Lab Notebook: Ecology of Marine Infectious Diseases

Sonia Singhal


22 Aug 2012
Labyrinthula lab:

Chasing after the (apparently non-specific) primers a bit...



21 Aug 2012
Labyrinthula lab:

Discovered that the GoTaq Flexi mix is only a buffer-- that is, it contains no MgCl(2) or dNTPs. Lisa got us fresh stocks of each and also mentioned that the ImmoMix was unused by anyone in the class (although it required loading dye for visualization). Sammi and I decided to hedge our bets and run a PCR from each mix. We autoclaved the remainder of the PCR tubes in preparation. Reactions were set up under the Lab 10 hood, and tubes and water were UV irradiated beforehand.

Same reaction set used as 20 Aug.

PCR mix 1:
Reagent
 µL for one reaction
Total µL
5x GoTaq flexi buffer
 4
 X 21
 84
BSA
 1
 21
MgCl2 (25 uM)
 3
 63
dNTP (10 uM)
 2
 42
P1
 0.5
 10.5
P2
 0.5
10.5
Taq
 0.2
4.2
PCR water
 6.8
142.8
Total
 18
378
18 uL of mix aliquoted per tube and 2 uL of the appropriate DNA added. Primers as per 20 Aug.

PCR mix 2:
Reagent
 µL for one reaction
Total µL
ImmoMix
 12.5
 X 21
 262.5
BSA
 1.5
 31.5
P1
 0.8
 16.8
P2
 0.8
 16.8
PCR water
 7.4
 155.4
Total
 23
483
23 uL of mix aliquoted per tube and 2 uL of the appropriate DNA added. Primers as per 20 Aug.

Cycle (both mixes):
Step
 Temperature (C)
 Time
1
 95
 10 min
40 cycles of:
2
 95
 30 min
3
 50
 30 sec
4
 72
 60 sec

5
 72
 10 min

Visualization on a 2% agarose gel (60 min at 65V) showed no bands for the Flexi mix, but there were bands for the ImmoMix!
labyGel_21Aug.png
Order of wells:
1. Phylloplesia head
2. Phylloplesia gonads
3. Phylloplesia gizzard
4. Phylloplesia digestive tract
5. Phylloplesia nidamental gland
6. Phylloplesia posterior
7. Lacuna diseased 1
8. Lacuna diseased 2
9. Lacuna diseased 3
10. Lacuna healthy 1
11. Positive control
12. Lacuna healthy 2
13. Lacuna healthy 3
14. CK Shallow Bay culture
15. FB Sh T-1-D culture
16. FB Sh T-1 culture
17. Beach Haven (extraction 1 Aug)
18. Vibrio RE22
19. Sandy's monoculture
20. Positive control
21. Negative control

Results:


20 Aug 2012
Labyrinthula lab:

Visualization of PCR products from yesterday, run on 2% agarose gel for 60 min at 65 V (done because we're using the bad ladder that smeared last time). Photo not included because there were no bands whatsoever to be seen, including for the positive control. (The ladder, however, looked gorgeous.) We thought that perhaps since the power had gone out in the middle of the night, the PCR hadn't finished properly.

Re-ran the PCR. I added in previous BH and monoculture extractions.
PCR mix:
Reagent
 µL for one reaction
Total µL
5x GoTaq flexi mix
 5
 X 21
 105
BSA
 1.5
 31.5
P1
 0.8
 16.8
P2
 0.8
 16.8
PCR water
 14.65
 307.65
Taq
 0.25
5.25
Total
 23
483
Primers:
Forward, 5’ GGAAGGCAGCAGGCGCGTAA 3’
Reverse 5’ GAATGTTTCCATCCTCAGTCGGTATAG 3’

2 uL of each DNA sample added to each reaction. Positive control: Pool Laby + (stock); Negative control: PCR water

Cycle conditions:
Step
 Temperature ( C)
 Time
1
 95
 10 min
40 cycles of:
2
 95
 30 min
3
 50
 30 sec
4
 72
 60 sec

5
 72
 10 min

Reactions removed from the PCR machine as soon as they finished. Another gel visualization still showed... no bands whatsoever.
Maybe our sterile technique was too sterile?


19 Aug 2012

Labyrinthula lab:
Redo of DNA extraction and PCR:

Sammi and I autoclaved all tubes we wanted to use (1.5-mL, 2-mL, PCR) and got new aliquots of all materials. Lisa gave us an untouched GoTaq Flexi mix to use for PCRs.

Extractions as per 18 Aug-- only CK and FB cultures redone. Sammi dissected different parts of a Phylloplesia to try to isolate which organs Laby might be present in. I added a Vibrio (RE22) extraction as a definite negative control and did it completely under the hood--not as a sterile space, but as a Laby-free space. PCR tubes and water UV'ed under the hood for 20 min before use.

PCR mix: Flexi Taq
Reagent
 µL for one reaction
Total µL
5x GoTaq flexi mix
 5
 X 19
 95
BSA
 1.5
 28.5
P1
 0.8
 15.2
P2
 0.8
 15.2
PCR water
 14.65
 278.35
Taq
 0.25
4.75
Total
 23
437
Primers:
Forward, 5’ GGAAGGCAGCAGGCGCGTAA 3’
Reverse 5’ GAATGTTTCCATCCTCAGTCGGTATAG 3’

2 uL of each DNA sample added to each reaction. Positive control: Pool Laby + (stock); Negative control: PCR water

Cycle conditions:
Step
 Temperature ( C)
 Time
1
 95
 10 min
40 cycles of:
2
 95
 30 min
3
 50
 30 sec
4
 72
 60 sec

5
 72
 10 min
6
 10
 overnight


18 Aug 2012

Labyrinthula lab:
DNA extraction and PCR of Labyrinthula cultures:
Plated cultures used:

Scraping of each culture put into 700 uL ASL; DNA extracted using the QIAgen Stool kit (protocol under 26 July). Sample #2 had the lid pop off during the centrifugation at Step 13, but no liquid was lost. All other lids remained closed.

Sammi extracted DNA from her Lacuna snails.

PCR mix: GoTaq
Reagent
 µL for one reaction
Total µL
GoTaq mix
 12.5
 X 14
 175
BSA
 1.5
 21
P1
 0.8
 11.2
P2
 0.8
 11.2
PCR water
 7.4
 103.6
Total
 23
322
Primers:
Forward, 5’ GGAAGGCAGCAGGCGCGTAA 3’
Reverse 5’ GAATGTTTCCATCCTCAGTCGGTATAG 3’

2 uL of each DNA sample added to each reaction. Positive control: Pool Laby + (stock); Negative control: PCR water

Cycle conditions:
Step
 Temperature ( C)
 Time
1
 95
 10 min
40 cycles of:
2
 95
 30 min
3
 50
 30 sec
4
 72
 60 sec

5
 72
 10 min

Visualization on 1.5% agarose gel:
LabyGel_18Aug.png
Order of wells:
1. Healthy Lacuna 1
2. Healthy Lacuna 2
3. Healthy Lacuna 3
4. Diseased Lacuna 1
5. Diseased Lacuna 1
6. Diseased Lacuna 2
7. Diseased Lacuna 3
8. * (previous Lacuna extraction?)
9. Positive control
10. Negative control
11. Laby culture 1
12. Laby culture 2
13. Laby culture 3
14. Laby culture 4
15. Beach Haven (extracted 1 Aug)
16. Positive control

...Because our negative control also shows bands, the results of the PCR are rather dubious. Since we were using the class reagents, something may have gotten contaminated...


17 Aug 2012

Bioinformatics lab:

Labyrinthula lab:
Seawater took a long time to sterilize--autoclave kept aborting! Finally realized that it needed more water. Also got more bleach for sterilizing clips.

See 15 Aug for general protocol. Notes of details and modifications follow.

1. Culture inoculum

2. Clip inoculum

The 1* culture from 6 Aug had grown over the inoculum leaves, so we additionally ran a second clip trial with that strain. Control plate looked uncontaminated, so 2 non-infected leaf-halves were included as negative controls.

Photos:
1*-1
1star-1_17Aug.JPG

1*-2
1star-2_17Aug.JPG

Control
blade_control_17Aug.JPG

Gregor and Courtney will be taking photos of all inoculated leaves ~ every 12 hours so that we can get time-series data on lesion development.


16 Aug 2012

Bioinformatics lab:

Labyrinthula lab:
Setup for next inoculation experiments:


15 Aug 2012

Bioinformatics lab:
Check over- or under-representation of annotated genes between the full dataset and the differentially expressed data set.
This means the analysis is complete!
The read headings can be expanded by clicking on the square to the left.

Labyrinthula lab:

Finalized the protocol for the reinfection experiments:

Protocol for Laby reinfection experiments

Approach 1: Blade inocula

Day 1: Preparation for plating

Day 2: Create the inocula

Days 3-5: Laby platings will be growing over the agar and autoclaved leaves. This may take anywhere from 3 to 6 days, depending on how quickly the particular strain grows. When it looks like at least one blade has been surrounded by Laby, prepare for the inoculations:

Day 6: Perform inoculations

Day 7-10: Track lesion progress
Photograph petri dishes at 12-hour intervals until clear signs of lesions. Lesions will appear as dark streaks radiating from the center of the blade, where the inoculum was placed. Temperature data can also be taken.

Day 11: Analysis
Use ImageJ to analyze all blades. Protocol to be completed when Courtney goes over it with us.

Approach 2: Liquid culture inocula

Day 1-7: Start cultures

Day 8: Setup for experiment

Day 9: Inoculations, including scratch trial

Day 10-14: Monitor progress of lesion growth
Photograph petri dishes at 12-hour intervals until clear signs of lesions. Ideally, lesions will appear first in the area where the blade was scratched and then expand. Temperature data can also be taken.

Day 15: Analysis in ImageJ.


14 Aug 2012

Bioinformatics lab:
Joining tables in Galaxy:

Galaxy’s setup is:

Protocol:


13 Aug 2012

Labyrinthula lab:
Reinfections:
Both Sandy’s culture and the 2* field culture show clear signs of lesions. We photographed all blade-inoculum plates both with and without the clips. The blades themselves were placed back into the same petri dish in the same place on the table, in case we want them for histology.

Liquid-inoculum plates will be left for a little longer to see if any infections develop later. We know that the cultures grow even more slowly than the plates, and not very many cells were inoculated into each plate.

Bioinformatics lab:

Downloaded the SwissProt database onto the lab computers (CL_14 à Users à Shared à EIMD_blast à db) and unzipped it. The database was then created through the terminal:

%% cd /Applications/blast/bin
%% ./makeblastdb -in [input file in .fasta] -out [output file name, no .fasta] -dbtype prot

Input: /Users/Shared/EIMD_blast/db/uniprot_sprot.fasta
Output: /Users/Shared/EIMD_blast/db/uniprot_sprot

Practice BLAST on the QPX transcriptome data, just to make sure it worked. Only the uniprot database worked; the viral database that was already loaded did not seem to.

%% ./blastx -query [input file in .fasta] -db [uniprot_sprot, no .fasta]


Started a BLAST of the full transcriptome:

First opened file (/Users/Shared/EIMD_blast/query/QPX_transcriptome_v1.fasta) in TextWrangler. (Say “No” to any options that come up when TextWrangler opens.) To prevent misreading of the sequence names/sequences, we did a reformat:

In Terminal:
%% ./blastx
-query [input file]
-db [database file, no .fasta]
-evalue 1e-20
-max_target_seqs 1
-outfmt 6
-out [outfile]
-num_threads 2

Notes:
*query used: /Users/Shared/EIMD_blast/query/QPX_transcriptome_v1.fasta
*db: /Users/Shared/EIMD_blast/db/uniprot_sprot
*out: /Users/Shared/EIMD_blast/out/qpx_transcr_swisspro.txt
*E-value of e^(-20) => only HIGHLY similar sequences will be selected
*max_target_seqs – only return the first (highest) hit for each contig
*outfmt 6 = tab-delimited
*num_threads = number of CPUs to use

Note that the command above should be written in a single line, with spaces instead of enters/tabs. I’ve written it out that way to make it easier to read all the arguments.

Let the program run on the terminal overnight. Final location of the resulting text file:
https://catalyst.uw.edu/sharespaces/download/17360/318727?html=1&url=https://catalyst.uw.edu/sharespaces/download/17360/318727

Headed text file, made from R:
https://catalyst.uw.edu/sharespaces/download/17360/318803?html=1&url=https://catalyst.uw.edu/sharespaces/download/17360/318803


12 Aug 2012

Noticed this evening that some of the plates from the reinfection experiment look like they have developed lesions – especially for some of the blade inocula, it looks like there is a dark lesion that extends to either side of the inoculum. This morning, that was not as evident (at least, at a glance).


11 Aug 2012

Vibrio lab:
Dropped our larvae with 10% bleach and did final total counts:

 1
 2
 3
 4
 Control
A
 21
 42
 51
 44
 35
B
 32
 40
 34
 45
 32
C
 34
 34
 33
 55
 41


Labyrinthula lab:
Made up new liquid cultures of several of the Laby cultures for the herbivore group:
All cultures were in ~ 10 mL of Laby broth in 50-mL Falcon tubes, with the exception of the two liquid cultures from 9 Aug that had been spun down and resuspended. All cultures except “Shallow Bay 1 CK” were made in triplicate.

Also replated several agar cultures that look like they’re starting to take over the plate. The False Bay samples were additionally replated (into separate plates) for the Herbivore group.


10 Aug 2012

Vibrio lab:
Plating results:
T1N5 plates from 8 Aug, with the dilutions of Vibrio, finally grew. Final counts:
Dilution
 Rep 1
 Rep 2
10^(-5)
 33
 4
10^(-6)
 5
 0
10^(-7)
 2
 1

TBS plates also grew, but:

Larval bacterial challenge:
Because Ashton and I both had a lot else going on, we decided that we would take our 48-hour counts today, then drop the larvae and do the total counts tomorrow. We went over alive vs. dead with Carolyn—basically, what had been skipped was moving to a higher magnification when it looked like a larva was dead, to verify. I think it is likely that my assignment of what was dead was way too conservative; sometimes ciliates or bacteria make it look like the velum is vibrating at low magnification, but at high magnification it is clear that something is wrong. The numbers this time matched much more closely.

Once again, I counted row A and row C, and Ashton did the controls and row B.

Table of larvae mortality after 48 hours:

 1
 2
 3
 4
 Control
A
 3
 40 +/- 1
 43
 38
 2 +/- 1
B
 5
 37
 31
 40 +/- 1
 0
C
 4
 21
 32
 27
 0

Notes:

Labyrinthula lab:
Reinoculation experiment:

We have two inoculation experiments going:
  1. Autoclaved blade inoculum (Protocol 1 under 9 Aug)
  2. Liquid culture inoculum (Protocol 3 under 9 Aug)

What we actually did in this experiment follows. A thorough protocol taking some of the problems into account will be posted later.

General:


1. Autoclaved blade inoculum
Pictures of the inoculum blades.
Both the Sandy and 2* cultures had blades that looked infected; the 1* culture is still growing so it was dropped from this experiment and left to grow more. Sadly, there was contamination in one of the controls (#2)!

Control #1
autocl_inoc_contr1_3.JPG

Control #2
autocl_inoc_contr2_3.JPG

Sandy #1
autocl_inoc_sandy1_3.JPG

Sandy #2
autocl_inoc_sandy2_3.JPG

1*-1
autocl_inoc_1star-1_3.JPG

1*-2
autocl_inoc_1star-2_3.JPG

2*-1
autocl_inoc_2star-1_3.JPG

2*-2
autocl_inoc_2star-2_3.JPG


Replicates:

4 leaves total per plate à take 2 leaves and halve them
X 2 plates
X 2 cultures
+ 3 controls

This was dropped down to 2 leaf-halves per plate, for a total of 4 replicates per culture, + 3 controls = 11 clip inocula plates total

Procedure:


2. Liquid culture inoculum

Replicates:
Dilution factors 10^0, 10^(-2), 10^(-4)
X 3 reps per factor
X 2 cultures
+ 3 controls that would receive sterile seawater

Because the liquid culture from Sandy’s monoculture did not have enough cells/mL to make inoculations worthwhile, it was dropped from the experiment, and instead we did a full dilution series by factors of 10 from 10^0 to 10^(-4) for the BeachHaven culture, = 3 x 5 + 3 controls = 18 plates total.

Procedure:
10am
11am
3:30pm
8pm

Hemocytometer counts in 10 µL of a 1:2 dilution of liquid culture:
Rep
 Count
 Calculation
 Total
1
 48
 48 x 2 x 10 x 1000
 9.6 x 10^5
2
 33
 33 x 2 x 10 x 1000
 6.6 x 10^5
3
 36
 36 x 2 x 10 x 1000
 7.2 x 10^5


Mean
 7.5 x 10^5 cells/mL
Formula for calculation: # cells/mm^2 x dilution factor x 10 µL in hemocytometer x 1000 µL per mL

Dilution factors:
10^0 dilution = (7.8 x 10^4 cells in 0.1 mL) / 25 mL = 3120 cells/mL
10^(-1) dilution = 312 cells/mL
10^(-2) dilution = 31.2 cells/mL
10^(-3) dilution = 3.12 cells/mL
10^(-4) dilution = 0.312 cells/mL



11 Aug 2012

Vibrio lab:
Dropped our larvae and did final total counts:

 1
 2
 3
 4
 Control
A
 21
 42
 51
 44
 35
B
 32
 40
 34
 45
 32
C
 34
 34
 33
 55
 41


Labyrinthula lab:
Made up new liquid cultures of several of the Laby cultures for the herbivore group:
All cultures were in ~ 10 mL of Laby broth in 50-mL Falcon tubes, with the exception of the two liquid cultures from 9 Aug that had been spun down and resuspended. All cultures except “Shallow Bay 1 CK” were made in triplicate.

Also replated several agar cultures that look like they’re starting to take over the plate. The False Bay samples were additionally replated (into separate plates) for the Herbivore group.


9 Aug 2012

Vibrio lab:
Gram staining to identify whether an isolated population of bacteria is Gram-positive or Gram-negative.
Plated culture: Add an inoculation-loop’s worth of a single colony to a small (<10 µL) drop of sterile water on a glass slide.


Serum agglutination test

…I went through the protocol, but I got interrupted for other things before I could view the slides. By the time I got back to them, the liquid had evaporated, leaving me with a lot of salt crystals and some swimming bacteria in the control slide and a long, branching, somewhat snowflaked structure in the test slide. Try again tomorrow!

Oyster larval mortality, day 1
Counted the number of dead oyster larvae in the 12-well plates set up yesterday (8 Aug). Larvae were pronounced “alive” if a) they were clearly moving, or b) their cilia or velum were moving rapidly even if the larvae were stationary. Larvae in which there was no sign of motion, or in which there were sporadic “pulses” of motion, were pronounced “dead”. I was (perhaps) extremely conservative with my death counts: if any part of the cilia or velum looked like they were vibrating, I counted the larva as alive. Only when the velum was a) still or b) pulsing sporadically did I count the larva as dead.

Ashton counted the control wells and row B. I counted rows A and C.

Table of larval mortality after 24 hours:

 1
 2
 3
 4
 Control
A
 3
 6 +/- 1
 7 +/- 1
 12 +/- 1
 1
B
 2
 20
 21 +/- 1
 28
 2
C
 1
 6 +/- 1
 4 +/- 1
 7
 0

Notes:

Plating results from the serial dilutions yesterday (8 Aug):
Sadly, nothing grew in the T1N5 or TBS plates. The T1N5 plates were parafilmed and moved to a 30 C incubator in hopes that they will grow a little faster. The TBS plates were left on the benchtop for another night.

The TCBS plate showed colony growth on the Vibrio side, but not on the Aeromonas side, demonstrating that the media is indeed selective for Vibrio growth. Colonies are fairly light colored.

Labyrinthulid lab:
Sandy’s Laby have grown well and are growing over 1-2 eelgrass. Our 2* cultures also appear to be taking off. Sadly, there is still nothing in the 1* cultures.
Pictures:

Sandy #1
autocl_inoc_sandy1_2.JPG

Sandy #2
autocl_inoc_sandy2_2.JPG

1*-1
autocl_inoc_1star1_2.JPG

1*-2
autocl_inoc_1star2_2.JPG

2*-1
autocl_inoc_2star1_2.JPG

2*-2
autocl_inoc_2star2_2.JPG

We discussed protocols for the reinoculations. We have 3 different approaches we could use:
  1. Inoculum from autoclaved blades (currently growing)—clip to healthy blades
  2. Dosed response: Inoculate autoclaved blades from (quantified) liquid culture, then clip to healthy blades.
  3. Dosed response II: Inoculate healthy blades directly with (quantified) liquid culture.

We decided to go for protocols 1 and 3 tomorrow.
Protocol 1:

Protocol 2:

Also to do tomorrow:

Finally, we discussed running the PCR test on some of the Laby plates/ liquid cultures. Drew would like to run one of the molds from our field samples as a negative control for the primers. Be sure to discuss this with her!


8 Aug 2012

Labyrinthulid lab:
Reinfections:
Checked on the eelgrass infection plates from yesterday (7 Aug). Both of Sandy’s cultures are growing well. One 2* culture may have some growth, but it’s not certain that that particular culture is a labyrinthulid.
Pictures of each plate:

Control #1
autocl_inoc_contr1_1.JPG

Control #2
autocl_inoc_contr2_1.JPG

Sandy #1
autocl_inoc_sandy1_1.JPG

Sandy #2
autocl_inoc_sandy2_1.JPG

1*-1
autocl_inoc_1star-1_1.JPG

1*-2
autocl_inoc_1star-2_1.JPG

2*-1
autocl_inoc_2star-1_1.JPG

2*-2
autocl_inoc_2star-2_1.JPG

See 6 Aug for full names of the plates.

Vibrio lab:
Results of streaking from yesterday, 7 Aug:

vibrio_t1n2.JPG


vibrio_tcbs.JPG

Serial dilutions of Vibrio and Aeromonas cultures:
Plated the following dilutions:
Media
 Bacteria
 Dilution
 Replicates
T1N5
 RE66
 10^(-5)
 2
T1N5
 RE66
 10^(-6)
 2
T1N5
 RE66
 10^(-7)
 2
TSB
 Am
 10^(-4)
 1
TSB
 Am
 10^(-5)
 1
TSB
 Am
 10^(-6)
 1

Tomorrow we’ll use the colony counts on these plates to estimate the concentration of the initial cultures.

We also made a streak of the initial Am and RE66 cultures onto TCBS to test growth/ metabolism.

Plates inverted and incubated at room temperature overnight.

Inoculation of oyster larvae:
2 plates made: Experimental and control.
1. Oyster larvae added to all wells of the experimental plate and to 3 wells of the control plate.
2. Wells filled with ambient, sterile seawater to 3.9 mL. (This involved keeping track of the volume of larvae that had been added. Math errors here were prevalent, and the volumes had to be recalculated for most wells. The end water level for all wells, however, looked roughly equivalent.)
3. Bacterial treatments: 100 µL of the appropriate bacterium at the appropriate concentration added to the wells of the experimental plate. Setup is recorded in the table below. Control wells did not receive any bacteria.
Calculations for dilution factors IN PHYSICAL NOTEBOOK, TO BE UPLOADED.

Setup of experimental plate. Notes:

 1
 2
 3
 4
A
 10^4 RE66
= 10^(-3) dilution
 10^5 RE66
= 10^(-2) dilution
 10^6 RE66
= 10^(-1) dilution
 10^4 Am = 10^(-2) dil +
10^5 RE66 = 10^(-2) dil
B
 10^4 RE66
= 10^(-3) dilution
 10^5 RE66
= 10^(-2) dilution
 10^6 RE66
= 10^(-1) dilution
 10^4 Am = 10^(-2) dil +
10^5 RE66 = 10^(-1) dil
C
 10^4 RE66
= 10^(-3) dilution
 10^5 RE66
= 10^(-2) dilution
 10^6 RE66
= 10^(-1) dilution
 10^4 Am = 10^(-2) dil

Initial larval counts per well:

 1
 2
 3
 4
 Control
A
 34
 41
 ~56
 ~55
 40
B
 33
 44 +/- 2
 36
 41
 32
C
 38
 34
 34
 55 +/- 2
 41

Note for the broader experiment: Each group has a set of plates to measure, in total the effects of

Microplate cultures allowed to grow overnight at room temperature.


7 Aug 2012:

Labyrinthula lab:
Reinfections:
From each of the BeachHaven CK, DH replate, and Sandy’s Laby cultures (first three plates from 6 Aug), 2 small slabs of agar were cut from the leading edge with sterile instruments and transferred to 2 fresh plates. 4 pieces of autoclaved eelgrass were then arranged around the agar cut in a roughly rectangular fashion. 2 plates served as contamination controls, and received eelgrass but no agar cut. Pictures:
IMG_0505.JPG

IMG_0504.JPG

IMG_0503.JPG

Note that although some of the plates are dated 8/6, all plates were in fact made today.

Also, a note for the future: I had cut up and autoclaved both young and old parts of the blades. It turns out that the younger (thinner, lighter green) segments do not do as well in the autoclave. Most of those pieces disintegrated, so they were used as controls. Next time, using exclusively older parts of the blade will prevent this problem.

Plates were parafilmed and put in Sandy’s lab with the heater running on low to provide a little extra heat for growth.

Vibrio lab:

Streaking practice from stock plates of Vibrio tubiashii (Vt) and Aeromonas media (Am).
Ashton’s and my strain: X00123

1. T1N2 plates:
Half 1: Probiotic test, a curved streak of Vt overlaid by a line of Am.
Half 2: Streak test: Vt streaked out to single colonies.
See diagram below.
vt_platesetup.png
2.TCBS plates: Selective for Vt; depending on the metabolism of the colony, it will turn a different color.
One TCBS plate was divided into six portions, and each member of the class made a streak from our Vt stock plate.

Protocol for spec pH:


6 Aug 2012

Vibrio lab:
Experiment preparation:

Went over the protocol for taking alkalinity measurements:

Labyrinthula lab:
Reinfections:
As a Stage 1, we will add autoclaved eelgrass to Labyrinthula plates and see if the Laby can infect the eelgrass. I cut putatively healthy blades of eelgrass from the tank cut into 3-cm pieces and autoclaved them. Tomorrow, slabs of agar with Laby will be put into new plates with 4 blades of eelgrass. We will have two plates each of:

Image of the game plan I put on the board (courtesy Courtney):
IMG_0496.JPGimg_0496

5 Aug 2012

Labyrinthulid lab:
The replate of Drew's culture (4 Aug) and the replate of the "feathery" section of one of Catherine's cultures (3 Aug) are both showing signs of growth. The other plates (from 3 Aug) are not. The agar chunks in these plates seem to be very wet--perhaps a problem with moisture? There may be something growing on the water, but it's hard to tell, even under the microscope.

4 Aug 2012

Labyrinthulid lab:
Located the good culture that Drew had made from a False Bay plant; because it seemed to be growing quite a bit, made a replate from part of the leading edge.

The room-temperature cultures for inoculation that Lisa had made (3 Aug) were in the sun, at a tangible temperature difference from parts of the room that were not exposed to sun. I moved the cultures to a bench top instead.

3 Aug 2012

Labyrinthulid lab:
Gel electrophoresis of the PCR products from 2 Aug. Visualization on a 1.5% agarose gel by SybrSafe using UV illumination. Because we had problems with the last ladder, a different, more reliable ladder was used instead. We used 7 µL of each PCR product and 5 µL of ladder.

Order of wells:
Comb 1
 Comb 2
Well number
 Sample
 Well number
 Sample
1
 Jamie’s sample
 1
 Pos. control for wells 3-4
2
 Maya’s sample
 2
 Neg. control for wells 3-4
3
 Sonia’s sample (2 µL DNA)
 3
 Jenna’s sample
4
 Pos. control for wells 1-3, 6
 4
 Ana’s sample
5
 Neg. control for wells 1-3, 6

6
 Sonia’s sample (5 µL DNA)

7
 Neg. control for wells 7-15

8
 PCH-12

9
 DC1-D12

10
 PD1

11
 PD2

12
 LD1

13
 LD2

14
 LH

15
 PH

16
 Pos. control for wells 7-15

gel_3Aug.png
Results:

A BLAST search with the primer sequences showed some hits for other (unicellular) organisms beside Laby, but nothing in particular that popped out. In addition, all hits for non-Laby species were only with the forward primer. The combination of forward AND reverse primer sequences ONLY appears in Laby species.

Ideally, if we wanted to find out why these bands are different, we would want to either extract the bands or re-do the PCR and send the product in for sequencing.

Inoculation experiment
Discussed protocols for the inoculation experiment further. Separate cultures of Laby are currently being grown at room temperature and 37 C.

Literature protocol: From Muehlstein LK, Porter D, Short FT. 1988. Labyrinthula sp., a marine slime mold producing the symptoms of wasting disease in eelgrass, Zostera marina. Marine Biology 99:465-472.
Day 1
Day 2
Day 3
Day 4 +


Lisa’s protocol:
Day 1-2
Day 3
Day 4-6

We discussed doing this with old blades vs. young blades (is there a difference in susceptibility?). Sammy suggested mixing the Laby in with the agar and allowing it to infect the blades in that way. There was some concern that an experiment like this would not have real ecological relevance, especially as regards the variables we measured in the field, but we seem to have decided to pose it as:
1) a test of Koch’s postulates (can we get reinfection),
2) a new method for inoculation, and
3) a way to test some basic hypotheses.
We would like to use both verified Laby samples from Sandy and a Laby sample that we isolated from the field.

Laby culture transfers
Lisa made new cultures in 50-mL Falcon tubes as well as in Petri dishes (to provide greater surface area, if that is better for Laby growth) and put them at 25 C and 37 C. I transferred, to separate agar plates, a block of agar from some restreaked plates:
  1. “Parent” plate from which I took the re-streak sample 31 Jul
  2. “Feathery” Laby from the re-streaked plate (made 31 Jul)
  3. “Smooth” Laby from the re-streaked plate (made 31 Jul)


2 Aug 2012

Morning: Resampling of eelgrass at False Bay.

As compared to Picnic Cove and Beachcomber Bay, False Bay seems to have generally less dense / more patchy eelgrass in the areas we surveyed. Shallow transect seemed to show smaller individuals and less disease than deep transects.

Afternoon: PCR on the DNA extracted 1 Aug.
Reactions:
  1. Jamie’s sample
  2. Maya’s sample
  3. My sample (2 µL DNA)
  4. Positive control (provided by Lisa)
  5. Negative control (water)
  6. My sample (5 µL DNA)

Stock mix:
Reagent
 µL/ reaction
 X 6 =
 µL/ stock
Go-Taq 2x master mix
 12.5
 75
Primer 1
 0.8
 4.8
Primer 2
 0.8
 4.8
BSA
 1.5
 9
Nuclease-free H2O
 7.4
 44.4
Total
 23
 138

Mixture was aliquoted in 23-µL increments into PCR tubes and 2 µL of the appropriate DNA (/water) were added to each tube.

I made up a separate PCR tube for my reaction with 5 µL of template:
Reagent
 µL/ reaction
Go-Taq 2x master mix
 12.5
Primer 1
 0.8
Primer 2
 0.8
BSA
 1.5
Nuclease-free H2O
 4.4
DNA
 5
Total
 25

Primers:
Forward: Laby WC F
3’ GGAAGGCAGCAGGCGCGTAA 5’
Reverse: Laby WC R
3’ GAATGTTTCCAATCCTCAGTCGGTATAG 5’

Cycling conditions:
Step
 Temperature ( C)
 Time
1
 95
 10 min
40 cycles of:
2
 95
 30 min
3
 50
 30 sec
4
 72
 60 sec

5
 72
 10 min

PCR products stored overnight at 4 C.

1 Aug 2012

Labyrinthulid lab:
Morning: Sampling of eelgrass in Picnic Cove. See protocol for 30 Jul.

Picnic Cove had extremely muddy water, and most eelgrass measured (in both the shallow and the deep transects) had lesions on them.

Afternoon:
Transmission experiments
Discussed with Sandy some alternatives to the mesocosms for running transfection experiments. We will most likely be using small, upright cylinders in the quarantine room to prevent infections from entering the bay with outflow water. Ashton suggested several ways to attach the cylinders easily to a base. We can also put small heaters into some of the cylinders to test the effect of temperature, and tidbits to record temperatures.
The transmissions will most likely be done as per [name of paper]: autoclave a section of eelgrass, infect in with Laby on a plate, and then clip in to a healthy, living eelgrass and count the number of lesions that develop after 4-5 days.

PCR on Laby
Used eelgrass samples previously collected as substrates for DNA extraction and amplification. See 26 Jul for protocol.

I extracted DNA for a positive control from Laby grown on plates:
2. 7-18-2012, Replate 7-25-2012

Data entry
Transcribed the data sheets that Amy and I recorded this morning into the giant spreadsheet:
https://docs.google.com/folder/d/0B5AIgISTKSN1V19DMDBObG8yN3c/edit?pli=1&docId=0ApAIgISTKSN1dHVJeUI1MWNSWjc5MlV4clZsRV9oUHc

31 July 2012

Labyrinthulid lab:
Morning: Sampling of disease prevalence in Beachcomber/ Beach Haven on Orcus. Modifications from the sampling protocol from 30 Jul:

Afternoon:
Game plan for what we can do with the rest of the lab:
  1. Field sampling: False Bay, Beachcomber, Picnic Cove à already well underway.
  2. Inoculation experiments: in a mesocosm or smaller environment. Possibility of crossing the inoculations with a variety of temperatures and/or herbivore presence.
  3. PCR on laby. Try isolating from herbivores as well.

Culturing Laby
Plated lesion sections from 6 plants collected today. Plates labeled with “7/31, BC” and the plant identification number. Key: SH1 = shallow transect 1; SH2 = shallow transect 2; D2 = deep transect 2; -# = plant ID number. Note that SH1 only has plants #1 and 4.

Photos:
IMG_6601.JPG

IMG_6600.JPG

IMG_6599.JPG

IMG_6597.JPG

IMG_6595.JPG

IMG_6593.JPG

Healthy sections of the same plants were put into desiccating agent for Sandy’s collection for microsatellite analysis.

I also replated (streaked) an older plating on which a fungus was growing (Plate: “Beach Haven CK, 1. 7-18-2012, Replate 7-25-2012”) onto a new plate (labeled “7/31 SS, Replate of Beach Haven CK”). Because there were two non-fungal growths, I took a streak of each one on separate halves of the plate and labeled them on the plate bottom (as “smooth” or “feathery”).

PCR on herbivores
Helped Sammy set up cages with an herbivore on infected or uninfected eelgrass blades from Sandy’s tank.

30 July 2012

Labyrinthulid lab:
Morning: Sampling of disease prevalence of Laby at False Bay. Link to complete sampling methods TO BE INSERTED. In brief:

Afternoon: Histology of Laby
Histology slides from diseased and non-diseased eelgrass from Picnic Cove had already been made up by Catherine. We were to provide independent analyses of the diseased state of the eelgrass by quantifying the number of Laby cells in each one.
Method:

Counts for the slides I looked at can be found in the Google Docs spread.

Culturing Laby
Purpose: Culture Laby for PCR, re-inoculation, etc.
Method:

Photo of the culture:
Smoot Culture.JPG

27 July 2012

Armina Lab
Visualization of the PCR products made yesterday (26 Jul) on a 1.5% agarose gel, visualized with SybrSafe and UV illumination.
gel_27Jul.png

Order of wells (see 26 Jul for details of samples and primer sets):
Number
 Primer set
 Sample
1
 Universal bacterial
 My sample
2
 Ashton’s sample (well punctured)
3
 Positive control
4
 Negative control
5
 Ashton’s sample (re-load)
6
Empty
7
 Ehrlichia
 My sample
8
 Ashton’s sample (well punctured)
9
 Ashton’s sample (re-load)
10
 Positive control
11
 Negative control
12
 WS
 My sample
13
 Ashton’s sample
14
 Positive control
15
 Negative control

Results:


26 July 2012

Armina Lab: DNA extraction and PCR

DNA extraction: QIAGen Stool kit
1. From the tissue samples stored in ethanol yesterday (25 Jul; acc 12-1-5), one piece of tissue was extracted using sterile dissecting tools, minced into small pieces, and placed into a 2-mL microcentrifuge tube. (The sample should be weighed before being minced, but the lab scale was not sensitive enough to record such a small mass.)
Break up tissue cells.
2. 700 µL of Buffer ASL added to the microcentrifuge tube and the contents vortexed for 1 min. Then a second set of 700 µL Buffer ASL was added and the contents vortexed until the mixture was roughly homogenous. (Note: Tube vortexed for several minutes without complete homogenization, but the quantity of tissue in solution did increase over that time.
3. Mixture heated for 5 min at 70-75 C
4. Sample vortexed briefly, then centrifuged at full speed for 1 min.
5. 1.2 mL of the supernatant was pipetted into a new 2-mL microcentrifuge tube.
Remove PCR inhibitors.
6. 1 InhibitEX tablet added to the sample and vortexed until the tablet had dissolved. Tube incubated for 1 min at room temperature.
7. Sample centrifuged at full speed for 3 min. Supernatant transferred to a new microcentrifuge tube and re-centrifuged for 3 min at full speed.
Digest cellular proteins.
8. A new microcentrifuge tube was set up with 15 µL of Proteinase K. 200 µL of the resultant supernatant from Step 7 was transferred into this tube, and 200 µL of Buffer AL was added.
9. Sample + Proteinase K + Buffer AL vortexed for 15 sec.
10. Tube incubated for 10 min at 70 C.
Wash steps.
11. Samples centrifuged briefly.
12. 200 µL of 95% molecular-grade ethanol added to the tube, vortexed, and spun down.
13. Mixture applied to a QIAamp spin column and centrifuged for 1 min at 8000 rpm.
14. Column transferred to a new collection tube and 500 µL of Buffer AW1 were applied. Column spun for 1 min at 8000 rpm.
15. Column transferred to a new collection tube and 500 µL of Buffer AW2 were applied. Column spun for 3 min at full speed (~16.3 rpm).
Elution.
16. Column transferred to a clean 1.5-mL microcentrifuge tube and 100 µL of Buffer AE were applied. Column incubated at room temperature for 1 min, then spun for 1 min at 8000 rpm.
17. Column discarded; elution stored at 4 C.

PCR of DNA samples to determine presence/ type of DNA from a disease-causing agent.
We are running three PCR reactions on our armina DNA samples:
Name
 Primer set
 Protocol
 Detection
Universal
 EUB A / EUB B
 Generic
 Any bacteria
Ehrlichia
 EHR16s F / EHR16s R
 Generic
 Rickettsia
WS
 RA 3-6 / RA 5-1
 WS-RLO
 Particular Rickettsia species from abalone

Reactions: (for each reaction set)
  1. My sample (12-1-5 DNA extracted 25 Jul)
  2. Ashton’s sample (a stock sample from a previous year)
  3. Positive control (provided by Lisa)
  4. Negative control (water)

Generic protocol:
Stock solution:
Reagent
 µL/ reaction


X 5 =
 µL/ stock
Go-Taq 2x master mix
 12.5
 62.5
Primer 1
 0.8
 4.0
Primer 2
 0.8
 4.0
BSA
 1.5
 7.5
Nuclease-free H2O
 7.4
 37.0
Total
 23
 115

Two sets of Generic stock were made up, one with the EUB A/ EUB B primer pair, the other with the EHR16s F/ EHR16s R primer pair.

23 µL from each stock were pipetted into 4 PCR strip tubes, and 2 µL of the appropriate DNA sample (marked above as reactions) were added to each tube.

Cycling conditions:
Step
 Temperature ( C)
 Time
1
 95
 10 min
45 cycles of:
2
 95
 15 sec
3
 60
 1 min


WS-RLO protocol:
Stock solution:
Reagent
 µL/ reaction




X 5 =
 µL/ stock
Go-Taq buffer
 4.0
 20.0
Primer RA 3-6
 0.1
 0.5
Primer RA 1-5
 0.1
 0.5
BSA
 0.8
 4.0
dNTPs
 0.4
 2.0
MgCl2
 1.2
 6.0
Taq polymerase
 0.32
 1.6
Nuclease-free H2O
 11.08
 55.4
Total
 18
 90

18 µL of stock were added to each of 4 PCR strip tubes, along with 2 µL of the appropriate DNA sample (reactions above).

Cycling conditions:
Step
 Temperature ( C)
 Time
1
 95
 3 min
40 cycles of:
2
 95
 1 min
3
 62
 30 sec
4
 72
 30 sec
5
 72
 10 min

Gels of all sets will be visualized tomorrow.


25 July 2012

Armina lab: Dissection of Armina californicum:

Accession number: 12-1-5
Total mass: 4.3 g
Total length: 36.4 mm
General observations:
armina ventral.png armina dorsal.png

(Note on the previous dissection: Animals had been used for neuroethology, so they had already undergone one operation to take measurements and samples from the brain.)

Digestive gland was extracted from a small transversal cut on the dorsal side behind the head and removed from the connective tissue. The digestive gland was cut transversally in three places to create four pieces, as pictured below:
armina cut locs.png

Each of the pieces was cut in half:

The histological cassette was immersed in Invertebrate Davidson’s fixative for 24 hours. The PCR tube was stored at 4 C.


24 July 2012

Invertebrate dissections:
Oyster:
Shell cracked open at hinge and pried apart; adductor muscle cut from the right-hand shell to expose the oyster in the left-hand shell. Two cross-sections were taken width-wise across the oyster, just above the labial palps. The heart was also exposed.

Diagrams from the dissection:
IMG_6317_oyster.jpg



Sea cucumber:
Organism was cut lengthwise along the tentacles, starting with the aboral end. Most of the internal organs spilled out before the cut was very large.

Diagrams:
IMG_6318_cucumber.jpg
Right-hand text reads, from top to bottom:
Direction of cut
Aboral cavity
Respiration trees
Attachment of guts, etc.

I also observed the dissection of the second sea cucumber, which had a larger mass of respiratory trees and a much lower mass of gonads than the one Ashton and I dissected. I examined the results of dissections of sea stars and a sea urchin. The sea urchin, in particular, was surprisingly empty, with gonads, Aristotle's lantern, and digestive ceca all located along the internal sides of the test.

Histological comparisons:
Using pre-made slides, we compared normal and diseased tissues for oyster, abalone, and Armina under a compound microscope.
Some diagrams:
IMG_6319_hist2.jpg

IMG_6320_hist1.jpg