The protocol describes a streamlined approach for the screening and isolation of a heavy metal resistant microbes from geothermal Springs. This approach represents a research area of growing interest worldwide for application in the field of biosensing and bioremediation. The method described therein can be easily modified to isolate microbes from diverse environmental sources such as water, food, soil, or sediment.
The minimal inhibitory concentration to identify a resistant microbes is a quick strategy to characterize the new species or strains. This protocol is easy to perform and only requires manual skill and experience with the basic microbiology techniques. To begin, inoculate two grams of collected sample into 50 microlites of freshly prepared Luria-Bertani medium with a pH of four or seven.
Incubate this sample at the temperature of the sampling site plus or minus five degrees Celsius. Plate 200 microliters of the sample on Luria-Bertani agar with a pH of four or seven and keep it in a static condition at 55 or 60 degrees Celsius for 48 hours. After the incubation, isolate single colonies and repeat this streak plating cycle at least three times.
To prepare one milliliter of frozen cell stock, add 20%glycerol to overnight grown cells. Use a mixture of acetone and dry ice for fast freezing. To prepare an inoculum from a glycerol stock, inoculate 50 microliters in 50 milliliters of Luria-Bertani.
To obtain a growth profile, dilute this pre culture in 10 microliters of Luria-Bertani. To adjust the optical density to 0.1 add 600 nanometers. Grow these cells for 16 hours at 55 or 60 degrees Celsius in the orbital shaker.
Measure the optical density at 600 nanometers at 30 minute intervals. Curve from this data with time on the X axis and optical density at 600 nanometers on the Y axis. Plot similar growth curves with varying pH of the culture medium, to determine the optimal pH for laboratory conditions, Inoculate the isolate streaked from the glycerol stock in 50 microliters of Luria-Bertani medium and incubate overnight.
Centrifuge this overnight grown culture for 10 minutes at 5, 000 times G then discard the supernatant and harvest the culture palette. Prepare 10 milliliters of bacteria lysis buffer composed of 20 millimolar Tris-HCL, two millimolar EDTA, 1.2%Triton X-100 and 20 milligrams per milliliter lysosome immediately before use. Re-suspend the pellet in 180 microliters of bacterialysis buffer.
Genomic DNA extraction as instructed in the purification kit and measure genomic DNA and its purity by UV-Vis. To assess purity, determine the 260 to 280 and 260 to 230 optical density ratios. Assess the integrity of the genomic DNA by loading 200 nanograms of each sample on a 0.8%agarose gel and comparing the size distribution to a height, weight molecular marker.
Grow the isolate from glycerol stock in 200 milliliters of Luria-Bertani optimal pH and temperature conditions. Dilute each pre culture in five milliliters of Luria-Bertani medium in 50 milliliter of polypropylene tubes containing increasing concentrations of heavy metals and antibiotics to obtain an optical density of 0.1 at 600 nanometers. Grow the cells for 16 hours on an orbital shaker with 180 revolutions per minute at 55 or 60 degrees Celsius.
Calculate the minimum inhibitory concentration for antibiotics or heavy metals by identifying the concentration values in the tubes where microbial growth does not occur. Check that the concentration is inhibitory and not lethal for the cells by plating 200 microliters of the culture grown at minimum inhibitory concentrations on Luria-Bertani agar plates and verifying the presence of colonies after overnight incubation. This protocol was used to test bacterial samples from the Piscarelli area, an acid sulfuric geothermal environment Phenotypical characterization of the isolated microorganisms showed that isolate one has a higher tolerance to arsenate and vanadate and is resistant to cadmium.
Comparative data suggests that isolate one has a strong resistance to pentavalent arsenic while isolate two has resistance to trivalent arsenic. Antibiotic resistance tests showed that isolate one is highly sensitive to all the tested antibiotics even when low concentrations were used. In contrast, isolate two is resistant to all the antibiotics tested except for chloramphenicol and tetracycline.
The microorganism probably has acquired antibiotic resistance due to random mutations or horizontal gene transfer representing a selective advantage in extreme environmental conditions. This study demonstrates the usefulness of such methods to select environmental microorganism that can inactivate the pollutants and convert them into harmless products.
