Grace’s Notebook: Tuesday, November 21, 2017

(1) I figured out a better way to image the larvae. I dump the ethanol in the tube into a 80ml beaker. Then, I dump it back into the tube. This dislodges all of the stuff at the bottom. Then I dump the ethanol back in the beaker, and it ends up containing the larvae, leaving behind the larger bits of junk. I wait for the larvae to settle, then pipette off the ethanol until I get to the larvae.

(2) Started the Analysis of the 2015 Oyster seed data. Emma performed the Pecan step, so I started in at Step 4, using the .blib file that she created. I am stuck at Step 4e . We need an isolation scheme file from Step 3b:

We asked Emma to help us out. And here is what she provided: GitHub Issue

(3) Titrator: I worked through the SOP to calibrate the pH probe. It seemed to work well. Then Steven and I tried out the CSUN method from Hollie on pH buffers – just to try out the method. It seems a little odd to me because it did each sample one at a time. I thought that it would run like that pH calibration method where it went from sample to sample automatically.

Grace’s Notebook: Monday, November 20, 2017

(1) I made pie charts for the sex ratios of the Pre- and Post-OA C. gigas histology classification. I am still unsure about the sex for some of them, but here is what I have so far:

I’m searching for histology examples of different tissue types in C. gigas to try to determine if what I think is gonad, truly is.

(2) The Titrator Temperature Probe arrived today, so I connected that to the Titrator. I assigned it as a sensor using the touchscreen. I’m not sure if it’s reading, so I’ll wait to check that when we re-calibrate the pH probe.

(3) I have been taking images of oyster larvae. It’s not going so well. It’s quite difficult to get 100 larvae imaged when there is so much other stuff and ethanol in the tube with them. I’ve tried lots of different ways to get the excess ethanol off the top, as well as try to avoid the junk at the bottom. But the larvae are floating in the ethanol and I always end up taking too much ethanol up… and it just takes a really long time. I’m going to have to think of other ways to do this so that I don’t waste time.

 

Grace’s Notebook: Friday, November 17, 2017

(1) Worked on updating the SOP a little bit more today, but I don’t think I really contributed much. I focused mainly on the first two sections. I read through the last two sections, and I think I’ll understand them better if I can work through it once we re-calibrate the probe. I think working through it will allow me to pinpoint which pieces of information I should add in order to make the process smooth in the future.

(2) I re-took some images from Pre-OA C. gigas histology slides. I was annoyed at myself for not mapping out the slides when I imaged them originally, so I did that today for future reference:

I re-imaged: 01, 04, 05, 07, 08, 12, 15, 18, and 20. I was able to add some new information to the classification spreadsheet I’m working on based on these new images.

I am having an exceptionally difficult time figuring out what Gigas-01 is. Here are some images I took of the only non-connective tissue in the sample:

Magnification: From right: 40x, 40x, 10x, and 4x.

(3) I received some more reading material for the Crab project in Juneau from Greg Jensen.

Grace’s Notebook: Thursday, November 16, 2017

(1) Worked more on updating the SOP. Still not nearly clear enough. Have to add more specifics.

(2) Worked more on the C.gigas histology classification.

Tomorrow I’ll retake some images because I think I took pictures of connective tissue rather than gonad. It’s harder to tell what is gonad when they are in the early stages of development.

I am comparing the gonad cells to those in these images from Fabioux et al. 2005:

Here is what I’ve done so far:

(3) Finally, I’ve been emailing with Pam Jensen about the crab work in Juneau coming up. She has provided more information on how that week will be laid out, as well as some references and papers to read. I have begun reading some today.

Grace’s Notebook: Wednesday, November 15, 2017

(1) I worked more on classifying Yaamini’s C. gigas gonad histology slide images. Here is the spreadsheet of the attempt at classifying I did today.

(2) Working on updating the titrator SOP from Hollie so that it reflects what our specific process will be.

Future work:

Dan dropped off the remaining OA Larval samples today, so when I have time, I’ll start imaging those. There’s quite a bit more tubes than I was expecting.

The priority is getting the Titrator going, so once the Temperature probe arrives, we’ll re-calibrate the pH probe and then start moving through the samples!

Grace’s Notebook: Tuesday, November 14, 2017

Scallop Gonad Histology: I organized all the cassettes because they were many samples that we didn’t need mixed in tubs with samples we do need. I sent out the gonad tissue samples that Katherine specified along with two extras (241 and 361) because there was not a good place to put them.

C. gigas gonad classification: I made a spreadsheet to organize the classification process. So far I’ve only determined the sex of about half of the samples. The classification is going to be more challenging because I haven’t been able to find a good paper detailing the classification of female C. gigas. And most of them so far seem to be females – at least the ones that are easier to sex.

Imaging Oly larvae: I spent some time on this today. However, I found out that all the tubes that I have don’t actually need images taken, because they already have data. I believe Dan will bring over some sample tubes for ones that need images taken sometime this week and then I’ll get that all finished up.

Titrator: The Mettler-Toledo buffers arrived today! So we can recalibrate the pH probe. I also purchased a Temperature probe, since we don’t have one.

Grace’s Notebook: Monday, November 13, 2017

Titrator:

A technician (Brian) from Mettler-Toledo came this morning from 9:30-11:45 to show us how to use the titrator. It was extremely helpful as he was very nice and informative. We used some buffers that we had in 209 to calibrate the pH probe, but they were expired. We’ll run the calibration again once the buffers from Mettler-Toledo arrive. He also showed us how we would run the method that Hollie sent us. It’s pretty straightforward. He is in this area often and said he’d be able to stop by and help if we ever have any questions. Once we calibrate, we’ll be able to start running samples!

Tanner crabs

I met with Steven, Sam, and Pam Jensen today to hear about the Tanner Crab and Hematodinium project. I will be joining Pam in Juneau the week after Thanksgiving to assist with the final sampling as well as breaking down the lab.

C. gigas histology images

I began looking into analyzing the Pre-OA and Post-OA C. gigas histology images that I took a while back.

This GitHub issue provides some links to papers that may be useful. Still in search of the best examples to compare our images to. The best description I can find does not include images:

Image OA oyster larvae (Dan Gillon)

I will continue taking images of the oyster larvae and organize them. Dan said that Joth will have a technician measure the larvae in the images.

Scallop histology

I missed the 2pm deadline to send out the samples today due to the meeting with Pam, but will send them out tomorrow. I am only sending out the samples that Katherine will be performing gene expression on:

 

Grace’s Notebook: Friday, November 10, 2017

Today I grabbed Scallop gonad histology samples from FSH and will send them out on Monday to be made into slides. Katherine provided me with the list of all the scallops she’s planning on doing gene expression on in this GitHub Issue. Which ends up being about half of the total number of cassette samples taken.

I also addressed a couple issues (#39 and #32) I had with Yaamini’s SRM protocol and everything seems to be looking good!

Some cleaning/tidying of labs/tidying cords connecting the titrator. Essentially just put all the cords on one power strip and then used zip ties to make them less messy looking. A technician is hopefully coming out Monday.

Grace’s Notebook: Wednesday, November 8, 2017

Today I made a 0.1 mol/L NaOH solution for testing out the titrator in the (hopefully) very near future. I weighed 4g of NaOH solid. Placed it in a 500ml beaker which a magnetic stirrer. I added 500ml of NanoPure water.

Stirred until dissolved. I then poured into a 1L graduated cylinder. Added 500ml more to reach 1L. Poured this final solution into the titrant bottle that is connected to the Titrator and Rondolino. I labeled the bottle with the date, contents, and my initials.

 

Grace’s Notebook: Wednesday, November 1, 2017

Still working on the same part of Yaamini’s SRM protocol. I am almost halfway done. It’s a bit mind-numbing, so I have to take breaks.

During those breaks, I looked over the titrator protocol some more, and tried to control the Rondolino using the touchscreen. I read through the manuals again to try to see if I was missing something. I probably am, because there are a lot of parts to this titrator. I created a GitHub issue outlining my troubles and troubleshooting. I think what I need is more brains working on it with me in person!

Waiting for more information on the next steps regarding this GitHub Issue pertaining to the 2015 oly larval proteomics project.