We finally got all our equipment for gathering metadata on the water, and decided to do some sample testing using tap water and saltwater from the tank. We ended up with the following:
Tap water –
NO2-N levels: 65 ppm
NO2 levels: 154.63 ppm
Phosphorus: 105 ppb
NO2-N levels: [TRIAL ONE] 65 ppm, [TRIAL TWO] 57 ppm
NO2 levels: [TRIAL ONE] 213.85 ppm, [TRIAL TWO] 187.53 ppm
Phosphorus: [TRIAL ONE] 48 ppb, [TRIAL TWO] 200 ppb
Some problems we encountered: When I did the trial one on the saltwater phosphorus test, I used the wrong reagent (phosphate reagent is used for the nitrite scanner, while phosphorus reagent is used for the phosphorus scanner). In addition, the phosphorus scanner maxes out at 200 (Andrew later confirmed this by doing additional scans on the saltwater), which means we’ll need a broader range scanner.
First off I hope everyone had a great Thanksgiving break!
If you read Andrew’s previous posts regarding the project, you would know that we have decided to scrap all of the samples we have extracted DNA from and start from the beginning. This is so we will have water chemistry data collected at the same time as DNA is collected thus providing the most consistent and accurate data.
On Tuesday, we received a portion of our water chemistry kit, which tests Hardness, Sulfites, Alkalinity, Iron, pH, and Chloride. We decided to do a practice run on a couple of the tanks so we can familiarize ourselves with the reagents as well as fine tune our sampling procedure. The results are listed below:
Freshwater Tank A
Hardness Test: 93 ppm CaCO3
Sulfite Test: 2 ppm Na2SO3
Alkalinity Test: 90 ppm CaCO3
Iron Test: No detectable amount
Chloride Test: 20 ppm Cl-
Iron Test: No detectable amount
We learned a couple of important points through this test run that will speed up our water chemistry sampling process in the future. For every single test we did, we started using the high concentration detection procedure, but found all of the concentrations in the tanks were extremely low, and had to redo it using the low concentration detection procedure. For our real samples that we will hopefully will be taking in the next week, we can save reagents and time and just jump right to the low detection procedures. We also noticed that the Hardness and Alkalinity tests detected the same molecule (CaCO3) and also had similar concentrations and have thus decided to use only one of the tests. (I will get back to you with the chemistry behind this reasoning, which I didn’t really understand). For both the Freshwater and Saltwater Test, we were not able to get a detectable amount of Iron and will likely scrap that water chemistry test. Lastly our pH meter results were a little different from Russell’s highly sensitive pH meter (pH=8.3) that takes continuous measurements and Tweets them. (Eisen is probably going to like the idea of that!) We will either scrap our pH meter and just use his or will have to verify if our pH meter is giving is accurate readings, by putting it in solutions of known low acidity. This is just an idea of mine, not sure if it’s a good way to check for its accuracy.
That’s where we are in the project as of Tuesday. I will get back to you about the differences between Hardness and Alkalinity and also update when we start taking samples again!
Well the theme of this week so far is summed up in the title here. Because we know these coral ponds were going to be set up next week we ordered all of our kits and probes with expedited shipping… and they’ve been trickling in over a long period since. We’ve been doing some initial experimentation with the measurements, but I’ll leave that story for the students.
Today we received colometric scanners that measure nitrate, nitrite, ammonia, phosphorus, and dissolved oxygen. Only it turns out that it’s really only the scanners… they don’t actually come with the reagents required to use them. Sort of like how the pH meter came on Monday with with instructions for activating and calibrating the electrode but none of those solutions either.
Any of course the day before Thanksgiving is clearly the best time to be putting in rush orders of reagents. Not to mention the various things that still haven’t arrived and are about to get eaten by the holiday.
So… assuming everything gets here by early next week, and that we can get it all to work properly, and that we even have the right equipment to measure the levels we’ll encounter in the first place, we should be all set.
After discussions on sampling methods, we have decided on new samplings methods. Since we are getting rid of all 18 current samples, our new sampling methods will hopefully be consistent through out the whole project.
For the water samples, we will take 3 x 1.5 Liters of water in one Coral pond.
For the sediment samples, we want to take less than we have in the past. We plan to use 2 mL centrifuge tubes and fill them to the 1 mL mark. We will make sure to get a mix of oxic and anoxic layers of sediment.
For the wall scrapings, we will use a Kim wipe to vertically scrape up and down in one location on the inside of the container. We will put a piece of tape on the top of the container where we sampled, so we don’t sample there again (because there won’t be anything there after we scrape it off). This is important because we will be doing a substantial amount of sampling for our study on succession of the Coral ponds.
On a side note, we are also discussing whether we should sample from both coral ponds or just one. (They are identical in terms of maintenance and what is put into them) We haven’t come to a decision yet.
Now that we have some of our new water chemistry equipment, we have discussed flaws in our first 18 samples. We collected all 18 samples with no tools to collect metadata (water chemistry). We have decided to take all the samples again and get rid of the 18 we already have. We still don’t have all the water chemistry equipment we need, but it should all be here tomorrow. It is crucial to determine the water chemistry at the time of every sampling. (During our study of succession on the coral ponds, this will be a lot of water chemistry testing!)
We have received a tentative schedule for the new ponds that we will be studying succession on.
Right now they (the people who set up and monitor the tanks) are rinsing the containers with freshwater as well as rinsing the sand with freshwater. The sand is going into the containers as they are getting rinsed. We will take a few samples here before anything else gets added.
Then they will fill the containers with new saltwater and run pumps for a few days with just the new seawater and sand. (too make sure everything is working well) This should be done by mid December. We will also take a few samples here.
Still in mid December (hopefully) they will add old sand, rocks and animals from old reef tank. This is where we will start extensively taking samples because we really want to see how the microbial communities become established. We will sample lots for the first couple days and slowly decrease the amount of sampling for the following few weeks. This is because we know most of the changes will happen within the first couple of days.
Then they are going to add live rock that have been curing. Curing basically gets rid of the dead stuff so the live and healthy organisms can thrive! We will again sample more extensively immediately after the rocks are added to catch the rapid changes happening to the microbial communities. We also might sample the rocks before they are put into the ponds.
After a short adjustment period due to all the new organisms, they will begin to populate the ponds with corals and other inverts. Once again we plan to sample a lot for the first few days but continue to sample, but less often after the first few days.
The next change that we predict will occur when students begin to reach into the ponds in the Spring. We will sample during this time too, to see how human interaction affects the microbial communities.
That’s the plan for now! 🙂
We’ve ordered and are recieving a series of equipment to measure Nitrate, Nitrite, Ammonia, dissolved Oxygen, pH, salinity, temperature, Phosphate, alkalinity, Chloride, hardness, Iron, and Sulfite in the tanks’ waters. Now we can gather more information about the environment these microbes are thriving in.
Because I was so late to introduce myself, I can also talk about what we’ve been doing for the last few weeks. We’ve gone to the aquariums and collected samples, done some DNA extractions, and done PCR on our samples. Our samples came from salt water and fresh water tanks and include water, sediment, and gunk from the walls of the tanks. Our latest issue has come after PCR, while running the Gel. It seems like our issue might be all the way back in PCR A. I am very excited for the new aquatic systems that we will start sampling in the next couple weeks. We are hoping to start sampling the minute they load the tubs. Our hope is to sample very frequently in the first couple days because we know much of the microbial community will develop in this time. So what’s the point of that, you ask? Well we would love to study the succession of microbial communities in these new aquatic systems. That’s all for now!
So the project is finally in full swing! After a couple of weeks of practice sampling and getting used to different protocols for extracting, purifying and amplifying the DNA, we have now moved on to working with our real samples. We have extracted DNA from 18 samples (includes replicates of 3) from the tropical tank and the cold water tank. Funnily, the hardest part of the project so far has been running gels. We initially hit a couple of problems such as thickness of the gel, a loose wire in a gel box and differences in loading buffers and dyes. However, now that Akshay is going to be coming in, hopefully he can give us a couple of hints from his experience from IGEM, cause god knows how many gels he had to make this summer 😛
So after a long and intense summer and first half of fall quarter, the iGEM international competition came and concluded. So now I get the chance to walk down the lab (towards the gel room!) a few more feet and start work on the aquarium project I am helping with in the Eisen Lab. I can now devote more time to the processes of the project, and not just be involved with going to retrive samples from the aquarium, and trying to figure out where we keep our Taq Polymerase. I will try and come in the lab in the next few days, and figure out the workflow and how to get this project going. I am very excited for the project as well as hoping for some actual success in our process. This is the first of many blog posts as well, so stay tuned for the next segment of our project!