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Monday, April 13, 2015

Update

Blood: I performed the DNA extraction on all samples and will run horizontal agarose gel to see if there is a significant enough amount to warrant PCR amplification.

Algae: I helped Tony harvest his algal growth. Algal water was collected into falvon tubes and centrifuged. The supernatant was drained and the algal sediment was collected using a micrpippette and deposited into a metal pan. The pan was placed in an oven set at 70 degrees F for 45 minutes. Dried algae was scraped out of pan and collected in a beaker. The dried algal powder was ground using mortar and pestal (to make sure the cell walls were broken) and added to DIH2O a 30 cc syringe was used to remove water from beaker and inverted for 48 hours. The syringe shows 2 phases, a water phase and an oil phase. Oil extraction complete. The fish are still alive and happy and were added to the aquaponics fish family.

Protein: I began a new protein extraction and will run PAGE gels as soon as I read a couple more protocols. This week has been really busy with papers to write and exams in Calc and Chem.

Fish: The aquaponic fish are still quite happy with their salad bar root buffet and the addition of the algae fish. Half of the water was removed and replaced with DIH2O. I will check the pH when I return to lab next week. The fish have eaten almost all of the roots so I am going to grow more in the other tank.  I noticed that there were a couple of plants that had roots stuck in the holes. I thought I would be helpful and pull the roots through. I grabbed a container out of the tank and it slipped out of my hand. I spent the next 30 minutes cleaning it up. No plants were harmed in the making of this blog.


Update on blood, protein, algal infection, and fish

Blood: A protocol has been created and now I am waiting on primers. I collected blood, scalp hair, eyebrow hair, fingernail, and spit from myself, my daughter, and a friend that wishes to remain anonymous. we will call him Mr. XY. I will need to extract the DNA from these samples and then run a gel to see if DNA is present. Hopefully there will be a significant amount and I can proceed to PCR amplification.

Protein: I loaded a duplicate PAGE gel to the one that contains bands with 2 different biomarkers. Lane 8 contains 5uL Biorad Precision Plus Protein Dual Xtra Standards. This will measure proteins that range from 250kDa to 2 kDa.
Lane 1 contain 5uL of Fisher Bio Reagent. This measure proteins that range from 116 kDa to 14.4 kDa.
Running buffer was prepared with 100 mL 10x tris/glycerine/SDS buffer to 100 mL DIH2O. Gel ran at 140 volts for 1 hour and 30 minutes. 
Neither biomarker banded. I am not sure why but there could be many explanations. Possibilities include improper technique or equipment malfunction. I will research protocols and try again.

Algla infection: I was curious as to whether the bacteria that killed the algae is harmful to fish. If it is not, then it could possibly be used to eradicate unwanted algal growth from fish tanks. Two feeder fish were purchased from a local fish store and placed in a bowl containing 60 mL of contaminated algal water and 1 L of DIH2O. The bacteria are present. They are cocci, gram positive with a spikey appearance. As this bacteria matures the spikes grow into fimbrae that detach and float in the water. I took a 20 mL sample of water and introduced 2 antibiotic discs (penicillin and clindimycin) to see if the bacteria were affected. 48 hours later there were no bacteria present and the fish seem to be happy. Antibiotic discs were added to the infected water. After 6 days I came into the lab to see new algae growing in the once infected tank.

Fish: The plants have matured and the roots are growing through the holes in the bottom of the container and into the water. I placed the new plants into the tank and the fish immediately started nibbling. The pH is still steady at 8. Josh suggested removing half of the water and replacing it with DIH2O. I will do this next week. Two more fish were added to the tank in hopes that their excrement will lower pH.











Protein Extraction:
Through creative optimism and a little hard work, I received 2 biomarkers this week (Thank you Josh). One is from Fisher (measures protein MW of 116.0-14.4 kDa) and one from Biorad (measures protein MW of 250-2 kDa). My plan was to run these two biomarkers on a 4-20% Tris-HCL PAGE and then use it to measure the bands on the 4-20% Tris-HCL PAGE that I ran last week (2 identical gels). I loaded to 10 uL of Fisher into lane one and 10 uL of Biorad into lane 8. Running buffer was prepared using 100 mL 10x tris/glycerine/SDS buffer to 900 mL DI water. The gel ran at 100 volts for 2 hours. What resulted was each biomarker produced one band about 1/4 from the top of the gel. I am not sure why this happened. The electrophoresis tank did not make the usual high pitch noise that it does when it reaches maximum voltage but it stayed at a steady 100 volts and I could see continuous bubbles  so I didn't really worry about it. There are so many possibilities aas to why this run was not successful (equipment malfunction, defective product, and technique are at the top of my list so far). I will research this more and run a few more gels next week.

Blood: I am preparing to do a DNA extraction in order to visualize the double genome for blood type that I have been told that I have. I am meeting with Anil today to brainstorm some possible ideas.

Fish: The fish are still happy but the pH is still very stable at 8. They are also dependent on fish food and I would like them to eventually obtain optimal nutrition from the roots of the plants in the tank. Jeremy suggested that they do not like the plants that are growing and did a bit of research on fish palate. He discovered that fish enjoy bok choy, kale, arugula, and swiss chard. I used the extra tank to plant these. we will see if the fish enjoy their new salad bar.

Algal infection:
So after taking a water sample and staining it, I discovered that there is indeed bacteria present. They are cocci,Gram positive bacteria that look pretty scary under the microscope. I streaked a McKonkey plate to make sure that they are gram negative and I also streaked a MSA plate to see if they are halophiles given the salinity of the water, although this may have been a byproduct from the algal cell lysis.  

Friday, March 13, 2015

The electron crowd experiment



What a busy but incredible week this has been. I received a phone call from a fellow intern Sunday night. His project revolves around a specific type of algae that can be grown, undergo an oils extraction which can be used for bio-fuel. As fascinating as this sounds I know very little about this concept. He stated that the algae he was harvesting "died" and that the project was a failure and that he was going to trash the water and start over. I explained that there is no such thing as "failure" in science and asked him to meet me in the lab Monday morning to discuss what else this could mean. He agreed to meet and talk with me before trashing the water. As I stood in front of his tanks that morning I saw  cloudy water with a light green hue, sediment at the bottom of the tank, and a lot of hard work that had been done. The brainstorming began. I started asking myself  a few basic questions:
1. Why did the algae die? Something killed it or the growing conditions were not optimal
2. What is the sediment on the bottom of the tank? Put it on a slide and look under a microscope.
3. What would kill the algae? Most likely a microbe, collect it, stain it, identify it.
4. Was the original algal sample tested for microbial growth when it arrived? No
5. If a microbe attacked the algae where did it come from? The lab or where it was shipped from
6. What can we do to prevent this from happening again? Identify the cause and implement a procedure to restore the tank.
7. Can we restore the algal growth? If we can identify the cause of death and eliminate it, it is possible to restore the algal growth.
8. What can we learn from this? Without a proper protocol and constant monitoring, algae will die. Also there could be a benefit of a microbe that kills algae.
9. What are possible benefits of a microbe that eliminates algae? A natural algaecide for fish tanks.

Now I was ready to meet my friend. When he arrived my excitement at all the new possibilities was contagious. A new project had begun, but there was a lot of work to be done and only two of us. Lucky for us,  a perfect storm entered (Jeremy) and the electron cloud was formed. Identifying the contaminate of the water was the first order of business. I collected water from the tank, the pure algal culture, and the bucket with the rest of the algal water that Tony brought back from the algal lab in Carlsbad, Ca. I placed each specimen in a centrifuge for 10 minutes at 16 RPM. I extracted the supernatant added more sample and centrifuged again. I repeated this two more times until I has a sufficient pellet at the bottom of the PCR tube. I placed a sample of the pellet on a microscope slide and viewed each sample under the microscope. I visualized algae and other microbes. I decided to do a quick gram stain which ensued in an argument between the three of us about the color of our new friend. I sad the color was purple but the boys were adamant that the color was pink (I was wrong). Because there was indecision, Jeremy streaked a TSA plate (to isolate a colony), and a macconkey plate (to see if it was indeed gram negative or gram positive). After 24 hours in the incubator, there was no growth. Jeremy asked if it were possible that the microbe was anaerobic. We decided to light a candle to see. Tony was busy testing the water sources for salinity, pH, and buying fish (to see if the bacterium would negatively impact them). He also learned how to successfully perform a gram stain (perhaps it is possible to teach an old dog new tricks).  A specimen of contaminated water was obtained (100mL) and 1 disc of clindamycin and 1 disc of penicillin was added to this water. 24 hours later there was zero microbial growth. Two feeder goldfish were obtained and placed in a bowl with a mixture of 30 mL of contaminated water with 1 liter of DI water. Salinity was 896 PPM. After the fish were added, the salinity dropped to 696 PPM.  the following afternoon an additional 30 mL of contaminated water was added to the fish bowl. 48 hours later, salinity was 1620 PPM, 30 mL contaminated water was added today and salinity is currently 2090 PPM. When the fish were first added the salinity decreased. That was the only time a decrease in salinity was observed.  MSA plate was streaked today with contaminated water to see if the bacteria is sensitive to salt.
This is what we have learned:
1. The bacterium are anaerobic, gram negative, cocci, that start as round spheres with spike-like projections and mature into what appears to be filaments that grow in a bunch and break away from one another and infiltrate water. Many other tests need to be done before identification can be made.
2. The algal water arrived and had a salinity of around 5 PPM. After contamination the salinity was so high that the meter read "error". Either the bacteria or the algal death increased the salinity in the water.
3. A combination of clindamycin and penicillin killed the bacteria in an isolated study.
4. The bacteria does not appear to harm fish.
This has been an amazing week. I have truly loved this experience with this electron cloud. You boys are the best! I look forward to working with you both when we return from holiday.



Thursday, March 5, 2015

Save The Fish!



FISH:
I decided to save the fish! I was sad to hear that nobody picked up Jenni's aquaponics project from last semester. I decided to take it on because they are the friendliest fish that I have ever seen. The water was really low and the plants were not looking very healthy. The pH is around 8 which is not healthy for the fish or the plants. The plants are happy with a pH around 7.5 and the fish will die at a pH of about 6.5 so pH 7 seems to be the happy number. I added water and waited 3 days to see if the pH would lower. It did not. Adding more fish (ammonium) will lower the pH but I was not sure how many fish to add. A second tank was set up to become a spare fish home. If it gets too acidic in the aquaponic tank they will have a safe place to go. 5 new fish were purchased and added to the spare tank. After acclimating, 2 of these fish were added to the aquaponic tank. Hopefully this will bring optimal pH to all of my new friends.





So many things

This week has truly been interesting . I found out that it is possible for a person to have two different blood types. It happens most often with twins exchanging genetic material in vitro and is commonly called chimerism. After receiving a letter from United Blood Services after a recent donation I was made aware that I may be one of these people. The letter also stated that they are not sure which blood I could be given if I ever required a transfusion because according to the test I have A and O blood. . I also am not a twin so this information was even more confusing. I decided to delve more into this and began by typing my blood. The test showed little to no agglutination in any of the serums. I called my mother to try to help me fill in the blanks. She stated that a fetus was removed through a D&C 27 months after my birth. After much research I learned that chimerism can be the result from one twin absorbing the genetic material from a nonviable fraternal twin fetus. I explained the situation and she confirmed that when I was 5 I had a minor surgery and she was told that I had a rare blood type and could not receive a transfusion if the need arises. She also confirmed that I was a very selfish child so the possibility of me absorbing a twin is highly probable. I would like to test my DNA to see if this is truly the case. I also want to mix different blood types with mine on a microscope slide in order to see if there is anyone that is compatible with me. A few brave souls volunteered this week but so far none of the RBC's got along with mine. I am currently looking for primers in order to get a final answer out of my DNA. 

PROTEINS:
With the help of Josh and his mad hot gun skills I was finally able to successfully pour an agarose vertical gel and it was really cool. I loaded to 50 mL syringes with smelly SDS agarose gel and loaded the vertical plate while Josh kept the glass warm with a heat gun. I am still trying to figure out the best way to place the gel and the proper voltage and running time for the vertical chamber before I perform an electrophoresis with this. I am also waiting on bio-markers before I run and electrophoresis with the denser PAGE gels.













It's all about the gels this week.

After reading a journal article titled SDS Agarose Gels for Analysis of Proteins I discovered that they were using mini-casettes (8x8x0.1 cm) that were loaded with agarose gel. I have previously been using polyacrylamide mini-cassette gels but the concentration was not dense enough to capture the specific proteins that I am trying to capture. I had some of these  mini-casettes in the refrigerator. I took them apart using the key (long flathead screwdriver), being very careful to only break the side seals in order to keep the seal at the bottom intact. I used liquid tape to secure the edges. 0.5 g of agarose was mixed with 50 mL of SDS on a hotplate. A 5 mL syringe was used to load gel. Unfortunately the liquid tape did not hold and the gel escaped enough to allow air bubbles into the cassette rendering it useless. 
I decided to try a horizontal gel to see what kind of results I got. SDS (yelloe in color) was used to pour this gel (whitish-clear in color) and the wells were loaded with brain,urine and blood. A bio-marker was loaded but will only register proteins >6. After 88 minutes of electrophoresis at 100 volts I noticed that the gel was changing color. The gel was changing from yellow to clear as the proteins migrated from the negative pole to the positive pole. The positive reservoir was also changing from clear to yellow. When electrophoresis began both wells were clear from the buffer solution. I cut this gel in half and stained half of it with comassie blue and half of it with fast blast. The comassie blue showed banding but again, the bio-marker was not adequate so I still can not calculate the molecular weight of these proteins. The fast blast was too dark to visualize any banding.
I became extremely frustrated and decided to attempt another vertical gel. I was pleasantly surprised to see a package from Genscript waiting for me. Inside was PAGE mini-cassete gels with the concentration needed to capture the 4 kda proteins. Next I will run the PAGE and attempt to successfully pour an agarose vertical gel.

Agarose gel stained with comassie blue

Brain storming



Reservoir well turns yellow

Agarose mini-cassette