The cryosphere offers access to preserved organisms that persisted under past environmental conditions. The overall goal of this procedure, is to uncover hidden diversity by successfully collecting and removing exogenous material from frozen cores. This method can help answer valuable historic questions about select time periods by identifying organisms recovered from glacial ice and permafrost.
Collecting an aspetic core in the field is challenging given the high probability of contaminate introduction. Therefore the main advantage of this technique is that contaminate removal occurs in a controlled laboratory setting. Visual demonstration of this protocol is critical as the scraping and decontamination steps can be kind of hard to learn.
It's really a two person job, so it requires a lot of patience and team work especially. Demonstrating this procedure will be two support scientists from my laboratory, Karen Foley, and Robert Jones. This protocol requires meticulous cleanliness.
Use an oven set to 450 degrees Celsius to bake the following for four hours. Sterile nucleic acid free metal racks, glassware, metal forceps, and glass wool. Use a wrap of heavy foil to prevent re-contamination.
Next, filter sterilize 85%ethanol and ultra-pure water using 0.22 micron filters. These liquids must stay at or below four degrees Celsius or they will crack the sample, making it easier to contaminate. Store the ethanol at minus 20 degrees Celsius.
And store the water at four degrees Celsius. Then, sterilize a plastic ruler with 70%ethanol, followed by DNA decontamination solution and RNase decontamination solution. Wipe each solution off immediately after it has been applied.
Then store the ruler in a sterile, nucleic acid free Whirl-Pak bag. Next, prepare the cold room space by first scrubbing the walls, floors and a metal work bench therein with 1%bleach. Then set the room temperature to approximately minus 11 degrees Celsius.
Once prepared, wear light-duty suits, nitrile gloves and masks when in the cold room to reduce any chance of contamination of the room or the samples. This procedure requires that two people work in tandem. This can be challenging considering the many opportunities for re-contamination.
To ensure success, we monitor each other, communicate, and change gloves frequently. With everything prepared, bring the sterilized materials and samples into the cold room, and set them up on the bleach scrubbed table. This includes prepared cultures of S.marcescens.
First the permafrost core is placed on a prepared sheet of aluminum foil. Next, the outside of the core is lightly inoculated with a dilute culture of S.marcescens using a sterile foam plug. Then, set the core on a sterile metal rack above a tray.
Now, the steel microtome blade is wiped sterile with 70%ethanol, DNA decontamination solution, and RNase decontamination solution. Next, researcher A cleans their nitrile gloves in the same manner. Then A holds the core at a 45 degree angle above the tray.
B gently scrapes about five millimeters off the outside of the entire core, including the ends, using the sterile blade, while A turns the core after each scrape. As needed, the blade must be re-cleaned with the three solutions. Next, B quickly and carefully pours filter-sterilized 95%ethanol over the core while A turns the core.
Then, the core is quickly rinsed with water. The metal rack over the tray is now replaced, and the core is set on the new rack. A now cleans their nitrile gloves and then places the core on a clean rack above the tray.
Meanwhile, B sprays the entire core with DNA decontamination solution, followed by a rinse with filter-sterilized water. Next, B sprays the entire core with RNase decontamination solution followed by another rinse with the water. Lastly, the core is placed in a sterile Whirl-Pak bag.
Begin with placing a metal rack over a glass dish in a bio hood with laminar flow. Then place the core on the metal rack. Allow two to five millimeters of the outer surface of core to thaw over about 10 minutes.
During this time, turn the core 90 degrees every two minutes. After the thawing, grip some glass wool with forceps and swab the entire surface of the core to collect bacteria. Then, inoculate the two plates using the glass wool.
Next, measure outer dimensions of the core using the ruler but do not touch the core. Also inoculate two additional plates with the original culture that was previously used to dope the outside of the core to verify the inoculae was viable. Then, transfer the core into a large sterile bag and store it at minus 80 degrees Celsius.
Incubate the inoculated plates at 23 degrees Celsius for one week, and then examine them for S.marcescens colonies which turn red at this temperature. Though this protocol has a series of critical steps, one of the most important ones is to check for the growth of Serratia Marcescens. If there are no visible S.marcescens colonies then proceed with the next thawing phase.
IF there are S.marcescens colonies, then scrape another five millimeters off the core and repeat the procedure. Check whether S.marcescens grew from the original culture exposed to the cold temperatures of the cold room. Samples were collected from ice and permafrost from a 110 meter tunnel located in Fox, Alaska.
The tunnel provided access to ice-rich silt and alluvium. Samples were collected in triplicate. At facilities in New Hampshire, the described protocols were carried out.
The 16S rRNA gene was sequenced from bacteria grown from the cores. Pseudomonas species dominated but members related to Planctomycetia were also present. And, of particular interest was the absence of Serratia species, which shows that the decontamination protocol sufficiently removed exogenous DNA.
After watching this video, you should have a good understanding of how to successfully decontaminate ice and permafrost cores. Once mastered, the cold room processing steps can be done in 30 to 45 minutes if it is performed properly. When trying this procedure, it's critical to be mindful of everything and everywhere that you touch.
When in doubt, just change your gloves and re-sterilize. Following this procedure, other methods such as amplicon sequencing and shotgun metagenomics can be performed to answer additional questions, like what was the species composition for that time period, or what functional attributes dominated under those environmental conditions.
