Aug 3, 2010
We tested for protease today, using the protocol at http://bio533.wikispaces.com/Lab_OA. We pipetted 1ml of the V.t. inoculated marine broth and the followed through the steps.

After the final step, our samples turned orange. This means that there are metallo-proteases present in the culture. If we did not know V.t. was in the broth, we would not be able to identify it because other bacteria make these. However, we could do an agglutination, which would tell us whether or not we have V.t.

We also ran a Differential Display, following the Sigma GeneSnare protocol. We received 3 libraries of sea fan cDNA (control, SPX, and Aspergillus) and we ran the PCR. Here is the master mix:

2 ul 5mM arbitrary primer (#19 from Steven's magic box)
1 ul 10uM dt-ACP2 (this sticks to the adapter on the library cDNA)
6 ul ddH2O
10 ul See Amp 2X Mix
_
19 ul soup
+ 1 ul cDNA

2 negative controls were run, for a total of 5 samples.

The cDNA libraries were made by performing a reverse transcription on RNA with a step that includes ligasing an adapter/anchor primer to the DNA for the dt-ACP2 primer.

Aug 2, 2010
Today Teamatode ran a traditional PCR on the L. sitkana DNA that was subjected to Restriction Enzyme. We also cleaned the class RNA with Ambion Turbo DNA Free kit. We used the protocol from http://genefish.wikispaces.com/FISH441. We then ran a qPCR.

We also cleaned out the oyster larvae capsules for the ocean acidification experiment. This involved filtering the larvae out of each 2 liter bottle (control first, followed by lowest to highest concentration of V.t. inoculation). We should have done the big bucket of extra larvae first, so remember that for next time. Samples are taken and placed in six well plates filled with 5ml of sterile sea water and in 500 ul RNAse later, and sterile sea water is replaced. There was a lot of algae in the tank, and we are not sure that the larvae are still alive.....

We did a serum agglutination test, using our marine broth inoculated with V. tubiyashi and V. t. antibody (see protocol at http://bio533.wikispaces.com/Lab_OA). The control showed the cells just hanging out, while the test sample had a lot of clumping. This is because the antibody is clinging to multiple reception sites on the V.t.

July 31, 2010
Today I joined forces with Carrie and James to form Tremateam Teamatode for the mini project. We will be looking at Littorina sitkana to see if there are any epigenetic effects based on the stressor of infectious trematodes from bird guano. We will be looking at differences in methylated DNA between populations at Rueben Tart (control) and Cattle Point (bird poop central).
James and Carrie already collected adults from Rueben Tart a few days ago, and today we went to Cattle Point to get some adults (and Carrie collected some bird guano to look at). We collected 60 adults and 15 juveniles, though we’re not sure if we’ll be using the juveniles. We will be processing them for RNA and DNA tomorrow.

Carrie and James ran a tradition PCR yesterday on the DNA we extracted in class. They ran a PCR with 3 samples for each individual (no restriction enzyme, one restriction enzyme that lyses DNA if it is methylated, and another restriction enzyme that lyses the DNA if it is not methylated. We ran the gel for their PCR today.

Making a gel (1.5% agarose):
0.75g agarose
50 mul TBE
microwave until it boils
add 6ul EtBr
swirl it and then pour it into the gel cast
~20 min for gel to solidify
load samples (7ul ladder, 10ul of each sample)
run at 120V for 20 min
visualize with UV

The first gel we ran had a lot of primer dimer, but it looked like there were potentially some bands before the dimer, so we decided to run another gel, this time loading 14ul of each sample. When we visualized it again, there were definitely several bands in some of the wells, though we are not sure what they mean…..

July 30, 2010
Today we went to Argyle Creek to collect some chiton (Mopalia). We were told some of them were looking sick, similar to Black Abalone Wasting Syndrome, so we wanted to check them out. We collected a few healthy and sick-looking. However, only 1 of the sick ones looked really bad. It was difficult to tell if they were healthy or not, and we chose unhealthy ones based on not clinging strongly to the rock. However, when they were placed in the bag, most of them exhibited similar behavior to the healthy ones, by grabbing onto each other or onto the rock in the bag.

Lisa Fong and I decided to plate some samples from 3 animals:
healthy
obviously sick (infected 1)
collected as sick but appears healthy (infected 2)

We washed each animal with sterile sea water, and then for each we swabbed the girdle and the foot, and we plated on both TCBS and marine agar. For the obviously sick one we also swabbed a leasion and healthy section of the girdle, as well as a lesion and healthy section of the shell. We then parafilmed all plates and incubated them at 37 degrees C overnight.

The obviously sick animal had very different behavior from the other two animals. It did not respond much when swabs were being taken, whereas the other animals balled up like a pill bug when swabs were being taken. The obviously sick animal also had 3 barnacles and 1 anemone on it. We are not sure if this is normal, though it seems slightly suspicious....

July 29, 2010

Protein gel run and Western Blot
We looked at our gels, and protein was shown in all wells. We decided that 30ul was best for each of the samples, and we ran another gel (duplicates).

I set up the rig with Loni, so we rinsed the gel because it's preserved in urea-type liquid. We then put it in the rig, put buffer in the rig, and rinsed the wells. We filled up the rest of the rig, loaded the samples and ran the gel. This process allows us to see that proteins are in our samples. It is not specific, it just allows us to separate all the proteins present (we visualized it with the dye first to make sure protein was present, although sometimes the target protein may not even be visible - that doesn't mean it's not there). We ran duplicates so that we can stain half the gel and make sure it worked, and then we can run a western blot on the other half. However, we decided to just blot the whole gel. We transfer the proteins from the gel to a membrane/paper, and then we hybridize the specific protein we are looking for, and we can quantify the level of expression through image analysis. It is important to be sure the proteins are loaded equally so if any upregulation is seen it is true upregulation, not just variable amounts of loaded protein. From this, we look at the relative amount compared to the total amount of proteins there. We will be targeting a heat shock protein today.

Setting up the western blot:
To transfer the proteins from the gel to the membrane, we need to make a filter sandwich that looks like:

Anode (+++)
filter paper
gel
membrane
filter paper
Cathode (---)

A current is run from top to bottom of the sandwich, and it brings the proteins from the gel and transfers it to the membrane. Before doing this the membrane has to be fully soaked in buffer, and the filter papers need to be pre-wet also (not too dry, not too wet).

The protocol for this is at http://bio533.wikispaces.com/Lab_Proteomic

An electric current is run from top to bottom of the sandwich, and the idea is for the current to bring the proteins from the gel to the membrane. Unfortunately after all that work, no magic happened and there were no bands. :(

We dissected some oysters today, some 4-5 years old and others about 2 years old. We used a butter knife to shuck the oyster, using the hinge as an entry point. It was important to keep the organism intact, so once the shell was open, we needed to keep the knife as flush as possible against the flat side of the shell and detach the adductor muscle. Once the shell was opened we took some hemolymph from the pericardial sack and put in on a slide. We fixed the cells onto the slide with methanol (about a minute), and then stained them with Giemsa stain (about 2 minutes) to visualize the cells. We also took the heart out and dabbed in on another slide, dehydrated/fixed them in methanol, stained them with Giemsa stain. After staining the slides were rinsed in water and looked at under the microscope. We looked for hemocytes and Bonamia. My slides were too lightly stained, so next time I need to be sure I leave the slides in the stain a little bit longer.

July 28, 2010
Morning:
Our slides that sat overnight showed staining, so we rinsed them and counterstained them, waited for them to air dry, and then coverslipped them with permount. Our slides were 08: 1-15, which have stippled RLO. We had a sample slide that was already stained for comparison.
Rickettsia occurs in the epithelium of the digestive gland. It was easy to see the nuclei of the hemocyte aggregates within the digestive gland, indicating an immune response. Hemocytes are generally in and around the digestive gland because they perform other functions such as nutrient transport, but the large aggregates often indicate something else is going on. Then there were larger nodules/spots within the epithelium, which were the Rickettsia.
We looked at our positive and negative slides, and found that there was no staining of any of the Rickettsia on either slide. This is a good thing, because the stippled Rickettisia was not targeted. However, no one else's slides showed any staining, so it is more likely that something went wrong with the batch. :(
RLO: new vs classic vs stippled
Here is a recap of the hybridization/detection process:

We also started our proteomics lab today. Morgan set up the experiment last night, heat stressing some anemones. She set the heat bath for 25 degrees C last night, but then this morning the heat bath was off, so she cranked it to 30 degrees C during breakfast and lecture. Then we started to follow the protocol from http://bio533.wikispaces.com/Lab_Proteomic for protein extraction from the anemones. I worked with Carrie and James. We had an anemone from the heat stress tank, and we took tissue that weighed 0.2g for protein extraction.

Afternoon:
We skipped the quantification of our protein and just went on to run a protein gel.

July 27, 2010
We continued processing our slides, following the Day 2 protocol at http://bio533.wikispaces.com/Lab_Withering.
Drew received the histology slides of the tissue sampled from the sick Armina, and it looks like a rickettsiale infection in the tissue under the epidermis.

July 26, 2010
Morning:
We processed 2 slides in groups today, and I worked with James and Carrie. We were looking at Withering Syndrome (WS) in abalone. We depariffinized (Xylene/SafeClear) and rehydrated (serial ethanol dilution) tissues in the morning.
RLO’s are only in the digestive epithelial tissue in abalone, though it may occur elsewhere in other animals.
Looking at the monitor, Caroline pointed out the difference between classic, stippled and new Withering Syndrome. Classic is a lighter blob, while new is darker, and often larger than the classic. Stippled has a slightly different pattern.
Looked at sea grass wells – growth in sample wells while controls were clear. Next step?
Afternoon:
We finished up Day 1 of the in-situ protocol, and left the probe to incubate overnight.
You can find the protocol for the in-situ experiment at: http://bio533.wikispaces.com/Lab_Withering


July 24, 2010
Today we looked at the gel from our traditional PCR, and there were bands for all samples, including the negatives. There was also some primer dimer.
We analyzed our data from the qPCR. We looked at the amplification curves, which tells us the Critical thresholds (Ct) of the reaction. We also looked at the Dissociation curve to see at which temperature the fluorescence went away, which tells us the melting point, which depends on the size and GC content of the gene.
There was contamination in pretty much all of the negative controls.
Here are my Ct values:
actin (-) = ND
actin (-) = 35.65
actin (TY2infected) = ND
actin (TY2infected) = 35.17
CJunk (-) = ND
CJunk (-) = ND
CJunk (TY2infected) = ND
CJunk (Ty2infected) = ND
Arbitrary expression value = 10^(-(0.3012*Ct)+11.434)
For the actin (TY2infected), this expression = 6.93
Normalized = gene of interest/reference gene, so CJunk/mean Actin for TY2infected = ND
Then we took the average normalized data from over the whole class, separating the infected from the uninfected, and then we did a simple t-test. We graphed it and it looks like there is a significant difference. However, we only had data for 3 infected and 3 uninfected.
The class data is at http://spreadsheets.google.com/ccc?key=0AtV_gF766XZAdDdxeGVVZWJNZkdCV3RTbjZnM3E3RUE&hl=en&authkey=CIfI6fwG#gid=0


C-jun expression by genefish.
C-jun expression by genefish.


July 23, 2010
Morning:
We checked the plates and media from the eel grass. Plates on marine agar and TCBS agar showed a lot of growth for the diseased plant, and some, but much less growth for the healthy plant. Kathy tried to isolate the microbe by plating from what was grown. Nothing noticeable was seen in the media – we are letting them sit another day.
Reverse Transcription – making cDNA from mRNA.
We took our RNA samples, thawed them on ice and then:
  1. Put 17.75 ul of RNA into a 1.5 microcentrifuge tube
  2. Heat sample to 75 degrees C for 5 min
  3. Put on ice for about 10 min
  4. Create a Master Mix (courtesy of Emma) and add 7.25 ul to RNA. Vortex and spin
  5. Incubate at 42 degrees C for 1 hour
  6. Transfer to 95 degrees C for 3 min
  7. cDNA!!
Master Mix ingredients:
· 5X MMLV buffer 5 ul
· 10 mM dNTPS 1.25 ul
· MMLV RTase 0.5 ul
· Oligo dT primers 0.5 ul
· Total mix to put into RNA sample – 7.25ul
Tritonia nudibranch suspected to have Labyrinthulid infection on its dorsal epidermis - white splotchy pattern. Caroline took samples to try to culture the fungus in QPX media (from clams). She also took samples for histology, and did some scrapings for Carrie and James to look for cells under the microscope. It was difficult to determine lesion sites vs healthy tissue sites because the animal has been dead for one day and the splotches were no longer
Afternoon:
Q-PCR
Gene of interest: C Jun Kinase (CJunk)
Reference gene: Actin 1 – this normalizes everything
We want to compare healthy and disease individuals
Master Mix ingredients (multiplied by 5 to accommodate 2 negative controls and 2 samples for each gene):

July 22, 2010
We checked our TCBS and marine agar plates from yesterday. My plates were clean, though James and Carrie had some yellow colony growth on the healthy tissue marine agar plates. Lani and Marie had streaked swabs from lesion tissue from a sicker animal, and they had growth on all of their plates, with both yellow and green colonies. Matthew also had some yellow colony growth on an all plates with healthy tissue from the same animal as Lani and Marie. Yellow colonies = can ferment sucrose. Green colonies = cannot ferment sucrose.

We also performed an RNA extraction today, following the protocol listed at:
http://bio533.wikispaces.com/Lab_Littorina#Day3
RNA is extremely sensitive, and there are RNAses everywhere, making this procedure very delicate. I processed TY2, and there was a pellet at the end of the day, which was suspended in 50ul of 0.1%DEPC-H2O.

Additionally, Sarah and I processed some of the sick eel grass we collected this morning at Picnic Bay to culture for Labyrintha, a fungus within the organism that is suspected of causing mass die offs of eel grass in the area.
Note: The QPX media is used for sea fans, so we are not sure it will work with the eel grass. We are keeping our fingers crossed.
Morgan and Kathy plated some samples from healthy and sick eel grass on marine agar and TCBS plates. All samples were incubated overnight at 37 degrees C (?).

July 21, 2010
Morning:
Plate streaking for microbes off of sick Armina nudibranchs - dilution streaking with 4 quadrants
The lab divided up into 5 teams. Carrie, James and I teamed up and worked on a nudibranch from the disease tank with disease, though it was the healthiest individual in the tank and it only had 1 minor lesion. Several signs that show an animal is sick is a change in behavior and/or morphology. For these animals, the sick guys were upside down, and they were lacking in stripes and ridges. It looks like something is eating away at its skin. This could be a lab phenomenon - stress from capture and containment could compromise their immune systems - not necessarily occurring in the wild....
Sterile technique must be used during this process.
We labeled the plates as: DD clean CK1, DD lesion CK1, DD clean KJS 2, DD lesion KJS2, DD clean TY3, DD lesion TY3. Carrie swabbed the left side of the lesion and the left side of the health tissue. James swabbed the anterior of the lesion and the left posterior for heathy tissue, and I swabbed the right side of the lesion and healthy tissue. After we did this, we realized we should have swabbed the health tissue first....
We scraped the lesion and the healthy tissue with a razor and put the cells on slides to look under the microscope. We found ciliates in the lesion tissue, which were absent in the healthy tissue.
Afternoon:
DNA extraction, PCR, Gram staining
Today I extracted TY2, a sample connected from a L. sitkana that was infected with Cercariae (0.01g). We extracted the DNA using a Qiagen Stool Kit following their protocol. We used the Stool Kit because it has an ingredient that binds to junk proteins that can act as inhibitors, so that we get cleaner DNA. Then we ran a PCR, in duplicates, adding 2 ul of DNA sample to 25 ul of PCR mix (courtesy of Carrie). We will gel the contents of the PCR tomorrow.
We also watched Caroline gram stain some of the slides from this morning.
Under the scope we saw Gram negative rods. Many Vibrio species are gram negative rods. We also saw a ciliate, which is Gram negative, but may have gram positive components within the cell. However, this may just be an artifact from not decolorizing long enough.

July 20, 2010
As a class we looked at histology slides to identify anatomical features. We all watched the monitor as Caroline pointed out key features
We also looked at histology slides on our own
We took our samples that we collected this morning of Littorina sitkana (bigger, further from water edge, striped, wider) and Littorina scutulata (smaller, along water edge, checkered, elongated) and processed them by doing the following:
  1. bleach everything
  2. crack open the shell and retrieve the organism
  3. put organism in petri dish and add some water
  4. under the dissecting scope, cut off the operculum, head and digestive gland
  5. cut a piece of the foot for DNA and put in freezer
  6. put the rest of the body (minus the head, DG and operculum) in a tube for RNA and put in freezer on dry ice
  7. look for parasites in the DG
  8. if parasites found, let Drew know so they can be pictured and/or videoed
I opened 6 L. sitkana and 1 L. scutalata. I found parasites in 1 individual, TY2, and it was exploding with Cercariae species in 2 different life stages, adult and redia. At least one other Cercariae species was found in L. scutulata, which had a tail and eyes
BTV JPEG 018 by genefish.
BTV JPEG 018 by genefish.
eyed scut 017 by genefish.
eyed scut 017 by genefish.


July 19, 2010
Dissections – invertebrates were sedated by placing them in magnesium chloride
Dissected a cockle, which is a bivalve.
· We shucked it with a butter knife, provided by Lisa
· Identified the mantle, which is the part that is responsible for the making of the shell
· Found the heart, which was tucked away under the gills, it was white, still beating, very primitive, just a more concentrated blood vessel
· Found the digestive system
· Saw the posterior adductor muscle, but couldn’t really find the anterior one
· The foot was really big!
Looked at a sea urchin
· Identified the gonads – uni sushi!
· Looked at the feet
· Looked at viscera
· Looked at calcareous ring
Looked at a geoduck
Looked at an oyster
Looked at a sea cucumber
Looked at a snail/gastropod under the dissecting scope
Looked at a sea star (Pisaster?)
Watched the sea anemone shoot out nematocysts when bleach was applied (on the camera microscope) – the mechanism is unclear, though it is speculated that it has to do with osmotic pressure