The overall goal of this procedure is to produce protest enrichment cultures from the Antarctic Lake Bonnie, and measure carbon fixation as part of an initial proxy for metabolic diversity. This is accomplished by first sampling lake water depths in Lake Bonnie that correspond to known peaks in protests and phytoplankton. The lake water samples are then inoculated into various growth media and incubated in a growth chamber equipped with a temperature regulator and lights for four to six weeks.
The cultures can be transported as needed at four degrees Celsius. The next step is to extract crude cell lysates from the enrichment cultures. The final step of this procedure is to assess carbon fixation potential in the cell lysate using a radioisotope assay for ribulose one five Bisphosphate, carboxylase, oxygenase, or Rubis co activity.
Ultimately, the pro enrichment cultures show a variation of carbon fixation potential in the natural protest community. This method can help us answer key questions regarding lake microbial ecology. For example, the role of microbial eukaryotes in carbon cycling at various steps in stratified Antarctic lakes.
Although water column sampling for subsequent generations of enrichment cultures is a frequently used technique, this method contains modifications to operate in an extreme cold environment where unforeseen complications can occur. I first had this idea for this method when I was thinking of ways to test the Redisco assay in natural lake samples. Visual demonstration of this method is critical as the sampling steps are difficult to imagine because the sampling sites are in an environment foreign to many researchers.
Samples are collected from Lake Bonnie, one of numerous permanently ice covered lakes located in the McMurdo dry valleys in Antarctica optica one day prior to sampling the water column, choose and prepare the sampling site. This will allow the stratified layers of the water column to reform after disturbance due to drilling and ice hole melting. Identify the location of the drill site by GPS to access the water column.
Begin by drilling a hole through the ice with a Jiffy Ice aga attached to a four inch Jiffy flight extension and cutting bit to prevent freezing of the drill in the hole. Try to avoid drilling into the liquid water by halting drilling approximately four to six inches above the top of the water column. The drill hole must be widened from a diameter of six inches to two feet for sampling.
This is accomplished by use of a hot sea model hole melter that circulates heated polyethylene glycol through a copper pipe hole. Melting typically takes up to 18 hours on the day prior to sampling the water column. Ensure that all required equipment and sample storage vessels are assembled at the sampling site.
Sampling materials include a NICU bottle of five to 10 liter capacity, a winch with depth calibrated cable, sufficient sample bottles for each sampling depth, a messenger and coolers for sample transport. Begin sampling early in the day at about 5:00 AM as lake water filtering should be completed on the day of sampling. Using a kin sampler attached to a calibrated hand winch.
Collect water samples of one to five liters from the desired depths for this study. The sampling depths are six meters, 15 meters and 18 meters measured from the PI symmetric water level in the ice hole. Store lake samples in coolers and transport from the sample site to the field laboratory.
Using a sled attached to an A TV samples should be processed or stabilized at the field lab. Immediately following sampling, the cultivation of enrichment cultures should begin once samples have been transported to the field laboratory. Concentrate 0.5 to one liter of lake water onto 47 millimeter 0.45 micron pore size.
POLYETHERS sulfone filters using gentle filtration. If the diversity of the natural community is desired, filter a second sample onto 47 millimeters. 0.45 micron pore size.
POLYETHERS sulfone filters flash freeze the filters in liquid nitrogen. Transfer each filter to a 50 milliliter sterile falcon tube containing around 20 milliliters of filtered lake water. Collected from the same sampling depth as the filter.
Transfer the cell suspension to a 25 centimeter square sterile cell culture flask for enrichments on different culture media. Set up multiple replicates for each sampling depth. Supplement each culture flask with 50 x sterile solutions of the following culture media, bold basil, medium, BG 11 and F two.
If cultivation of mix atrophic organisms is required, set up two F two supplemented vessels and add two to three grains of sterile rice to one of the incubate culture flasks at low temperature and lower radiance in a temperature controlled growth incubator for at least four weeks prior to shipment to the US laboratory for shipment to the US.Transfer enrichment cultures to sterile 50 milliliter Falcon tubes. Request shipment requirements of do not freeze and keep cool on the shipping request. Upon arrival in the us, transfer five milliliters of each enrichment culture into 45 milliliters of appropriate growth media in a 125 milliliter.
Sterile erlin may flask capped with a sterile sponge. Maintaining enrichment cultures as standing cultures in a temperature controlled environmental growth chamber of four degrees Celsius and lower radiance transfer cultures to fresh media every 30 days. To begin the procedure for cell lysate extraction, filter five liters of enrichment culture onto a 25 millimeter watman GFF filter using gentle vacuum.
Next, cut the filter into four to five sections with sterile steel scissors. Then use sterile forceps to transfer the pieces to a two milliliter screw cap tube filled. One fifth of the way full with 0.1 millimeter diameter.
Zirconia silica beads. Add 1.25 milliliters of filter extraction buffer to the tube and then disrupt cells using a mini bead beader bead for 30 seconds on a speed setting of 48 and then ice for one minute. Repeat the 32nd bead beating two more times with one minute ice incubations in between bead beating to prevent heating of the sample centrifuge.
The filter slurry for two minutes at 3000 times G at four degrees Celsius. To form a loose palt of GFF filter fibers and cell wall debris. Remove a 200 microliter quat of the snat and immerse in 800 microliters of ice cold.90%acetone.
Transfer the remaining snat to a 1.5 milliliter micro centrifuge tube. Measure extractable chlorophyll A in a spectrophotometer at wavelength values of 6 64 nanometers and 6 47 nanometers centrifuge. The remaining SUP natant for two minutes at 15, 000 times G at four degrees Celsius.
Transfer the S to a clean 1.5 milliliter micro centrifuge tube while avoiding the pellet of insoluble cellular membranes and remaining GFF filter fibers. This soluble cell lysate may now be used in the rubis carboxylase activity assay, which will be demonstrated next. To begin this assay transfer 125 microliters of each soluble lysate to a 1.5 screw cap micro centrifuge tube that has been chilled on ice.
For negative control samples, add 125 microliters of soluble lysate to a 1.5 milliliter screw cap micro centrifuge tube, but do not add substrate. Later run duplicate reactions for all samples and negative controls. Prepare 10 milliliters of assay buffer, fresh and chill on ice.
Iqu enough of the assay buffer for all of the samples and negative controls to a five milliliter tube. From here onward, all steps must be performed under a fume hood. Add C 14 labeled sodium bicarbonate to the allotted buffer and keep the buffer on ice.
Remove 10 microliters of the assay buffer containing C 14 and dilute it in one milliliter of assay buffer invert. To mix, add 100 microliters of the diluted mixture to three milliliters of bios safe two scintillation counting cocktail in a scintillation vial and invert to mix. Measure C 14 counts with a Beckman LS 6, 500 counter scintillation counts should be over 18, 000 counts per minute before continuing with the assay.
When enough C 14 labeled sodium bicarbonate has been added to the assay buffer, move the reaction tubes to a dry bath, preset to 25 degrees Celsius and incubate for three to five minutes. Next, add 100 microliters of assay buffer to the lysates and incubate at 25 degrees Celsius for five minutes. After five minutes, add ribulose bisphosphate to each sample and allow the reaction to proceed for five to 10 minutes a 25 degrees Celsius.
To stop the reaction, add 100 microliters of 100%probiotic acid centrifuge for one hour at 2000 times G to exhaust unincorporated C 14. Finally, transfer the total volume of the samples to scintillation vials containing three milliliters of bios. Safe two scintillation counting cocktail determine the counts per minute of acid stable.
End products by scintillation counting. Enrichment culturing was used to isolate novel cold adapted phototrophic and mix atrophic protests from the Antarctic Lake Bonnie at three depths. Various growth media types were used to select for a greater diversity of organisms, bolds, basil medium or BBMF two si, marine medium, and BG 11 medium.
The nutrient concentration and photosynthetically active radiation or PAR shown in this table represents conditions at the time of sample collection. Visual inspection of the enrichment cultures by light microscopy revealed that the cultures were dominated by phototrophic protests as indicated by the presence of chlorophyll pigment in most cells. The cultures also harbored a variety of cell morphologies depending on the sampling depth from which the inoculum was taken and the type of media used in the culture.
Maximum rates of carboxylase activity catalyzed by the enzyme rubis were measured in the enriched cultures as a proxy for carbon fixation. Potential chlorophyll. A abundance was also monitored as an estimate of phototrophic protest biomass as shown here.
Despite cultivation of all cultures under the same temperature, light regime, carbon fixation potential and phototrophic biomass vary dramatically between the enrichment cultures. Maximum rubis O activity was observed in enrichment cultures four and six growing in BBM media and enrichment cultures 13 and 14 growing in BG 11 media. In contrast, cultures 1921 and 23 enriched on F two growth medium exhibited maximum carboxylase activities that were four to 34 fold lower.
These differences were not due to lower biomass levels in the F two cultures as carboxylase activity was expressed on a chlorophyll a basis. Moreover, a Pearson correlation analysis revealed that rubis co activity did not correlate with chlorophyll a concentration. The BBM cultures inoculated with lake water from six meters and 15 meters.
Enrichment's forum five had the highest chlorophyll A levels, while all other cultures exhibited relatively low chlorophyll a regardless of rubis enzyme activity Once mastered, the lake sampling techniques take about two hours to perform. Depending on how many samples are taken, the carboxylase assay takes two hours as well when performed properly Following this procedure, other methods such as the heterotrophic enzyme activity assay can be used to answer additional questions such as which depth heterotroph occurs. Okay, After watching this video, you should have a good understanding of how to collect lake samples, enrich them for produce and assay for rubis carboxylase activity.
