 78 and 36 isolates showed negative responses, respectively. Of the 36 isolates tested, it was found that 14 of them were able to promote the growth of tomato seedlings. In in vitro test, 14 endophytic bacteria showed 73-93% mortality rate of Meloidogyne spp J2. The results at greenhouse showed that 3 bacterial isolates, namely, AL21, AL44, and AL53 suppress the number of root-knot nematodes up to 50-74% and improve tomato plant growth by 32%. These results indicate that endophytic bacteria isolated from Tagetes sp. have the potential to act as biocontrol agents of parasitic nematodes and a plant growth promoter.

Biosurfactant Screening of <i>Halomonas meridiana</i> BK-AB4 for Microbial Enhanced Oil Recovery

Biosurfactant is produced by a certain microorganism to reduce surface tension. Microbial enhanced oil recovery (MEOR) is one of the many applications of biosurfactant. However, the biosurfactant for MEOR needs to be able to withstand the extreme environment of oil reservoirs with high temperature and high salinity. Halomonas meridiana BK-AB4 is a halophilic bacterium obtained from the Bledug Kuwu crater in Central Java, Indonesia. The similarity of both environment condition indicates the potential to produce suitable biosurfactant. This study evaluates the potential of Halomonas meridiana BK-AB4 in producing biosurfactant compared to several bacteria isolated from the crater. The blood agar test of Halomonas meridiana BK-AB4 exhibited greenish discoloration around the colony, indicating the ability of type α-hemolysis. The interfacial tension was measured using the Du Nouy ring method to represent the biosurfactant activity, with the results of 0.014 dyne/cm. These results showed that Halomonas meridiana BK-AB4 has a very good potential for producing biosurfactant for MEOR. The optimum time of the culture starter for production is 6 hours, as determined from the bacterial growth curve.

Evaluation of various methods of selection of B. subtilis strains capable of secreting surface-active compounds.

The aim of the study was the evaluation of a three-step method for the selection of bacterial strains capable of producing surfactin. The procedure consisted of the following steps: 1.blood agar test, 2. measurement of the surface tension (ST) of the medium using the du Nouy method before and after submerged culture, 3. qualitative and quantitative assessment of surfactin by HPLC. Forty five Bacillus subtilis natto strains producing haemolysis zones (≥3mm) were selected. Nineten of them reduced ST of the medium to ≤ 40 mN/m; in six cases, the reduction was as much as 50%. All indicated strains produced surfactin. Positive correlations (p <0.5) between the percentage reduction of ST of the medium and surfactin concentration (r = 0.44), indicate that this parameter is determinant of the ability to synthesize this compound. The blood agar test has been shown to be useful only as a pre-selection criterion for surfactin producers (18 strains selected by this method reduced ST by only ≤30%). The proposed selection strategy proved effective and made it possible to select the BS15 strain that reduced the ST of the medium to 30.56 ± 0.15 mN/m and simultaneously provided a high concentration of surfactin compared to other strains.

Designing a light-activated recombinant alpha hemolysin for colorectal cancer targeting.

Introduction: Colorectal cancer (CRC) is one of the main health burden worldwide, which can cause major economic and physiological problems along with relatively high rate of mortality. It is important to develop new methods for the localized delivery of recombinant protein therapeutics, in large part due to the failure of conventional therapies in most cases. Since E. coli Nissle 1917 (EcN) does not produce any virulence factors, here we used these bacteria with the light-activated promoter system to deliver therapeutic agents in the desired location and time. Methods: In this study, Staphylococcus aureus alpha hemolysin (SAH), after codon usage optimization, was cloned into blue light activating vector (pDawn) and transferred to EcN strain. Then, the functionality and cytotoxicity of secreted alpha hemolysin was evaluated in the SW480 colon cancer cell line by using different experiments, including blood agar test, flow cytometry analysis, and DAPI staining. Results: Our findings revealed that EcN can produce functional SAH under the blue light irradiation against SW480 cancer cells. Moreover, cytotoxicity assays confirmed the dose- and time-dependent toxicity of this payload (SAH) against SW480 cancer cells. Conclusion: Based on our results, EcN is proposed as an appropriate light-activated vehicle for delivery of anticancer agents to the target cancer cells/tissues.

Isolation and Identification of Symbiont Microorganisms from Bioluminescent Marine Life

Bioluminescence means the ability of animals or plants to naturally produce light. The three known ways by which bioluminescence is produced are through specific cells called photocytes, bioluminescent glands in tissues and symbiotic bioluminescent microorganisms. Bioluminescence in Loligo duvaucelii is known to be caused by the presence of symbiotic microorganisms in bioluminescent sacs. There is a need to compile more information on bioluminescent symbiotic microorganisms on marine life in Indonesia and their potential. This study aims to determine the species of bioluminescent microorganisms on squid and fish, namely Loligo sp. and Loligo edulis from the waters of Jepara and the Bombay duck (Harpadon nehereus) from the Strait of Malacca, Indonesia and their potential. The samples were collected by isolating the microorganisms from the luminescent organs, after which the bioluminescent microorganisms were used in the research. This research consisted of antimicrobial tests against pathogenic microorganisms which were conducted qualitatively. The bioluminescent microorganisms were identified using biochemical assay and molecular assay (16S rRNA PCR). Tests results from Loligo sp. symbiotic microorganisms found two isolates which showed antimicrobial activities against pathogenic Multi Drug Resistant (MDR) microorganisms, namely uncultured bacterium clone 1P-1-G05 against Escherichia coli with 32.59 mm of inhibitory zone and Uncultured bacterium clone 3g10a against Enterobacter sp. with 28.44 mm of inhibitory zone. The bioluminescent symbiont microorganisms in Loligo edulis, which was identified to be Photobacterium phosphoreum, showed antimicrobial activities against Vibrio harveyi, E. coli, Staphylococcus aureus, and Bacillus sp. Bioluminescent symbiotic microorganisms on H. nehereus identified Alteromonas macleodii, which showed gamma hemolysis on the blood agar test.

Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis)

Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylLl and cylLs could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

EVALUATION OF DIFFERENT SCREENING METHODS FOR BIOSURFACTANT PRODUCERS ISOLATED FROM EGYPTIAN FRESH WATER SAMPLES CONTAMINATED BY OIL SPILLS USING BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS

In all countries, oil exploration and use threatens the health of the environment and living creatures including humans. An oil spill is the release of petroleum hydrocarbon into the environment. One of the most applicable and safe method is the bioremediation treatment, using microorganisms .This work aims at treating oil spills in fresh water and to compile information on types and properties of biosurfactant. It also describes factors affecting these biosufactants production. Different screening methods e.g. oil spreading assay, Emulsification index (E24), Drop collapse assay, Blood agar test, Hemolysis, MaCconkey agar test were tested. Two isolates gave highly Emulsification tests named O3, O11, which were identified as Bacillus subtilis and Bacillus licheniformis respectively, were isolated from petroleum hydrocarbon contaminated water. Optimization of two isolates were done and highest biosurfactant activity at temperature ranges from 10 oC to 50 oC and pH ranges from 3 to 10 and salinity ranges from 0%to 30%. Also, the biosurfactant was characterized by Fourier transform infrared spectroscopy (FTIR) and surface tension measurement. FTIR showed the production of biosurfactant from Bacillus subtilis similar to surfactin, while the biosurfactant from Bacillus licheniformis similar to lichenysins. The use of microbial biosurfactants significantly decreases the hydrophobicity and increases the rate of hydrocarbon biodegradation. Biosurfactants obtain demonstrated good surface tension reduction capacity by Bacillus subtilis and Bacillus licheniformis up to 30 and 36Nm/m and have emulsifying activity 71% and 65% respectively.

Potential of Brevibacillus sp. AVN 13 isolated from crude oil contaminated soil for biosurfactant production and its optimization studies

In this study, a hydrocarbon degrading organism Brevibacillus sp. AVN 13 was isolated and utilized for biosurfactant production. A total of twenty four isolates were obtained from crude oil spilled soil and screened for biosurfactant production using different methods such as blood agar test, cetyl trimethylammonium bromide methylene blue agar test, hydrocarbon overlay agar test, emulsification test and surface tension. Among the different biosurfactant producers, the isolate AVN13 was noted as high yielding strain which reduced the surface tension up to 36 mN/m with 72% emulsification activity. By 16S rRNA analysis, the high yielding isolate was identified as Brevibacillus sp. AVN13. The effect of pH, temperature, carbon source and nitrogen source on biosurfactant production by Brevibacillus sp. AVN13 was investigated. It was observed that the optimum pH and temperature for biosurfactant production were 7 and 40 °C respectively. Similarly, used engine oil and potassium nitrate were found to be the preferred carbon and nitrogen sources at a concentration of 1% v/v and 0.5% w/v respectively. The kinetics of biosurfactant production was studied which clearly indicated that the biosurfactant production was associated with biomass growth. Furthermore, the produced biosurfactant was undergone stability analysis of pH and temperature and reported. The antimicrobial properties of biosurfactant produced were also analyzed. The biosurfactant produced was characterized using FTIR (Fourier transform infrared spectroscopy) and found to be lipopeptide type of biosurfactant. These results suggested that Brevibacillus sp. AVN 13 could be effectively employed for biosurfactant production.

Antifungal Lipopeptides Produced by Bacillus sp. FJAT-14262 Isolated from Rhizosphere Soil of the Medicinal Plant Anoectochilus roxburghii.

This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7mNm-1. Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.

The antifungal mechanism of Bacillus subtilis against Pestalotiopsis eugeniae and its development for commercial applications against wax apple infection

The inhibition zones of three Bacillus subtilis isolates, BS-99, BS-23857 and BS-33608 against Pestalotiopsis eugeniae were, 13.5, 0, and 0 mm, respectively. BS-99 showed the strongest inhibitory activity, whereas, no inhibitory activity was reported for BS-23857 or BS-33608. The spore germination rates of P. eugeniae were not significantly inhibited by n-hexane, ethyl acetate (EtOAc), or methanol (MeOH) extracts of the fermentation broths of BS-99, BS-23857, and BS-33608. However, the hyphae of P. eugeniae became swollen and malformed after 12 h and stopped growing after 30 h of treatment with the MeOH extract of the BS-99 fermentation broth. Analysis by polymerase chain reaction (PCR), blood agar test plates, and high performance liquid chromatography (HPLC) indicated that, BS-99 produced the antibiotics, iturin A and surfactin, whereas, BS-23857 and BS-33608 only produced surfactin. These data suggest that, the antifungal activity of the B. subtilis BS-99 isolate against P. eugeniae is activated only when both iturin A and surfactin are present and not by surfactin alone. Unveiling the antifungal mechanism of B. subtilis, BS-99 could promote its commercial development and application as a biofungicide for controlling P. eugeniae infections of the wax apple, which is a highly valued fruit in Taiwan.

Incidence of Kanagawa phenomenon-positive and -negative Vibrio parahaemolyticus strains isolated from traveller's diarrhea and their relation to tdh and trh genes

A total of 1,319 strains of Vibrio parahaemolyticus isolated from traveller's diarrhea were analysed for Kanagawa phenomenon (KP) with the Wagatsuma blood agar test and the results were also compared with those of analyses of tdh and trh genes which encode thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). The majority of the strains (1,152 strains) counting 87.3% had positive KP, among which 1,049 and 103 strains were only tdh and both tdh and trh-positive ones, respectively. However, 167 strains counting 12.7%, which is quite high compared to the previous report, were found to have negative KP, among which 94 and 24 strains were only trh and both tdh and trh-positive ones, respectively.

Bacterial interference in vitro

The aim of the present study was to compare two methods for estimation of bacterial growth interference between various bacteria using a Bioscreen robot analyzer, allowing kinetic documentation, and a cocultivation test on blood agar plates. Six laboratory strains with different virulence and growth requirements were used: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Staphylococcus aureus, and Staphylococcus epidermidis. The interference activity was correlated with a reference system of Streptococcus sanguis (strain alpha 89) and Streptococcus pyogenes (group A streptococci, GAS serotypes T 9 and T 22). The methods used and results obtained were as follows: 1. Estimation of synergistic and antagonistic bacterial interferences using a Bioscreen robot analyzer. Suspensions of viable bacteria were added to microtiter plates with different concentrations of UV light-killed bacteria in liquid media. The Bioscreen analyzer monitored bacterial growth every 10 min for 24 h giving kinetic data during the growth period. Synergisms as well as antagonisms were demonstrated between the tested bacterial strains which have not earlier been reported in the literature. However, the antagonistic effect observed between the six strains was less than that induced by the S. sanguis strain on the two strains of S. pyogenes. 2. Cocultivation of bacterial strains on blood agar surface with precultivated or simultaneously stamped interfering bacteria indicated no detectable interference between the six tested bacterial strains, while the S. sanguis strain inhibited the growth of S. pyogenes strains as well as the hemolysis around the colonies. The Bioscreen method was found more sensitive for testing bacterial interference compared to the commonly used blood agar test.