June 10, 2014

Filtering EST and mRNA BLAST results to get final figures-- breaking up each table into 3 aspects (cellular components, biological processes, and molecular function)
EST taxID6580 all results

link to BLAST RESULTS

mRNA-- http://goo.gl/JrJDzl
ESTs-- http://goo.gl/WWQXmA

pie GoSlim terms-- biological process
should be about 13

May 30, 2014

qPCR of geoduck larvae
Well/rep
 Sample
 Stage
 Primer Set
 Developmental Process
A1/B1
 T1
 Trocophore
 Rp_RNF66_F
 Rp_RNF67_R
 multicellular organismal development
A2/B2
 T2
 Trocophore
A3/B3
 V1
 Veliger
A4/B4
 V2
 Veliger
A5/B5
 S1
 Settler
A6/B6
 S2
 Settler
A7/B7
 NTC
 --
C1/D1
 T1
 Trocophore
 Mm_flilCG1484_F
 Mm_flilCG1485_R
 nematode larval development
C2/D2
 T2
 Trocophore
C3/D3
 V1
 Veliger
C4/D4
 V2
 Veliger
C5/D5
 S1
 Settler
C6/D6
 S2
 Settler
C7/D7
 NTC
 --
qPCR mix used: two master mix/cocktails-- one for each primer set. Each larval sample ran in duplicate

 Rp_RNF66/67
 Mm_flilCG1484/5

[Stock]
 ul/rxn
 Cocktail
 '# of NTC
 ul/rxn
 Cocktail
 '# of NTC
Water
7
 98
 2
 7
 98
 2
Primer - F (uM)
 10
 0.5
 7
 '# of Unknowns
 0.5
 7
 '#of Unknowns
Primer - R (uM)
 10
 0.5
 7
 6
 0.5
 7
 6
Sso Fast EvaGreen Supermix (2X)
 1X
 10
 140
 Replicate
 10
 140
 Replicate




2

2
cDNA
2
Total # Samples
 2
Total # Samples
Total volume (ul)
 20
 20
 280
 14
 20
 280
 14

Results:


Larvae qPCR output

PLATE SET UP

1
 2
 3
 4
 5
 6
 7
A
 T1 Rp
 T2 Rp
 V1 Rp
 V2 Rp
 S1 Rp
 S2 Rp
 NTC
B
 T1 Rp
 T2 Rp
 V1 Rp
 V2 Rp
 S1 Rp
 S2 Rp
 NTC
C
 T1 Mm
 T2 Mm
 V1 Mm
 V2 Mm
 S1 Mm
 S2 Mm
 NTC
D
 T1 Mm
 T2 Mm
 V1 Mm
 V2 Mm
 S1 Mm
 S2 Mm
 NTC
JBqpcr_all.jpg
Quant. plot for all samples. Peak for V2 (column 4) may have something to do with amount of RNA-- notes in Sam's notebook that during RNA extraction there was a "goo" and the yield was very high.
JB_qpcr_neg.jpg
Quant plot for Negative controls only. May be contamination.. which means that maybe false positives for larvae.



JB_qpcr_mc.jpg
Melt curve for all samples-- plate colors need to be re-uploaded from original file-- I think I copied over the wrong plate layout-- colors don't match above.
JB_meltcurve_pos.jpg
Melt curve for positive signals with one solid peak-- corresponding wells are depicted in plate picture below
JB_mc_pos_plate.jpg
jb-mc-ntc.jpg
Melt curve for negative controls--- need to ask Steven how to interpret this.. looks like not one product so maybe not contamination that would make a false positive?







May 29, 2014

qPCR Results:
jessica_qpcr_primerwells.jpg
Well
 Working label
 Primer Set
 Developmental Process
D4/E4
 NTC
 N/A
 N/A
 N/A
D5/E5
 3
 Rp_RNF66_F
 Rp_RNF67_R
 multicellular organismal development
D6/E6
 4
 Rp_flilCG1484_F
 Rp_flilCG1485_R
 muscle fiber development
D7/E7
 5
 Mm_flilCG1484_F
 Mm_flilCG1485_R
 nematode larval development
D8/E8
 9
 Mm_HCA57_F
 Mm_HCA57_R
 embryonic development ending in birth or egg hatching

Jessica_qPCR_primer_quant.jpg
jessica_qpcr_primer_melt.jpg


Quantification Output

Well / Set
 Dye
 Content
 Description
 Efficiency
 C(t)
 Quantity
 Avg C(t)
















D4
 SBGnew
 Sample
 NTC
 N/A
 N/A

D5
 SBGnew
 Sample
 Rp_RNF66/7
 1.48
 38.13

D6
 SBGnew
 Sample
 Rp_flilCG1484/5
 N/A
 N/A

D7
 SBGnew
 Sample
 Mm_flilCG1484/5
 1.77
 31.44

D8
 SBGnew
 Sample
 Mm_HCA57
 N/A
 N/A

E4
 SBGnew
 Sample
 NTC
 N/A
 N/A

E5
 SBGnew
 Sample
 Rp_RNF66/7
 N/A
 N/A

E6
 SBGnew
 Sample
 Rp_flilCG1484/5
 N/A
 N/A

E7
 SBGnew
 Sample
 Mm_flilCG1484/5
 1.97
 30.68

E8
 SBGnew
 Sample
 Mm_HCA57
 N/A
 N/A









Results:
selecting primers 3 and 5 to apply to larval stages. Mm_flilCG1484/5 appears to give a strong signal with one solid product. Primer 3 only had one replicate amplify-- will apply to larvae anyway.

Made cDNA out of RNA extracts Sam White isolated 5/27-5/28
6 samples total:
T1, T2 (trocophores)
V1, V2 (veligers)
S1, S2 (setters interphase)

 Min. Per Rxn (ul)
 Total / Sample (ul)
RNA
 17.75
 35.5
oligo dT
 0.5
 1


70C for 5 min


Ice
Master Mix

Per Rxn (ul)
 Total (ul)
M-MLV RT buffer (5X)
 5
 60
dNTPs (10mM)
 1.25
 15
M-MLV RT
 0.5
 6
Once combined RNA+oligodT with Master mix, set in "Ernie" at 42C fo 1 hr, 95C for 3 min, and 4C hold for 9 hours, then rested in thermalcycler overnight before running qPCR.


May 27, 2014





[Stock]
 ul/rxn
 Cocktail (per primer)
 Total Volume
 -# of NTC
Water
10
 20
 100
 2
Primer - F (uM)
 10
 0.5
 1
 5
 -#of Unknowns
Primer - R (uM)
 10
 0.5
 1
 5
 4
Sso Fast EvaGreen Supermix (2X)
 1X
 10
 20
 100
 Replicate





2
cDNA
2
 4
 20
 Total # Samples
Total volume (ul)
 20
 23
 46
 230
 10






Thermalcycler conditions
 Cycles


95 C
 30 sec
 1


95 C
 5 sec
 30-40


55-60 C
 10 sec


65-95
 10 sec
 1


Working qPCR table:
https://docs.google.com/spreadsheet/ccc?key=0An9mivRT-woWdGk1bWtjZG9SSTVBS2h1M1BwMURMSVE&usp=sharing
Working label
 Primer Set
 Developmental Process
3
 Rp_RNF66_F
 Rp_RNF67_R
 multicellular organismal development
4
 Rp_flilCG1484_F
 Rp_flilCG1485_R
 muscle fiber development
5
 Mm_flilCG1484_F
 Mm_flilCG1485_R
 nematode larval development
9
 Mm_HCA57_F
 Mm_HCA57_R
 embryonic development ending in birth or egg hatching

May 12, 2014


Gel electrophoresis of PCR products.
Used 12 ul sample per well -- 5 ul of HyperLadder II where indicated in table below
set to 120 V for 45 min
Gel Set up:
Lane
 Primer
1
 Ladder (2000 bp)
2
 1
3
 2
4
 3
5
 4
6
 5
7
 6
8
 7
9
 8
10
 9
11
 10
12
 NTC
13
14
 1
15
 2
16
 3
17
 4
18
 5
19
 6
20
 7
21
 8
22
 9
23
 10
24
 NTC
25
 Ladder (2000 bp)
26
27
28

Working Label
 Primer Set
 Product Length
 Gene function
1
 Rp_dlgCG1725_F
 Rp_dlgCG1726_R
 162
 determination of adult life span
2
 Rp_diaCG1768_F
 Rp_diaCG1769_R
 373
 multicellular organismal development
3
 Rp_RNF66_F
 Rp_RNF67_R
 951
 multicellular organismal development
4
 Rp_flilCG1484_F
 Rp_flilCG1485_R
 527
 muscle fiber development
5
 Mm_flilCG1484_F
 Mm_flilCG1485_R
 319
 nematode larval development
6
 Rp_skoCG3937a_F
 Rp_skoCG3937a_R
 332
 nervous system development
7
 Rp_skoCG3937b_F
 Rp_skoCG3937b_R
 346
 post-embryonic development
8
 Rp_ACVR_F
 Rp_ACVR_R
 874
 sarcomere organization
9
 Mm_HCA57_F
 Mm_HCA57_R
 610
 embryonic development ending in birth or egg hatching
10
 Rp_CSRP2BP_F
 Rp_CSRP2BP_R
 425
 embryonic development ending in birth or egg hatching

Results

Very bright positive for Primer 5-- Meretrix flilCG1484 (nematode larval development)
looks like some contamination in negatve control-- but product is same length as well next to it (used primer 10 in neg control), most likely from loading the gel
Some of these are not the correct product length... Need to get a picture of the ladder key.
It looks like the bands from Meretrix are brighter-- but if two products or not the right length not sure what this really means.

20140512_164056.jpg

Notes and next steps:
really wish I had kept the gel a little longer to take a better pic. These are really blurry. Maybe make another
only need 100 V, 15 min. next time.
qPCR! check out melting curve make sure there's one product.
RNA extraction of larvae? if time allows..

May 9, 2014


Made 1.5% agarose gel-- 150 ml gel tray, 2.25 g agarose, 12 ul Ethidium Bromide.
28 wells-- will need 1000 bp ladder
most likely 15 ul in each well.
Stored in 209 fridge over weekend

May 8, 2014

Conventional PCR for all 10 primer sets in duplicate

 stock concentration
 volume per reaction (ul)
 volume for cocktail
      • of samples
 Neg. Control
 Total
 Notes**
Water
 --
 8.5
 187
 20
 2
 22
 *5 primer sets in duplicate
Primer - F (uM)
 10
 1
 22



Primer - R (uM)
 10
 1
 22



GoTaq Green Master Mix (2X)
 1X
 12.5
 275






0



cDNA
2
 42



Total volume (ul)
25
 550











Thermalcycler conditions


Used "Ernie"
Step
 Temperature
 Time
 Cycles



Denaturation
 95C
 5min
 1



Denaturation
 95C
 30sec
 40



Annealing
 55C
 30sec



Extension
 722C
 90sec



Final Extension
 72C
 3min
 1



Hold
 4C
 ∞
 1





Working Label
 Primer Set
1
 Rp_dlgCG1725_F
 Rp_dlgCG1726_R
2
 Rp_diaCG1768_F
 Rp_diaCG1769_R
3
 Rp_RNF66_F
 Rp_RNF67_R
4
 Rp_flilCG1484_F
 Rp_flilCG1485_R
5
 Mm_flilCG1484_F
 Mm_flilCG1485_R
6
 Rp_skoCG3937a_F
 Rp_skoCG3937a_R
7
 Rp_skoCG3937b_F
 Rp_skoCG3937b_R
8
 Rp_ACVR_F
 Rp_ACVR_R
9
 Mm_HCA57_F
 Mm_HCA57_R
10
 Rp_CSRP2BP_F
 Rp_CSRP2BP_R

Created working primer stocks-- dilution from 100 uM to 10 uM. stored in FTR 209 freezer

May 6, 2014

Goals:
Conventional PCR to test three primer sets
Reverse transcription of more RNA

Only had enough cDNA for three primers in duplicate (6 wells)
http://replygif.net/t/facepalm

Three cocktail mixes-- one for each primer set.
Forgot a negative control --> will run these primer sets with others in next PCR.
http://replygif.net/t/facepalm (another)

 stock concentration
 volume per reaction (ul)
 volume for cocktail
 number of samples
 Notes
Water
 --
 8.5
 17
 6
 *3 primer sets in duplicate
Primer - F (uM)
 10
 1
 2

Primer - R (uM)
 10
 1
 2

GoTaq Green Master Mix (2X)
 1X
 12.5
 25







cDNA
2
 4

Total volume (ul)
25
 50







Thermalcycler conditions

Step
 Temperature
 Time
 Cycles

Denaturation
 95C
 5min
 1

Denaturation
 95C
 30sec
 40

Annealing
 55C
 30sec

Extension
 722C
 90sec

Final Extension
 72C
 3min
 1

Hold
 4C
 ∞
 1

Primers used:
1. Rp_dlgCG1725
2. Mm_HCA57
3. Rp_skoCG3937b


Reverse transcription:

 Per Rxn
 Total used
RNA
 17.75
 177.5
oligo dT
 0.5
 5


70C for 5 min


Store on Ice
Master Mix
M-MLV RT buffer (5X)
 5
 50
dNTPs (10mM)
 1.25
 12.5
M-MLV RT
 0.5
 5
Once added the master mix to RNA/dT mixture, incubated at 42C for 1 hour and 70C for 3 min. Stored in -20C.

Primer Database:
https://eagle.fish.washington.edu:5001/fbsharing/z5naeMot

April 1, 2014


"Genomic resource development for shellfish of conservation concern"
http://onlinelibrary.wiley.com/doi/10.1111/1755-0998.12052/full
^ Link to Emma's paper as an example/resource of how to incorporate BLASTx into methods and materials
When citing BLASTx algorithm:
Altschul SF, Madden TL, Schäffer AA et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.

To search for key terms within a column in SQLshare:



March 11, 2014

Running 2 BLASTs on clam database taxonomic ID #6580
1. EST (Meretrix meretrix)
2. mRNA (Ruditapes phillippinarum)

http://nbviewer.ipython.org/url/eagle.fish.washington.edu/scaphapoda/Jessica/ipython_nb/Geoduck_hunt_EST.ipynb
http://nbviewer.ipython.org/url/eagle.fish.washington.edu/scaphapoda/Jessica/ipython_nb/Geoduck_hunt%20_Ruditapes.ipynb

Joined via SQLshare
https://sqlshare.escience.washington.edu/sqlshare/#s=home
to UniProt/Swiss-Prot database to annotate genes

March 10, 2014

To Do:



March 4, 2014

Reverse Transcription Protocol:
A single reaction volume = 25uL.
  1. Aliquotted 17.75uL RNA to 0.5mL snap cap tubes.
  2. Add 0.5uL of Promega oligo dT for total volume of 18.25uL.
  3. Heat samples at 70C for 5 min in thermocycler.
  4. Place samples on ice IMMEDIATELY.
  5. Make Master Mix:
PER RXN
5 uL 5x Buffer (M-MLV RT Buffer)
1.25 uL 10mM dNTPs
0.5 uL M-MLV RT
*Processed one reaction. For future reactions perhaps double the volumes for each sample used (may be pipetting errors with small volumes)
7. Mix well.
8. Add 6.75 uL of master mix to RNA
9. Mix well, but do not vortex.
10.Incubate @ 42C for 1hr in thermalcycler
11.Heat inactivate @ 95C for 3 min.
12.Spot spin.
13.Store @ -20C.

qPCR :


 Volume
 Concentration
Sso Fast EvaGreen Supermix


Primer F


Primer R


Water


Template




Primers in Boxes 11 and 12; Laternula ellipticca HSP and HSC
1561
 Le_HSP70A_F
 CTGTCTTGAGCGATGGTGGC
1560
 Le_HSP70A_R
 TTTGTTACGGTCTTTCCTAAGTA
1559
 Le_HSP70B_F
 AAGCTTGTCAACCACGGCGG
1558
 Le_HSP70B_R
 CCTTGACCCTTTGGCCAAGG
1557
 Le_HSC70_F
 CAATGACAACACTCGCCCCA
1556
 Le_HSC70_R
 TGTTGACAGTCTTTCCGAGGTA

February 27, 2014


Treating RNA stock with TURBO DNA-free-- treating 25 ul RNA stock to practice with procedure in case of error and for qPCR testing to assure no DNA.
5 x 50ul reactions, 5.052 ul RNA stock per reaction

  1. 5.052 ul RNA (5ug) + 5 ul DNA-free buffer + 0.5 ul DNase reagent + 41.443 ul water in 0.5 ml tube
    • *note* this volume of water is incorrect for the reaction here-- should be 34 ul. Next round of DNA-free treatment needs to be the reaction volume (48 ul) minus the volume RNA and 0.1 vol DNase buffer, 0.1 vol inactivation reagent. (45-5.052-5-1)
  2. incubate for 30 min at 37 C (water bath)
  3. Add 0.5 ul DNase reagent
  4. incubate for 30 min at 37C
  5. Add 0.5 vol DNase Inactivation reagent (5 ul)
  6. Incubate RT for ~2 min
  7. Centrifuge 10,000 x g for 1.5 min
  8. Transfer to new tube-- combined tubes into one

Next steps:

February 23, 2014


Laternula elliptica --
Primer Set
 Gene
Primer Sequence
 RSq
 PCR Efficiency
2F and 2Rev
HSP70A
 Lel2F
 CTGTCTTGAGCGATGGTGGC
 0.998
 116.80


Lel2Rev
 TTTGTTACGGTCTTTCCTAAGTA

4F and 4Rev
 HSP70B
 Lel4F
 AAGCTTGTCAACCACGGCGG
 0.975
 107.20


Lel4Rev
 CCTTGACCCTTTGGCCAAGG

3F3 and 3Rev
 HSC70
 Lel3F3
 CAATGACAACACTCGCCCCA
 0.996
 84.30


Lel3Rev
 TGTTGACAGTCTTTCCGAGGTA

http://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctg_time=1393878159&job_key=f2SXa_phU0Vsd2h7CVVaBRJ4aBQBZ3UR
95°C, 5 min; 35 cycles of 95°C, 20 s; 55°C, 20 s; and 72°C for 40 s elongation step of 72°C for 5 min

HSC = heat shock cognate
From Clark et al 2008 -- Antarctic marine mollusks do have HSP70 response


February 14, 2014

Geoduck capstone

RNA isolation


followed lab protocol on RNA isolation bench (will attach picture)
clipped off about half the siphon of one juvenile, remainder is still in RNALater

500 ul Tri Reagent
homogenize
500 ul Tri Reagent
-- took total of about 25 minutes
200 ul Chloroform
vortex
-- very cloudy/milky
centrifuged at 12000 g 15 min 4 deg C
aqueous in new tube
500 ul isopropanol
rest at room temperature about 8 min
centrifuged 12000 g 8 minutes 4 deg C

Nanodrop results
lambda
 230
Abs.
 10.884
A-260 10 mm
 224.743
A_280 10 mm
 13.819
260/280
 1.79
260/230
 2.27
ng/ul
 989.7



need units

To Do:
Proposal
check in with Taylor (have trocophores at least!)
Order primers (sheet to Greg, give to Robin)
reverse transcribe to cDNA
-- when doing next RNA isolations, try new kit from Sam in parallel


Experimental set up

brainstorm

larvae-- sent from Taylor at various stages of growth (easiest for them, most direct)
juveniles( all at same age, would be good subjects for environmental stressors)
adult-- diferent gonadal development stages, might be difficult to work with, limits tissue types can use (mostly siphon, nonlethal)

Set Up:
15 individuals at each development stage -- primarily interested in pre and post settlement mortality events and effects of metamorphosis
(summary- 105 individuals...)

Analysis:

RNA extraction
qPCR-- look for HSP70 at each stage

To Do (besides everything):
Email Brent with project plan to send to Mexico contact for primers
Order TriReagent (email sent to Greg, waiting to hear back)
Extract RNA from sample juvenile
Research primers -- NCBI
continue lit search



http://www.pac.dfo-mpo.gc.ca/science/species-especes/shellfish-coquillages/geopath/develop-eng.htm

http://protectourshoreline.org/DNR/ComprehensiveLitReview.pdf (Brent co-authored)

January 27th, 2014

Capstone; Geoduck Panopea abrupta



http://wdfw.wa.gov/fishing/shellfish/geoduck/
http://wdfw.wa.gov/fishing/commercial/geoduck/

http://wdfw.wa.gov/fishing/shellfish/geoduck/geoduck_references.html
References on Geoduck Clams (from wdfw.wa.gov)
  1. Bradbury, A. and J.V. Tagart. 2000. Modeling geoduck, Panopea abrupta (Conrad, 1849) population dynamics.II. Natural mortality and equilibrium yield. J. Shellfish Research, 19(1):63-70.
  2. Bradbury, A., B. Sizemore, D. Rothaus and M. Ulrich 2000. Stock assessment of subtidal geoduck clams (Panopea abrupta) in Washington. WDFW, Technical Report Number FPT00-01. 57 p.
  3. Goodwin, C.L.1976. Observations on spawning an growth of subtidal geoducks (Panope generosa, Gould). Proc. Nat. Shellfisheries Assoc. 65:49-58.
  4. Goodwin, L. and W. Shaul.1984. Age, recruitment and growth of the geoduck clam (Panope generosa, Gould) in Puget Sound, Washington. Wash. Dept. Fish. Progress Rpt. No. 215. 30 p.
  5. Goodwin, C.L. and B. Pease.1989. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (Pacific Northwest) -- Pacific geoduck clam. U.S. Fish. Wildl Serv. Biol. Rep. 82(11.120). US Army Corps of Engineers, TR EL-82-4. 14 p.
  6. Hoffmann, A., A. Bradbury and C.L. Goodwin.2000. Modeling geoduck , Panopea abrupta (Conrad, 1849) population dynamics. I. Growth. J. Shellfish Research, 19(1):57-62.
  7. Sizemore, B. and C. Blewett.2006. Geoduck studies in Hood Canal. Progress work associated with House Bill 1896. Report to the 2006 legislature, House Select Committee on Hood Canal. January 1, 2006. DNR, Aquatic Resources Division. 54 p.
  8. Washington Department of Natural Resources2000. The Puget Sound Commercial Geoduck Fishery Management Plan. December 2000. DNR, Aquatic Resources Division. 15p.
  9. Washington Department of Natural Resources.2001. Final Supplemental Environmental Impact Statement (FSEIS) for the State of Washington Commercial Geoduck Fishery. May 23, 2001.DNR, Aquatic Resources Division. 135 p.

Preliminary GenBank searches result in many Pab1-9, 12-19 etc cloned microsatellites, 18 S and 28S rRNA.

=

=

=

=

September 23rd 2013

Oyster Histology

Tissue catalog completed with notation for questions and photo notes.
When in town, the last three weeks have been spent completing cuboidal metaplasia count for all new slides (8 slides of 1000 uatm, 8 slides of 1000 uatm + mech, 3 slides 400 uatm, 2 slides 400 uatm + mech, 6 slides 2800 uatm, 2 slides 2800 uatm + mech). Latest metaplasia and tissue data have been added to "Oyster Histology" Excel file on personal computer and uploaded to Eagle.

http://eagle.fish.washington.edu/scaphapoda/Jessica/Oyster%20Histology.xlsx

Metaplasia worksheet:


Treatment
 Slide #
 Tubules open
 Total tubules
 Percentage open
400 uatm
 H2.74*
 16
 266
 6.02%
400 uatm
 H2.80
 172
 1,339
 12.85%
400 uatm
 H2.90
 46
 159
 28.93%
400 uatm
 H2.95
 189
 575
 32.87%
400 uatm
 H2.96
 291
 1,268
 22.95%
400 uatm
 H2.93
 8
 150
 5.33%
400 uatm
 H2.73
 42
 192
 21.88%
400 uatm
 H2.75
 130
 727
 17.88%
400 uatm
 H2.76
 148
 1257
 11.77%
400 uatm + mech
 H2.82*
 58
 426
 13.62%
400 uatm + mech
 H2.84*
 20
 203
 9.85%
400 uatm + mech
 H2.86*
 0
 199
 0.00%
400 uatm + mech
 H2.87*
 15
 324
 4.63%
400 uatm + mech
 H2.88*
 9
 131
 6.87%
400 uatm + mech
 H2.85
 63
 654
 9.63%
400 uatm + mech
 H2.81
 148
 984
 15.04%
400 uatm + mech
 H2.83
 100
 663
 15.08%
1000 uatm
 H2.121
 16
 289
 5.54%
1000 uatm
 H2.122
 54
 923
 5.85%
1000 uatm
 H2.123
 174
 503
 34.59%
1000 uatm
 H2.124
 29
 610
 4.75%
1000 uatm
 H2.125
 113
 716
 15.78%
1000 uatm
 H2.126
 142
 378
 37.57%
1000 uatm
 H2.127
 46
 341
 13.49%
1000 uatm
 H2.137
 42
 886
 4.74%
1000 uatm + mech
 H2.129
 0
 834
 0.00%
1000 uatm + mech
 H2.130.
 68
 799
 8.51%
1000 uatm + mech
 H1.131
 124
 1190
 10.42%
1000 uatm + mech
 H2.132
 73
 1007
 7.25%
1000 uatm + mech
 H2.133
 169
 741
 22.81%
1000 uatm + mech
 H2.134
 0
 451
 0.00%
1000 uatm + mech
 H2.135
 155
 1210
 12.81%
1000 uatm + mech
 H2.136
 245
 1337
 18.32%
2800 uatm
 H2.19
 110
 1,025
 10.73%
2800 uatm
 H2.21
 0
 613
 0.00%
2800 uatm
 H2.4
 0
 0
 0.00%
2800 uatm
 H2.5
 140
 707
 19.80%
2800 uatm
 H2.7
 0
 0
 0.00%
2800 uatm
 H2.17
 140
 686
 20.41%
2800 uatm
 H2.18
 429
 1016
 42.22%
2800 uatm
 H2.20
 285
 678
 42.04%
2800 uatm + mech
 H2.13
 15
 473
 3.17%
2800 uatm + mech
 H2.14
 0
 256
 0.00%
2800 uatm + mech
 H2.16
 135
 939
 14.38%
2800 uatm + mech
 H2.11
 159
 757
 21.00%
2800 uatm + mech
 H2.12
 13
 181
 7.18%

Photos of new slides:
Name
 Position
 Objective
 Notes
H2.17.1
 13 x 106
 x10
H2.17.2
 13 x 106
 x4
H2.18.1
 33 x 106
 x10
H2.18.2
 34x105
 x10
H2.11.1
 14 x 102
 x4
H2.12.1
 18 x 104
 x4
H2.12.2
 18 x 104
 x10
 possible example of dense hemocytes
H2.71.1
 39 x 101
 x10
 comparative to normal mantle?
H2.83.1
 16 x 105
 x4
 Intestine, sampling artifact
H2.83.2
 20 x 107
 x4
-- Need to take photos as representative images

Notes on Microscope:

To Do:



=

=

August 23rd 2013

Oyster Histology

Updated "Oyster Histology 1" Excel file on personal computer with the new slides data and rearranged it in an easier format to look at (finally figured out how!) and added "Metaplasia" worksheet so all data is on one document in 2 sheets. Adding stats data to worksheet next once finished with metaplasia data for new slides.

August 21st 2013

Oyster Histology

Samples are in!

Preliminary cataloging for tissue types present, updating Excel file on SR lab desktop under Oyster Histology 1(1). Will upload new/cleaner file later on (after returning from work)
New table should include latest samples with tissues present, which treatment groups all slides belong to, and additional notes. Cleaner version needs new layout-- present layout is very wide and not easily read.

As of right now it looks like only two slides do not include digestive gland. Unfortunately, both are from the 2800 uatm CO2 group, but at least we now have 6 slides to work with for that group instead of the original 2.

Ended day on counting dilated digestive gland lumen in slide H2.122-- took AutoCapture photos to start next round of data collection: AutoCapture 20102338p109--120.tif



August 14th 2013

Oyster Histology

Blog post up on monday the 12th following lab meeting
Selected and shipped off new samples to be stained
Slides kept in 70% ethanol, from Feb. 2012

Shipping Samples (in ethanol) for Central Histology Facility

(R. Strenge)
http://friedmanlabuw.wikispaces.com/Protocols

Inspected by Sam White, shipped with appropriate labels:


external image Excepted-Quantities-Paper-Label-D1779.gif


Slides Used

Treatment Group (uatm)
 Slide # (H2.__)
400
 73, 74, 75, 76, 80, 90, 93, 95, 96
400 + mech
 81, 82, 83, 84, 85, 86, 87, 88
1000
 121, 122, 123, 124, 125, 126, 127, 137
1000 + mech
 129, 130, 131, 132, 133, 134, 135, 136
2800
 1, 3, 4, 5, 6, 7, 8, 17, 18, 19, 20, 21
2800 + mech
 9, 10, 11, 12, 13, 14, 15, 16,

Treatment and corresponding sildes (all)

From Feb. 2012 sampling
Treatment Group (uatm)
 Slide # (H2.__)
400
 73, 74, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92, 93, 94, 95, 96
400 + mech
 81, 82, 83, 84, 85, 86, 87, 88,
1000
 121-128, 137-138
1000 + mech
 129-136
2800
 1, 3, 4, 5, 6, 7, 8, 17, 18, 19, 20, 21, 22, 23, 24
2800 + mech
 9, 10, 11, 12, 13, 14, 15, 16,

=

=

August 10th 2013

Oyster Histology

Makeshift Box and Whisker plot of Control groups with and without stress... next is trying to make this more distinct, simple, and in an easier way.
1: without stress 2: with stress
When I tried adding axes labels the box portion went away..
external image Jessicaboxandwhisker.png



Ran ANOVA test with single variable and repetition in Excel

One with high pCO2 running ANOVA between stressed and unstressed:
Anova: Single Factor










SUMMARY




Groups
 Count
 Sum
 Average
 Variance

Hi pCO2
 2
 0.1073
 0.05365
 0.005757

Hi pCO2 + stress
 3
 0.1754
 0.058467
 0.0057















ANOVA





Source of Variation
 SS
 df
 MS
 F
 P-value
 F crit
Between Groups
 2.78E-05
 1
 2.78E-05
 0.004868
 0.948765
 10.12796
Within Groups
 0.017156
 3
 0.005719









Total
 0.017184
 4





And another for the controls, stressed and unstressed:
Anova: Single Factor












SUMMARY





Groups
 Count
 Sum
 Average
 Variance

Control
 6
 1.0895
 0.181583
 0.01391

Control + stress
 6
 0.446
 0.074333
 0.002247















ANOVA





Source of Variation
 SS
 df
 MS
 F
 P-value
 F crit
Between Groups
 0.034508
 1
 0.034508
 4.271406
 0.065642
 4.964603
Within Groups
 0.080788
 10
 0.008079









Total
 0.115295
 11





August 7th 2013

Oyster Histology
1:45-3:30

Met with Carolyn Friedman to go over slides and use of the microscope

Other things to look into:
How long after being spun were the oysters sampled?
How were they sampled? -- how long were they out of the water, were some out of the water longer than others etc.
What about using "JMP" instead of Excel? -- Check out what kind of learning curve that would be

To Do:
Blog Post
Look into hemocyte accumulation
Review control slides
Make Box and Whisker plot
Get R code from Emma?

August 2nd 2013

Oyster Histology
10:00-11:30, 3:00- 4:30

Met with Emma
Treatment
 Slide #
 Tubules open
 Total tubules
 Percentage open
Control
 H2.74*
 16
 266
 6.02%
Control
 H2.80
 172
 1,339
 12.85%
Control
 H2.90
 46
 159
 28.93%
Control
 H2.95
 189
 575
 32.87%
Control
 H2.96
 291
 1,268
 22.95%
Control
 H2.93
 8
 150
 5.33%
Control + stress
 H2.82*
 58
 426
 13.62%
Control + stress
 H2.84*
 20
 203
 9.85%
Control + stress
 H2.86*
 0
 199
 0.00%
Control + stress
 H2.87*
 15
 324
 4.63%
Control + stress
 H2.88*
 9
 131
 6.87%
Control + stress
 H2.85
 63
 654
 9.63%
Hi pCO2
 H2.19
 110
 1,025
 10.73%
Hi pCO2
 H2.21
 0
 613
 0.00%
Hi pCO2 + stress
 H2.13
 15
 473
 3.17%
Hi pCO2 + stress
 H2.14
 0
 256
 0.00%
Hi pCO2 + stress
 H2.16
 135
 939
 14.37%


Problems:

To Do:


July 31st 2013

Oyster Histology

http://ac.els-cdn.com/S0022201196946261/1-s2.0-S0022201196946261-main.pdf?_tid=2791f4aa-fa05-11e2-810d-00000aab0f26&acdnat=1375291262_d7402c4609fc3471ee57bf9e85df59ea



July 30th 2013

Oyster Histology
3:15-6:30 PM

Completed Digestive Tubule Count!

Both open and total with percentage to make sure it's not just a sampling error (some slides had more of the digestive gland included than others)

Slide #
 Tubules open
 Total tubules (7/24/13)
 Percentage open
H2.13
 15
 473
 3.17%
H2.14
 0
 256
 0.00%
H2.16
 135
 939
 14.37%
H2.19
 110
 1,025
 10.73%
H2.21
 0
 613
 0.00%
H2.74*
 16
 266
 6.02%
H2.80
 172
 1,339
 12.85%
H2.82*
 58
 426
 13.62%
H2.84*
 20
 203
 9.85%
H2.86*
 0
 199
 0.00%
H2.87*
 15
 324
 4.63%
H2.88*
 9
 131
 6.87%
H2.90
 46
 159
 28.93%
H2.95
 189
 575
 32.87%
H2.96
 291
 1,268
 22.95%
H2.85
 63
 654
 9.63%
H2.93
 8
 150
 5.33%

Other Notes/ What's next:

Heard back from staining company-- should be able to see chloride cells in gill tissue using current stains. Check this with Dr. Friedman and review the paper.
Hemocyte accumulation: try using Image J to see if it will distinguish hemocytes (and chloride cells?) -- review Roberts paper
Run some stats! Let's see if any of this data is significant



July 29th 2013

Oyster Histology
10:00-11:30 AM

Lab meeting--
Next unseminar 8/22?
Research symposium 8/26 for lil interns
need another blog post!

Image J tutorial wiki -- might start using Image J for hemocyte accumulation. Need to become a little more familiar with it first though.
http://imagejdocu.tudor.lu/doku.php?id=howto:start


July 26th 2013

Oyster Histology
10:30-12:30 AM


Recounted degestive tubules expressing metaplasia
- testing methods

For slides H2.16 and H2.19 I used the count tool while keeping the camera feed live to re-count metaplastic cells
For slides H2.21-H2.80 I used the live feed to mark cells in a single screen, right click to total and to turn the markers red (rather than blue). Then I went through the same screen to count open lumen (while the markers were blue) and totaled that count. I then moved through the slides in rows doing my best not to overlap the photos.

Completed slides H2.16-H2.74. H2.80 had many more digestive tubules than the previous slides; I took pictures using Auto Capture and used the count tool for both total and open tubules.
I did not have time to complete slide H2.80 before having to go to work, ended on photo 20102338po32 (last one in autocapture folder)

To Do:
Finish slide H2.80- pics 26-35
Count digestive cells on remaining slides
Count open lumen on remaining slides
Calculate percentages
Review running some stats
Meet with Dr. Friedman about answering scope questions
Play with Image J for hemocyte accumulation
Review staining article-- Find out if histology company can stain for chloride cells
Blog post

Digestive tubule count updated to below table from July 18th



July 24th 2013

Oyster Histology
3:45-6:00 PM

Counting Digestive cells


Methods:
using Friedman microscope to take pictures of all digestive tubules, then use count tool to mark each tubule.
Later used "AutoCapture" tool for easier access to pictures once taken. After using capture initially, NIS Elements became unresponsive. I reopened it and used autocapture and it worked without crashing again.

Problems:

Questions:
To Do:
Play with Image J- for hemocyte count and for metaplasia
Run stats on metaplasia percentages
Review Roberts paper for hemocyte accumulation


Totals added to below table.

July 18th 2013

Oyster Histology
9:45-10:30 AM; 3:30- 5:30 PM

Metaplasia search

What I was calling "possible metaplasia" yesterday is most likely a slice of intestine (now corrected). So, today the goal was to go through each slide looking specifically for open digestive gland lumen as an indicator of metaplasia.

Notes: amount of digestive gland present varies pretty drastically between slides (leading to sampling errors) maybe use as percentage of total tubules present; requires count tool on Friedman microscope. See if there's a way to count two at once, to differentiate those "open" while counting total.
Intestine tissue tends to have more mucous cells and defined epithelial cells-- looks almost striated (use to differentiate between the two)

Slide #
 Tubules open
 Total tubules (7/24/13)
 Percentage open
H2.13
 15
 473
 3.17%
H2.14
 0
 256
 0.00%
H2.16
 135
 939
 14.37%
H2.19
 110
 1025
 10.73%
H2.21
 0
 613
 0.00%
H2.74*
 16
 266
 6.02%
H2.80
 172
 1339
 12.85%
H2.82*
 58
 426
 13.62%
H2.84*
 20
 203
 9.85%
H2.86*
 0
 199
 0.00%
H2.87*
 15
 324
 4.63%
H2.88*
 9
 131
 6.87%
H2.90
 46
 159
 28.93%
H2.95
 189
 575
 32.87%
H2.96
 291
 1268
 22.95%
H2.85
 63
 654
 9.63%
H2.93
 8
 150
 5.33%
*very few total digestive gland tubules
Open tubule count may change soon using the Friedman microscope.

Other miscellaneous tasks done today:
Fixed picture upload error from Eagle to wikispace; no more "#" in folder titles. Re-embedded photos from Eagle to July 17th table.
Conferred with Emma and Lisa about tissue types seen in July 17th table and whether or not metaplasia was present. Modified below table to reflect corrections (ie. identifying heart and kidney tissues, confirming germinal follicles)

To do:
Prepare slides for staining on Monday
Count total digestive tubules present in each slide to calculate percentages
Review stats notes/book to run preliminary stats on metaplasia


=

=

July 17th 2013

Oyster Histology
2:30-5:00 PM

Exploring stress response detection methods

Goals:


Picture #
 Picture
 Notes
H2.13.1
 external image H2.13.1.jpg
 Possible metaplasia
slices of intestine
H2.13.2
 external image H2.13.2.jpg
 A few open lumen-- metaplasia?
center long tissue probably intestine..
H2.13.3
 external image H2.13.3.jpg
 Germinal follicles
H2.13.4
 external image H2.13.4.jpg
 Unknown tissue
H2.13.5
 external image H2.13.5.jpg
 Closer view of unknown
H2.13.6
 external image H2.13.6.jpg
 Test of hemocyte counting:
Took area of mantle between digestive gland and intestine
zoomed in to 20x (546.6px X 1913.3 px)
used "count" tool to click on each hemocyte
counted 505 hemocytes...
It's possible that what I was counting may or may not be hemocytes
(more beneficial to do smaller or larger area?)
H2.14.1
 external image H2.14.1.jpg
 possibly metaplasia -- far left open lumen
center of photo most likely slice of intestine
H2.14.2
 external image H2.14.2.jpg
 left side might be different tissue
looks like accumulation of hemocytes but surrounded by some other tissue as well..
H2.14.3
 external image H2.14.3.jpg
 possibly spermatids (better pic than "unknown" table)
H2.14.4
 external image H2.14.4.jpg
 Mantle, with reproductive follicles
H2.14.5
 external image H2.14.5.jpg
 Intestine (shot of mucous cells)
H2.14.6
 external image H2.14.6.jpg
 Heart
Overall
good examples of mucous cells!


Notes:
Captured photos saved under Desktop folder = "Jessica"
Ran into some issues with the slide H2.14.2 picture upload-- continue later
All photos are uploaded to Eagle (C.gigas histology photos #2)
http://eagle.fish.washington.edu/scaphapoda/index.php?dir=Jessica%2FC.+gigas+histology+photos+%232%2F


Miscellaneous tasks done:
E-mailed Dr. Friedman
Blog post
Re-ordered slides-- far right is the start of the slides containing mantle, intestine, digestive gland, AND gill.
H2.13-H2.96


July 15th 2013

Oyster Histology
10:00-11:20; 3:30-4:45

Lab meeting notes: up the blog posts, keep an eye out on the Google + community for lab meeting themes?

Slides containing mantle, gill, digestive gland, and intestine:

Representative photos and unknown tissue photos

Slide number listed on the left (if no slide number than it's a continuation of the previous slide)
It's likely that these are the slides to be used in tissue analysis given the consistency of tissue type
Corresponding notes are in the Excel file below
http://eagle.fish.washington.edu/scaphapoda/index.php?dir=Jessica%2F

H2.13
 external image 28.JPG
 external image 93.JPG
H2.14
 external image 29.JPG
 external image 30.JPG
H2.16
 external image 33.JPG
 external image 32.JPG
H2.19
 external image 97.JPG
 external image 34.JPG
H2.21
 external image 36.JPG
 external image 37.JPG
H2.74
 external image 46.JPG
 external image 47.JPG
H2.80
 external image 49.JPG

H2.82
 external image 50.JPG
 external image 51.JPG
H2.84
 external image 52.JPG
 external image 53.JPG

external image 54.JPG
 external image 55.JPG
 external image 56.JPG
H2.86
 external image 58.JPG
 external image 59.JPG
H2.87
 external image 60.JPG
 external image 61.JPG

external image 63.JPG
 external image 64.JPG
H2.88
 external image 67.JPG
 external image 65.JPG
H2.90
 external image 69.JPG
 external image 70.JPG
 external image 72.JPG
H2.95
 external image 82.JPG
 external image 83.JPG
 external image 84.JPG
H2.96
 external image 85.JPG
 external image 86.JPG
 external image 87.JPG

external image 88.JPG
 external image 89.JPG

Next:
Draft e-mail to Dr. Friedman about unknown tissues and how to proceed.
Inflammation analysis:

Restain for chloride cells around gills

July 10th 2013

Oyster Histology
2:00-5:00 PM

It's official: my computer is too slow to embed pictures. Will be doing so from lab computers from now on...
Embedded pictures into below table
Figured out hot to post to the tumblr, next is OA blog
Reviewed Friedman binder to compare to unknown tissues and analyze metaplasia

(Sidenote/ tangent thought: wondering how histology of Ulcerative Colitis would relate to acidic environments and act as an example of what to look for in inflamed intestine cells; primarily bacteria caused but ulcers usually related to acidic environment... how similar? http://www.sciencedirect.com/science/article/pii/S1542356513004606 <-- markers)


July 8th 2013

Oyster Histology
3:00-7:30 PM

Tutorial on how to use Friedman microscope with Emma and Claire.

Double check How To section to embed pictures from Eagle

Updated Excel file to sort comparable data and isolate unknown tissue types (not sure if this will translate into the previous link)



Unknown Tissue Pictures:

Slide number on the far left, tissue sample will be on the right, most likely one pic at 4x, another 10x.
Red text indicates that particular slide doesn't contain all four tissues (gill, intestine, digestive gland, mantle)-- so it's likely not to be included in comparative analysis

H2.14
 external image 29.JPG
 external image 30.JPG

H2.16
 external image 32.JPG
 external image 96.JPG

H2.19
 external image 34.JPG
 external image 35.JPG

H2.74
 external image 44.JPG
 external image 45.JPG
 external image 47.JPG
H2.88
 external image 65.JPG
 external image 66.JPG
 external image 67.JPG
H2.90
 external image 69.JPG
 external image 70.JPG
 external image 71.JPG
H2.95
 external image 83.JPG
 external image 84.JPG

H2.10
 external image 25.JPG


H2.9
external image 68.JPG
 <-- bottom and top tissues surrounding the mantle; just mantle epithelium?

H2.93
 external image 73.JPG
 external image 74.JPG
 external image 75.JPG
H2.93 (con)
 external image 76.JPG
 external image 77.JPG

external image 79.JPG
 external image 78.JPG


external image 80.JPG
 external image 81.JPG




July 7th 2013

Oyster Histology

Continue literature search:

Acute stress alters intestinal function of rainbow trout (Olsen et al)
http://www.sciencedirect.com.offcampus.lib.washington.edu/science/article/pii/S004484860500178X
-- food deprivation as stress but looked at different biochemical markers of stress (haemacrit, blood glucose, lactate, cortisol) and changes in permeability of mannitol
-- electron microscopy of enterocytes at different sections (hindgut, midgut etc)

Tissue Adaptation to physical stress (Mueller and Maluf)
http://web.ebscohost.com.offcampus.lib.washington.edu/ehost/detail?sid=9aa32740-1ecf-4c3b-8cd9-406ad0b982fb%40sessionmgr114&vid=1&hid=124&bdata=JnNpdGU9ZWhvc3QtbGl2ZQ%3d%3d#db=a9h&AN=6406638

This will probably be a good resource:
"Histological techniques for marine bivalve mollusks" (Howard and Smith)
http://permanent.access.gpo.gov/gpo7399/tmfnec25.pdf


July 3rd 2013

Oyster Histology
10:00-11:30 AM; 2:00-5:00PM

Uploaded Excel file and histology slide photos to Eagle server--> Scaphopoda--> Jessica
http://128.95.149.81:5000/webman/index.cgi

http://eagle.fish.washington.edu/scaphapoda/index.php?dir=Jessica%2F


(renamed photo files, struggled with iPhone connections, followed Roberts lab on tumblr and Facebook)

Continued literature search...

Next Wednesday, 8;30AM, will be learning how to use C. Friedman microscope to measure different tissue sizes- looking for signs of inflammation
Still should get a better idea of what histology looks like in response to stress, become familiar with identifying tissues and noticing differences

June 28th, July 1st and 2nd 2013

Oyster Histology
Literature Search

Sites to go back to and find full text articles and read in more detail:
Still need: more histological examples of heart, kidney, post-esophagus, inflammation comparison


June 27th 2013

Oyster Histology
3:20-5:45 PM

I continued to catalog the tissue types contained in the rest of the slides today; a grand total of all 24 slides.
I'm not sure if the whole table will fit, it's pretty wide.

 H2.1
 H2.10
 H2.13
 H2.14
 H2.15
 H2.16
 H2.19
 H2.21
 H2.3
 H2.6
 H2.74
 H2.8
 H2.80
 H2.82
 H2.84
 H2.85
 H2.86
 H2.87
 H2.88
 H2.9
 H2.90
 H2.93
 H2.95
 H2.96
Gill
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
Intestine
 x
 x
 x
 x
 x
 x
 x
 x
 ?
 x
 x
x
 x
 x
x
 x
 x
 x
 x
x
 x
Digestive Gland

x
 x
x
 x
 x

x
x
 x
 x
 x
 x
 x
 x
x
 x
 x
 x
Mantle
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
Adductor
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
x
 x
 x
 x
 x
 x
 x
 x
Other
Heart?
premature sperm?
see pic 32
 heart?


see pics 43&44






heart? Kidney?
 see pic 68…
 kidney or post esophagus?
 spermatids and oocytes?? (pic 77)
 see pics 83-84






















post-esophagus?

Picture #
 27(10x), 90(4x), 91(10x)
 25(4x), 92(4x),
 28(4x), 93(10x)
 29(4x), 30(10x)
 31(4x), 94(10x), 95(40x),
 32(4x),96(10x), 33(4x)
 34(4x), 35(10x), 97(4x)
 36(4x), 37(4x),
 38(4x), 39(10x), 40(4x)
 41(4x), 42(4x), 43(10x)
 44(10x), 45(40x), 46(4x), 47(40x)
 48(4x)
 49(4x)
 50(4x), 51(4x)
 52(4x), 53(10x), 54(4x), 55(10x), 56(40x)
 56(10x), 57(10x)
 58(4x), 59(10x)
 60(4X), 61(10X), 62(4X), 63(10X), 64(40X)
 65(10x), 66(40x), 67(10x),
 68(4x),
 69(4x), 70(10x), 71(40x), 72(10x),
 73(4x),74(10x), 75(40x), 76(10x), 77(40x), 78(40x), 79(10x), 80(10x), 81(40x)
 82(4x), 83(10x) 84(40x)
 85(4x), 86(4x), 87(4x), 88(10x), 89(4x)
Notes:
 pic 27 might be mantle but looks like dense muscle, adductor muscle in visceral mass sample
 pic of what might be heart
 intestine appears torn/sliced, adductor dispersed/broken, 93 is closer of 28
really small sample, diffuse
 large slice in adductor muscle, intestine torn/sliced
 majority of tissue is digestive glad, potential heart tissue is on adductor sample
 generally difffuse, intestine torn/sliced, pic 36 is on adductor sample
 appears to be mostly mantle, can't tell if there is intestine or not, much of it is diffuse
small blue dots in clusters throughout mantle
 diffuse adductor
 lots of gill and digestive gland
pics 54-56 correlate to the same tissue/structure
 see pic for intestine?
diffuse mantle tissue, possibly reporoductive follicles throughout
 diffuse adductor
 not much intestine, what's there is pretty torn
 spread out sample, lots of tearing
 79 is a zoomed out shot of 78. There seems to be a lot more hemocytes (or at least the small purple dots) throughout the mantle than in other slides
 possibly a good representative slide of wanted cross section
 much of intestine sliced/torn, lots of digestive gland; pic 89 is adductor w/other tissue?

I also added the objective used for early slide photos and took a few more representative pictures. As I got to the later slides I was a little more comfortable with noticing other tissues and thinking of what they could look like, causing me to go back through slides from yesterday and there were a few similar structures. The slides seem pretty inconsistent when it comes to what kind of tissue is actually there; some of the cross sections are from different areas of each oyster and some are pretty torn/spread out.

To Do:

Planning on coming back into the lab in the morning, though may end up doing a literature search from home instead depending on car troubles I'm anticipating in the morning.



June 26th 2013

Oyster Histology
10:30-11:10 AM; 2:15- 5:15 PM

Today was mostly fiddling with set-ups, figuring out the Google calendar for the lab, setting up the wikispace to correlate with the Roberts Lab page and beginning analyzing histology slides. I set up the iPhone we are using for a camera, which took more time than it should have but ended successfully! *Remember to turn on the camera first then mount the holster to the scope, directly mounting the lense to the iPhone ends up cutting off much of the field of view. I then organized Emma's slides in numerical order and set up an Excel file to include the tissue types I was looking for: gill, intestine, digestive glad, mantle, and adductor muscle, as well as space for other tissues, additional notes, and a corresponding picture number. About halfway through I started adding what objective the pictures correlated to and I probably should have done that from the start. This may mean going back through earlier pics and assigning objectives to have an accurate scale.

Things to think about:
If there is "mystery tissue", how likely is that to be kidney? Example histology pics indicate close proximity-- take a closer look at what that looks like
Reproductive structures-- maybe double check for oocytes and spermatids
Heart tissue may be present
Some tissue torn or sliced-- not sure how much of that is from the live oyster and how much was transferring into slides

The 17 slides I have so far:

 H2.1
 H2.10
 H2.13
 H2.14
 H2.15
 H2.16
 H2.19
 H2.21
 H2.3
 H2.6
 H2.74
 H2.8
 H2.80
 H2.82
 H2.84
 H2.85
 H2.86
Gill
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
Intestine
 x
 x
 x
 x
 x
 x
 x
 x
 ?
 x
 x
x
 x
 x
x
Digestive Gland

x
 x
x
 x
 x

x
x
 x
 x
 x
 x
Mantle
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
Adductor
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
 x
x
Other
Heart?
premature sperm?
see pic 32
 heart?


see pics 43&44























Picture #
 27
 25
 28
 29(4x), 30(10x)
 31
 32, 33
 34, 35
 36, 37,
 38(4x), 39(10x), 40(4x)
 41(4x), 42(4x), 43(10x)
 44(10x), 45(40x), 46(4x), 47(40x)
 48(4x)
 49(4x)
 50(4x), 51(4x)
 52(4x), 53(10x), 54(4x), 55(10x), 56(40x)
 56(10x), 57(10x)
 58(4x), 59(10x)
Notes:
 pic might be mantle, but looks like dense muscle
 pic of what might be heart
 intestine appears torn/sliced, adductor dispersed/broken
really small sample, diffuse
 large slice in adductor muscle, intestine torn/sliced
 majority of tissue is digestive glad
 generally difffuse, intestine torn/sliced
 appears to be mostly mantle, can't tell if there is intestine or not, much of it is diffuse
small blue dots in clusters throughout mantle
 diffuse adductor
 lots of gill and digestive gland
pics 54-56 correlate to the same tissue/structure
 see pic for intestine?


June 25th 2013

Oyster Histology
9:00-10:25 AM

This was my first day working in the Roberts lab with Emma. I became more oriented with the lab and Emma lead me through some example histology pictures. We then looked at about 5 slides of hers and Mac's to give me a better idea of what kinds of tissues I was actually looking at. I will be primarily cataloging what types of tissues are in Emma's slides as they seem to be inconsistent in what tissues were included across the samples. The main tissues I am checking for are intestine, digestive glad, mantle, and gill-- the cross section of the oyster extending dorso-ventrally.
Other things to remember seeing: light blue dots (larger than hemocytes) might be mucous cells, spermatids can be dark and tightly clustered, oocytes may look similar to digestive tissues but without "star" shape, open digestive tubules may indicate metaplasia, signs of stress could be flooding of hemocytes