Microbes mediate the degradation of numerous dissolved organic carbon substrates or DOC compounds, such as Racine and aquatic environments. To fully understand the role of microbes in DOC transformation, simultaneous identification of their taxonomy and function is necessary. This video demonstrates a procedure for the taxonomic identification of free living bacteria or bacterial plankton that transform specific pools of DOC and aquatic environments.
Water samples collected from a test environment are processed and incubated with a compound of interest and bromo deoxy uridine, BRDU, which incorporates into newly synthesized DNA and actively growing cells. Next BRDU labeled bacteria plankton are labeled with ZI conjugated anti BRDU antibodies. Bacterial plankton with high levels of BRDU labeling are isolated by fluorescence activated cell sorting.
DNA is then extracted from the sorted cells and taxonomic identification is performed based on results of PCR based 16 s ribosomal RNA gene analysis. Hi, my name is Steven Robbins from the laboratory of Dr.Chen Mao in the Department of Biological Sciences at Kent State University. Today s lu and I will be showing you a culture independent method for the identification of DOC degrading bacterial plankton in aquatic environments.
The main advantage of this technique over existing methods like nutrient amendments, followed by analysis on bacterial community DNA, is that it focuses analysis on highly active bacteria and excludes dormant and slow growing cells. We show here the identification of bacteria plantain that use one specific DOC compound. But this method can also be applied to study the bacteria plankton that use other single DOC compound or group of DLCs such as algal exudates.
So let's get some water samples and get started. Before asay water samples for DOC degrading bacterial plankton, the water samples must be appropriately processed. The samples shown here were collected from the coast of Georgia to remove big particles and ER VREs from the water sample.
Use a peristaltic pump to transfer water from the collection carboy to a filtration unit with a one micrometer pore size filter. Next transfer 36 milliliter aliquots of the filtrate into three sterile 50 milliliter polypropylene tubes. Each containing four milliliters of freshly prepared 10%Paraform aldehyde solution Incubate for two hours at room temperature to preserve the cells following the incubation.
Pour the water into the filtration tower of a glass vacuum filtration unit with 0.22 micrometer pore size white membrane filters and apply the vacuum to draw the solution through. Wash the filter by applying 10 milliliters of PBS through the vacuum filter. Label the filters as negative controls and store them at minus 20 degrees Celsius.
To establish an experimental ecosystem or microcosm, begin by filling six one liter pre rinsed glass flasks with 800 milliliters of the pre-filtered water sample. Add BRDU to each microcosm to a final concentration of 10 micromolar. Rapidly rotate the flask by hand to mix.
Each sample will be assayed and triplicate. So to three of the microcosms, add the model DOC compound. Here reine is added to the microcosms at a final concentration of 100 nanomolar.
Then to the other three microcosms add sterile PBS. These will serve as non DOC controls place all microcosms in a 100 RPM shaking incubator in the dark at the insitu temperature, which is 25 degrees Celsius. Here at time points of 0 8 16 and 24 hours.
Collect 36 milliliters from each microcosm in a 50 milliliter sterile polypropylene tube containing four milliliters fresh 10%paraform aldehyde incubate for two hours at room temperature to fix the cells. Next filter preserved cells through 0.22 micrometer pore size white membrane filters. Apply 10 milliliters PBS through the vacuum filter to wash off the residues of PFA.
Proceed immediately to INS situ immuno detection or store the filters at minus 20 degrees Celsius to assay for BRDU incorporation into bacterial plankton. Begin by throwing the sample filters at room temperature to compromise bacterial cell wall. Place the filter on a filtration unit, then pipette one milliliter of lysozyme solution onto the filter to cover the cells and incubate room temperature for 30 minutes.
Following the incubation, apply a vacuum to remove the solution. Once the solution passes through the filter, wash the cells by adding 10 milliliters of PBS and apply a vacuum. Then to further compromise cell wall, add one milliliter of proteinase K solution to the filter to cover the cells.
Incubate room temperature for 30 minutes. Wash with PBS to produce single strata DNA. Add one milliliter exonuclease solution to cover the cells on the filter.
Incubate at 37 degrees Celsius for 30 minutes. Following the incubation, wash the filter by passing 10 milliliters PBS through the vacuum filter. Next, trim off the edges of the filter to remove the portion that does not have cells.
Then on an alcohol cleaned dry surface, use a sterile blade to slice the filter into eight equal sized pieces. Eight frame seal incubation chambers are needed for each sample place an eighth of the filter into the assembled frame seal incubation chamber sample side up. Once the chambers are assembled, use the Roche Intu cell proliferation kit flus to detect A-B-R-D-U labeled cells in situ.
Once the standing procedures complete, remove the filter section from the incubation chamber and place it on a sterile surface using a sterile blade, slice the filter section into small pieces. Transfer the filter pieces into a two milliliter micro centrifuge tube containing one milliliter PBS tightly close the tube and seal it with paraform. Then incubate the tube in a shaking incubator at 37 degrees Celsius and 200 RPM for 10 minutes.
Following the incubation, secure the tube and vortex it at the maximum speed. For five minutes, pipette the SUP natant into a sterile 15 milliliter polypropylene tube. Then resuspend the filter in PBS and repeat the vortex and incubation steps two more times each time adding the snat to the same 15 milliliter tube for each sample.
Store the resuspended cells at four degrees Celsius. The sample should be analyzed by fax within two days. Finally, sort the cells to obtain the population with high level of BRDU incorporation.
Here, a BD fax area flow cytometer and corresponding software are used. Once the cells have been sorted, the BRDU enriched population can be assayed to determine its taxonomic identity. To amplify the 16 s ribosomal RNA genes of sort of bacteria filter PCRs performed, pipette the sorted cells onto a white 0.22 pore size polycarbonate membrane filter and turn on the vacuum pump using a sterile blade trim off the cell free edges of the filter.
Slice the filter into four equal sized pieces for each condition, place a single filter piece in each of three PCR reaction tubes with the cells facing inward of the tube. Add 45 microliters PCR grade water. Let the filter section completely submerge in the water.
Next, add two Roche illustra pure T ready to go PCR beads briefly vortex to help dissolve the beads. Then add two microliters each of forward and reverse 16 s ribosomal. RNA gene primers.
Add either one microliter BSA to help absorb amplification inhibitors or one microliter of water. Perform PCR amplification on a thermal cycler. A touchdown PCR program is recommended, which has the A kneeling temperature sequentially decreasing from 62 to 52 degrees Celsius by one degree Celsius per cycle, followed by 15 cycles at 52 degrees Celsius.
Confirm PCR amplification by electrophoresis on a C of bromide stained 1%Agros gels excise the PCR amplicons from the gel and clean with a KaiGen cayo quick gel extraction kit. After PCR gel purification, pull the amplicons of the same sample together. Purified 16 s ribosomal DNA amplicons are now ready for a number of molecular analyses.
Samples were collected from a coastal site of Georgia and processed to study Racine degrading bacteria following the procedures described in this video. Racine is a polyamory compound that can be ubiquitously found in marine environments. It can serve as a nutrient and energy source for marine bacteria as shown here.
Facts analysis of samples revealed that after a 24 hour incubation, a group of bacteria with higher level of FXI fluorescence intensity, which was positively related to extent of BRDU incorporation was developed in Racine amended microcosms. These cells were designated as high BRDU incorporation cells or HI and were expected to contain mostly Racine degrading bacteria. No hi.
Cells were found in the no addition controls, which only contained cells with lower levels of BRDU incorporation referred to as lii cells were expected to mainly contain bacterial plankton that were not able to use. Added Racine HI cells were sorted into separated tubes and then collected onto membrane filters. Partial 16 ribosomal RNA gene amplicons were obtained after filter PCR and confirmed by gel electrophoresis.
Following sorting filter, PCR amplification of functional genes can also be performed to examine functional diversity among sorted cells. We've just shown you how to physically separate and identify functional bacterial cells in aquatic environment using an approach that couples fluorescent activated cell sorting and BRDU incorporation. Remember, BIDU is head earth, a lab coat and a glos should always be worn while performing this procedure.
So that's it. Thanks for watching and good luck with your experiments.
