By “calcium confirmation”, I mean that we have determined intracellular calcium is NOT involved in our algicidal compound’s mechanism of action. Sometimes that’s how it goes in science, especially in a field where so little is known; you have to weed through many negative results to find the positive hits.
This is the case with my phytoplankton bioassays. Each new crude extract has the potential to contain an algicidal compound, but many crudes are not active against the phytos or even enhance phyto growth (which is cool too!).
The element of chance in my work is one of my favorite aspects. When the crudes are spun down, they look pretty much the same. But when cell counts for a particular crude come back 10 times lower than they started in an experiment, I think to myself, “Wow, whoa, what makes this one so special?” Another exciting aspect is the fact that we have the technology to find out exactly what compound makes them “special,” and then we can go a step further and determine exactly why they function in this “special” way in the ocean.
My campaign to elucidate this mechanism of action continues next week, as I test the cells for reactive oxygen species.
Back in Georgia, Week 3 was off to a great start. The phytoplankton culture looked strong, there were fresh crudes to test in my phytoplankton inhibition assay (PIA) like the one above, and we had a solid game of pickup soccer among the lab members.
But by Wednesday, the abundance of phytoplankton in my PIA was dropping across the board. This is not the type of cell death I am looking for, because even the phytos who hadn’t been treated with possibly algicidal crude extracts were disappearing.
Then I remembered what a helpful lab technician had told me about my phyto culture.
—-“They typically don’t like this kind of bottle, and the cap was on a little too tight,” she said as she showed me her beakers of phytoplankton strains, which were covered in loose-fitting aluminum foil.—-
Even though they appeared to be doing OK, I was suffocating my phytoplankton!! Just like land plants, my phytos can’t grow without CO2, and these stressed out cells were not reproducing as they normally do, which is about doubling daily.
The only way to rescue them was to…well, I couldn’t really rescue them because the experiment was flawed…so I poured the remaining ones into a container of 10% bleach labeled “Unhappy Phytos” and restarted the experiment with a new strain borrowed from the technician.
The experiment is now back on track, and the only consequence is having to come into the lab a few times on the weekend. Luckily my barracks is only 200 yards from the lab.
It’s 2:45 am. I’m 80 kilometers off the coast of Bermuda. I’ve been awake for close to 20 hours. I’ve eaten at least 10 cookies and muffins from the galley. And I’ve watched 180 minutes of Season 6 of Game of Thrones, a show I’ve never seen before so I have no idea what’s going on, to pass the nighttime before 3 am.
“It’s up!” I hear from down the main passageway. Now’s my time to shine.
I trudge out on the deck, zip up a lifevest, twist on a hard hat, and grab some rope. My first job is to retrieve the ship’s yellow-framed CTD, a pretty darn cool instrument that measures conductivity, temperature and depth of the ocean water.
“Hold the rope farther from the cleat,” the marine technician on the other rope warns me. “They look innocent, but I knew a guy who lost a hand doin’ what you’re doin’.” Enough said; I took three healthy steps back from the cleat.
We lowered the CTD down snug onto it’s landing pad and I started filtering water from its ring of grey Niskin bottles. Dr. Harvey’s experiments need someone to take saltwater samples every six hours, so they can be diluted and measured for grazing of phytoplankton. Since I’m more of a night person than a morning person at sea, I have the 3 am shift, and Dr. Harvey has the 9 am.
It was a whirlwind research cruise. Not a lot of sleep, but a lot of good samples and a lot of fun! The rest I’ll leave to the pictures:
There was safety (first as always)…
There were sunsets…
There were colleages/friends/jokesters…
There was a sloth…
And at the end of it all, the were two delicious chickens (not pictured) inside a pig (pictured)…
Thank you to the crew of the R/V Atlantic Explorer!
My first week stationed at the Skidaway Institute of Oceaniography (SkIO), affiliated with the University of Georgia, was an exciting one!
Nothing beats waking up and running some fresh new experiments on phytoplankton. Within just three days of testing, I determined which fractions of my crude bacterial extracts were algicidal, because the phytoplankton populations plummeted when treated with these compounds.
This new intel was sent straight back to Haverford, where more fractionation will help us crack the phytoplankton-killing code.
In the meantime, my SkIO advisor, Dr. Liz Harvey, and I prepared for a four-day cruise in the North Atlantic off the coast of Bermuda! Dr. Harvey has been wanting to study the night-day differences, or “diel” variation, in rates of phytoplankton grazing, that is, other microorganisms eating phytoplankton. And I have the privilege of helping her on the voyage, which will take us to the Bermuda Atlantic Time Series (BATS)!
BUT FIRST, SAFETY…
I volunteered to model proper immersion-suit entry. Immersion-suits are often called “Gumby Suits.” Can you see why?
We arrived at the Bermuda Institute of Ocean Sciences on Thursday, met with our colleagues from Oregon, Virginia, California, and Bermuda, and gathered materials like deionized water and 10-liter carboys for taking samples.
When the ship, the R/V Atlantic Explorer, is fully loaded and the tide is right, we will set sail on Saturday!
Experimenting with ionomycin is about as close as one can get to playing with explosives in microbiology. And the Forth of July may be next week, but Mia and I saw a few fireworks this week when we triggered calcium release in our phytoplankton cells using this chemical trigger, or “agonist”. The algicidal compound of our research focus also caused some calcium release, but it didn’t deliver the same kind of boom we saw in our positive control. Here is how it all happened:
1. We stained our cells with a dye that becomes fluorescent in the presence of calcium ions.
2. We anchored our cells to a microscope cover slip so they wouldn’t move.
2. We flushed dilutions of ionomycin and our algicidal compound over the cells.
3. We used a microscope camera and its software to measure their change in fluorescence intensity.
4. We took our data into an Excel spreadsheet and saw that when we added these compounds, the cellular calcium went BOOM!
It wasn’t always easy to do these four steps smoothly; actually, we had plenty of not-so-shining moments and went through our share of duds. On some days, the cells seemed to be playing hide and seek with us, and when we did find them in a good cluster, they were often fluorescing too dimly to measure. We also ended up having a love-hate relationship with the microscope-camera-software conglomerate. Yes we needed it for data, but on more than one occasion (15 is more that one, right?), the cells and “junk” and everything would disappear from view completely. It was the dreaded “Diver Error”, and the only way we found to consistently fix this problem was to shut down the whole conglomerate, precisely in reverse order mind you, and then restart it again in reverse-reverse order.
As a result, I sometimes wake up in the night because I think there’s a “Driver Error” in my room but I turn on the light and it’s just…! No, that doesn’t happen but it was really annoying. And in all honesty, I’m not sure I would have held it together through this full week of microscopy without a lab partner toughing it out with me.
But I’ll have to leave Mia and the rest of the Whalen Lab behind as I ride the rails down the East Coast to Savannah, Georgia, where I’ll begin “Part 2” of my campaign at the Skidaway Institute of Oceanography.
This new post is pretty out there^, but I’ve been there once before and know where not to go to avoid the gators.
Goodbye Haverford until the Fall; new correspondence will come from the Harvey Lab!
The stage of an epifluorescent inverted microscope can be a hopeless place, but we did it! Mia and I hit a major milestone this week by successfully staining phytoplankton cells with a fluorescent dye and visualizing them under UV light. This accomplishment let us further develop our microscopy protocol that will be used in the following week.
We did suffer our first loss this week. After transferring it from our small-scale “starter” culture, our beloved bacterial strain, B297, refused to grow in the larger, more nutritious culture we had so carefully made for it. Any surviving cells were bleached and a new strain was grown. (This one looks like it is doing better, although the startling smell it gives off fills the hallway).
Preparations for Georgia also continued, as we diluted the crude extracts to realistic concentrations and prepared them for battles against phytoplankton. Soon, the samples will be loaded up in dry ice and I will be ridin’ the rails down to Savannah.
My campaign with Mia took us back into the chemical hood, where we separated the chemicals within the bacterial extracts based on their polarity using two column techniques: silica flash chromatography and C18 solid phase extraction chromatography. Solvents were flying left and right—everything from water to acetonitrile and isooctane to ethyl acetate—but we tamed the vacuum pump well enough to capture 13 total fractions from the crudes of two bacterial species. Just when we thought we were in the clear and could let the SpeedVac spin down our fractions, we neglected to check if the vacuum pressure had started on the instrument, and we would have shot ourselves in the foot had it not been for the careful eyes of Kristen. I learned an important lesson that day: always follow the ENTIRE instrument protocol (even the small steps, and especially on the expensive instruments).
Chromatography wasn’t the only action we saw this week. We helped professor Rob Fairman with dredging the Duck Pond, we transferred our “naked” and “lith-forming” strains of phytoplankton to new culture tubes, and we started an ambitious initiative to image the effects of known algicidal chemicals on phytoplankton cells using fluorescence microscopy.
Next week I may be writing from the darkness of the microscope room.
Wish me luck!
The week was highlighted by the arrival of another lab member, Mia O’Reilly, to my post. Mia and I were in the same squadron for Fall Superlab, so we are battle-tested together and we hit the ground running.
In preparation for the big bacteria-phytoplankton showdown in Georgia, where I will be transferred in July, we started off the week culturing marine bacteria and extracting their metabolites. A few of the culture shakers were not cooperating, but we put our heads together, and with the guidance of our principal investigator, Dr. Kristen Whalen, we were able to fix them into order.
Following an extended day on the rotary evaporator, we discovered that an unidentified bacterial strain (essentially a spy) had slipped into our culturing ranks. This called for immediate DNA isolation and sequencing! But Mia’s deft pipetting hands took care of the job, and we made plans to culture the appropriate bacteria later in the month.
The courage shown in lab this week reminds me of a song: www.youtube.com/watch?v=j6gZefW4yEA
Here’s to a smooth-sailing second week.