7/19/10

Day 1. Invert anatomy - Dissected an oyster (most likely Crassostrea gigas)
Points of interest:
  • particles (food, pseudofeces, other debris) travels along the cilia of the gills & is sorted by size on the way to the "food groove" at the base of the gills. stuff is passed along to the labial palps (function like a tongue in terms of descriminating b/w desirable and discard-able stuff). unwanted materials sent back out to be expelled by the excurrant siphon (i think). <-- 7/20 update - siphons are really just holes in the mantle in oysters, rather than the muscular protruding siphons found in clams, etc.
  • particles enter the digestive "tract" (in the visceral mass area) and are ground and partially digested by the actions and enzymes of the proteinaceous crystalline style (grinds particles against the gastric sheild) - precursor to the thyroid!
  • 3-chambered heart is located just before the adductor muscle, in the pericardial sac - *this is a good place to aim for if trying to withdraw hemolymph* stick syringe into muscle and then pull back across the heart (muscle juice is a good source for hemocytes too)
  • took a photo of oocytes - initially it seemed like the oyster must be male and full of sperm (chock full of milky gamete material!), but under the scope the gametes look more like eggs, though very oddly (and irregularly) shaped. <-- 7/20 update - apparently the oocyte shape we observed is totally normal, and sex is pretty much impossible to distinguish unless looking at gametes under the scope
  • <-- additional 7/20 notes on oyster anatomy - should have observed the anus near the anterior adductor muscle. The cup-shaped valve is the Right valve.



7/20

Day 2.
Part 1. General histology
Summary
  • Important to be familiar with the 3D organization of the organs/tissues of the whole animal before trying to interpret cross-sections!
  • Also important to be familiar with what "healthy" looks like before trying to work on potentially diseased individuals
  • lots of helpful info on general procedure for creating histological samples in Carolyn's lecture, her along with other ppt's with bivalve anatomy
Details
  • looked at C. gigas hemocytes - couldn't see actual granules in granulocytes w/out proper staining, but was able to distinguish granulocytes (more fibro-blast looking) from hiolinocytes (spelling?) that were more rounded and regularly shaped - both cell types are involved in phagocytosis, though they may be used differently against different types of foreign particles.
  • C. virginica (with SSO, sea shore organism unknown) - dilated digestive tubules are often an indicator that the organism isn't healthy (aggregations of hemocytes in non-normal places would be another tell-tale sign)

Histology - Things to work on
  • not fully comfortable with recognizing different tissue types in any of the organisms we looked at today - particularly not comfortable distinguishing the types of kidney tissue, or sections of the esophagus
  • would like to actually go through the process of making slides for histo analysis - seems like it would take practice!

Part 2. Littorina sitkana and L. scutulata trematodes
Summary
  • cracked 6 individuals of L. sitkana (no trems) and 1 of L. scutulata, and only found 1 infection in the L. scut.
  • saved most of the body (aside from digestive gland/gonad, head, and operculum) for RNA extraction (straight into the dry ice box), and a piece of foot tissue for DNA analysis - this required sterilizing instruments between each individual, so cracking took longer than expected
  • need to access a guide to marine trematodes - would love to know what each of the species (there were at least 3 different ones found today as far as I could tell) is, and what other hosts they utilize



7/21/10
Day 3
Part 1. Armina diagnostics
  • 5 nudibranchs were brought in from the neuroethology class – 2 currently healthy individuals from the “healthy” tank, 2 diseased individuals from the “healthy” tank, and 1 diseased individual from the “diseased” tank. Our group swabbed the diseased-diseased individual’s lesion (1 lesion in the mid-dorsal area, fairly small), as well as healthy tissue from other parts of the animal onto TCBS and marine agar plates (3 total plates of each agar type for both the lesion and the healthy tissue – 1 plate of each was streaked by each team member)). Each team member targeted a different section of the “leading edge” of the lesion
  • swabbed using plastic loop instead of cotton swab, and also used plastic loop instead of metal loop for streaking the plates. dipped the loop in ethanol to sterilize and then in distilled water to remove ethanol between streaking each quadrant
  • Also used a razor blade to scrape the surface of the epidermis around the lesion and on healthy skin. When observed at 40x, ciliates were observed in the lesion scrapings, but not in the healthy-skin scrapings. Possible that the ciliates colonize the lesions, or that they are a causative agent?

Part 2. DNA extraction and PCR
  • Used Quiagen stool kit to extract DNA from an infected L. scutulata (CK.scu.1) – had very small amount of tissue (<.01mg), so unsure whether amount will be sufficient for amplification
  • Protocol for extraction here

PCR (I mixed the master mix, and am hoping I did a good job!)

Per 25ul reaction:
12.5 Prometra GoTaq green master mix (contains MgCl, dNTP’s, Taq, green dye)
1.5 BSA
1 HCO
1 LCO
7 H20
2 DNA



7/22
Day 4

Part 1. Armina plates

  • My plates - no growth from streaks of lesion on either plate type (TCBS or Marine agar), or from healthy tissue
  • - suspect that the I might not have gotten all the bleach off of my plastic innoculating loop
  • James had some whitish raised colonies on his Marine agar lesion+ plate, and also on non-lesion plate - colonies were the same color and shape (ish) between plates
  • Other groups got a little growth (Vibrio) on TCBS plates from healthy animals, and much greater growth from lesions

Part 2. Labyrinthula from Picnic bay (eel grass and crabs)
  • A couple of groups shredded sections of eel grass tissue containing the dark possible-Labyrinthula scars, and sections of healthy tissue
  • placed some tissue parts on TCBS and marine agar just to see if anything showed up
  • for possible Labyrunthulid culture, placed shreddings into 6-well plates w/ QPX agar/broth (developed in Sandy's (?) lab when they had Labryinthulid-infected-? can't remember what type of organism they had before...)
  • I took a few swabs of a blackish lesion on the telson of one of the large Dungeness crabs we brought back from Picnic bay, and plated on TCBS and marine agar - not much had shown up as of 24 hours at room temp.
  • also looked at scrapings of the lesion, but didn't see any obvious fugal stuff - still would be interested to stain some scrapings to differentiate fungal cell walls if present

Part 3. RNA extraction - trematode-infected and uninfected L. scutulata and L. sitkana
  • my sample is an infected L. scutulata
  • Performing RNA extractions w/ Tri-reagent chemistry - nasty chemicals like phenol and chroloform
  • See protocol here
  • couldn't quantify RNA b/c no access to Nanodrop (but might be able to use plate reader for indiv. work later if right consumables are accessible)



7/23
Day 5
Part 1.
Reverse transcription PCR - to turn unstable RNA back to stable cDNA
General notes/general protocol (see Wiki for deets):
  1. Using oligo DT primer (lots of T's) - will lay down on the poly-A tail of expressed genes
  2. Heat blocks to 75c, 37c, 95c
  3. Put 17.5ul of RNA into clean RNA-ase free-tube (don't vortex, just tap to mix and put in centrifuge for a few sec)
  4. heat to 75 for 5 min , put on ice for 5 min, add 7.25ul of master mix (mixed by Emma),
  5. sit at 42 for 1hr, kill reaction by heating to 95degrees for 3 min
  6. store new cDNA on ice!

MasterMix (made by Emma)
PER RXN:
  • 1 ul Oligo dT Primer
<-- this binds to the poly-A tails of the mRNA, helps us just select expressed genes. If we were doing bacteria (no poly-A tails), could just put in non-selective hexanes(?) that would prime everything, not just expressed stuff
  • 1 ul RNase free water
  • Total = 15 ul


Part 2. qPCR
Target genes:
1. actin (reference/housekeeping gene that isn't usually differentially expressed between individuals or under diff circumstances
2. C-jun Kinase gene (an alternative might be p38 MAP kinase) (both are imp in NFKappaBeta signaling pathway, which is generally involved in the immune response, but in other stuff too)
  • Made master mix for both genes (primers for each)
  • Plate locations: b9,10 = actin(+) b11,12 = actin(-) c9,10 = c-jun (+) c11,12= c-jun (-)
  • Used lower amounts (.5 instead of 1ul per 25ul reaction) of primers compared to yesterday's PCR, b/c extra primer can form "primer dimer" which will increase fluorescence and mess up our interpretation of our results - we're really looking for differences in the amount of expression between two states (so, RELATIVE expression is what's quantified)
  • *possibility that I didn't add c-jun F primer (was distracted while adding and am not sure I put in in the c-jun master mix before discarding the tip!)

Per reaction:
  • 12.5 2xSybr… (contains Taq, dNTP's, MgCl; degraded by light, so important to add last)
  • 1.5 BSA
  • .5 primer R
  • .5 primer F
  • 8 H2O
  • + 2ul of our cDNA (don't have to add 2ul of water for -controls, these can just have a volume of 23)



7/24
Day 6
Notes on qPCR data processing/interpretation/ways to improve:

  • Genomic DNA carryover (inluding introns) - if you know you have introns in your DNA, you could design primer around it so you could tell if some was amplified w/ intron or without (would see 2 bands - large band w/ intron). If you don't know if there's an intron, you could do qPCR (in case its at a non-detectable level) on your RNA (can use pretty much any DNA primer), and should not get a product … if you do (and thus have genomic DNA) DNA-ase it a couple of times (maybe just do this anyway while setting up reaction)
  • 10-fold dilution - difference in Ct should be 3.32 (2^n = 10) - can use this to see if PCR is efficient - would normally normalize amount of RNA after quantifying, but w/out Nanodrop or plate reader, can just try dilution method
  • Normally, bring up concentrations of RNA to same amount by quanitfying and ajusting - quantify b/c nucleotides have spe
  • For expression - only relative amount matters (number of copies is meaningless, we don't know how much is around at a pariticular time)
  • For pathogens - probably are interested in absolute amount - tell how much of a pathogen is in a sample.
Data analysis - free program - Miner

For gene expression analysis
  • Arbitrary expression value =10^(-(0.3012*Ct)+11.434) - to test this formula, input a Ct of 30 and 33.20, should get expression value ~10fold difference
  • Normalize by dividing Arb. Exp. Val of gene of interest by AEV of actin (housekeeping gene) -
  • End product is normalized c-jun data - would just take average of inf and non-inf individs, t-test to look for significance
  • Genes could be being translated at a higher rate, genomic carry-over, could be used up at a higher rate b/c translated faster or have more or less storage of mRNA…. If you verified w/ protein (western blot) you could say more about this potential source of difference (expressed to protein faster or slower - why a difference in the availability of the mRNA)
  • Step 1 from now - try PCR'ing RNA to check for genomic DNA


Part 2. Setting up some new reactions to look for differences in constitutive expression of immune genes (which ones?) in L. scutulata (brooder) from high and low trematode prevalence sites

L. scutulata groups (can use class extracted RNA for High prev adult snails)
High prev. Adults
Low prev. Adults
High prev. Juveniles
Low prev. Juveniles

Steps:
1. Collect ~10 each of large and small L. scutulata (maybe increase numbers in case some of these turn out to be L. plena) from Reuben Tarte Park (Drew's low prev site), and ~10 of each size class from Cattle Point (might only need to extract from the juveniles, but should get adults too in case existing extractions are problematic
2. RNA extractions - protocol on class Wiki
3. set up PCR on extracted RNA (incl. class samples) using universal primers, to detect genomic DNA - be sure to include one of the positive DNA extractions from class as a positive control
4. if gel is free of bands in the RNA wells, move on to reverse transcription!
5. qPCR

Notes:

4. if gel is clear,




Monday 7/26
Part 1.
In Situ Hybridization: this procedure lets us visualize places in a piece of tissue that are expressing a gene of interest using a probe (RNA or DNA - DNA in our case I think) specific to a sequence of target DNA, which is then tagged with antigens that will react w/ a particular dye to label target DNA in tissue samples

We are looking for 3 different types of Rickettsia cells involved in abalone Withering Syndrome


Overview of procedure (detailed procedure here) and misc notes:
  • Deparaffinization of tissue samples, via xylene and drying w/ gradient of EtOH
  • Permeabilization of tissues with Proteinase-K and Tris-buffer
  • Prehybridization (we used the "alternative" method on the protocol page)
  • Hybridization of the DNA probe - Lisa heated the probe at 93c for 3 min to denature, then put on ice for 30 min
  • We drew circles around tissue w/ Pap pen before adding probe, but also used the Dnase free cover slips (does a better
  • job of keeping the probe soln on the tissue samples)
  • Stringency washes to remove probe and block attachment of antibodies to non-target sites




7/27/2010

Part 1. In Situ Hybridization Day 2

Detection steps cont'd (see protocol)

Slides had some color in the morning, but when we counterstained we didn't have anything...


Part 2. Littorina sitkana dissections
After recording spire height and body whorl width, large snails from Reuben Tarte park were dissected in sterilized petri dishes w/ a sterile hammer (yep, a sterile hammer) - we removed the head (tentacles/brain/as much radula as possible) and operculum. Gonad was examined for trematode infection, and 3 pieces of tissue were stored as follows: small piece of foot muscle preserved in 100% EtOH (a); most of body (minus dig. gland/gonad) preserved in RNAlater solution(b); gonad/digestive gland preserved in EtOH (c).

Label
Spire ht/whorl width(mm)
Infected?
Notes
Rt.L.1
11.2/10.1
N

Rt.L.2
12.1/10.8
N
disintegrated gonad
Rt.L.3
14.1/12.9
N

Rt.L.4
11.3/10.0
N

Rt.L.5
14.2/12.4
N
disintegrated gonad
Rt.L.6
12.0/11.0
N

Rt.L.7
12.1/10.7
N

Rt.L.8
11.8/11.6
N






7/28/2010

Part 1. Protein extraction

Copied from lab Wiki, and including some annotations:
PROTEIN EXTRACTION PROTOCOL
Extraction:
1. Add 0.5mL of CelLytic MT solution to a 1.5mL snap cap tube.
2. Using a clean razor blade, cut a piece of frozen tissue weighing 25mg and add to tube containing CellLytic MT solution.*
*to save time, this step has been performed for you.
3. Homogenize the tissue with a disposable pestle.
4. Close the tube and invert the tube several times.
5. Spin the tube in a refrigerated (the whole centrifuge is put into the 4c fridge for this part) microfuge for 10mins. @ max speed.
6. While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date.
7. Carefully transfer supernatant to labeled tube and store tube on ice. (tubes of spun supernatant are in a rack in the fridge so that we can return to them after lunch w/out having to freeze-thaw (freeze-thaw is especially bad for proteins apparently) We also saved the tissue pellet for potential DNA stuff later)
Quantification: (We don't have the Bradford assay reagent as of 7/28 so we're skipping quantification and just running the SDS-PAGE - I'm guessing we'll have enough of the extracted protein left to run the assays for quantification later…)

Part 2. Running protein extracts on SDS-Polyacrylamide Gel - per procedure on lab wiki
We loaded 3 different volumes of our extract to figure out whether we all had successfully extracted, and whether any needed to be diluted or concentrated to make the concentrations more even for the next step (Western blotting)

(incomplete entry - need to come back and finish!!! plus catch up on the past couple of days!!)





8/1/2010

Part 1. Larval care:
  • classmates changed larval water by pouring larval water through a 70um mesh filter, taking subsamples of larvae for mortality counts and RNA-later, and then rinsing remaining larvae back into their container w/ new filtered seawater.
  • added 1ug/ml of 1% neutral red to 5ml of oyster larvae at 11:50am. Below pics taken at 5:50pm for % mortality counts:
  • Dye note effective - some larvae that were still swimming appeared clear after 5 hours. Neutral red was supposed to only stain living tissue... For future counts, individual movement is being assessed, and the dye is not longer being used.

Part 2. Littorine project - see group page here.




8/2/2010

Part 1. Oyster larvae - innoculation w/ Vt and plate counts - need to get some additional notes on this stuff

Part 2. Serum agglutination assay

Used an antibody specific to Vt (made in a pet chicken!)

Part 3. Littorine project - see group page here.




8/3/2010

Part 1. Oyster larvae - most larvae were dead in high Vt treatment, and most were alive in control treatment (prelim results)!

Part 2. Azocasein Protease Assay

  • Started w/ a 1ml aliquot from class sample of Vt grown overnight in marine broth, then followed procedure as found on lab wiki page.
  • Result: product was bright orange, indicating protease activity!

Part 3. Littorine project - see group page here.