A Developing Plating Disaster

Over the past few days I’ve been watching my agar plates enact Lord of the Flies. The organisms descended into chaos.

Don't mistake this for a serene picture. It's chaos.

It appears that an aggressive Pythium species was in my experiment soil. I’m not concerned about its role in the actual experiment, but it has caused problems in the plating exercises and isolations I’ve been trying to do post-experiments. This is what many of the plates look like upon closer examination:

Mycelium on the plate surface.

A rapid growing white hyphae dominated many of the plates (both the negative and positive controls included). Pythium will usually grow faster than Phytophthora during plating, but I’ve never had it dominate so thoroughly.

The guilty party, which looks like a Pythium to me.

Right now I am watching my root plates, hoping the same organism does not appear. I’ve seen a little of it, but not the same level the soil plates had.

All quiet on the root plates.

If one facet of my 2010 summer fir experiments had to go wrong the plating is the best area for that to happen. I can continue to retest the soil and roots, it’s not a one and done situation like many other aspects of the experiments. The downside is that it is time consuming and I am getting short on plating supplies.

The sad thing about this entire process is how minor a part it is in relation to the experiment as a whole. Sometimes small things can create big problems.

A Burrito, Fir Roots, and Phytophthora

Today I spent a long day in the lab (aside from a short diversion thanks to my Hort 138 TA responsibilities). I plated roots and leaves. Tomorrow I have another day of set up work, then the transfers will probably start on Saturday. That should put me on pace to start morphological identification of samples by the middle of next week.

The Rhododendron catawbiense leaf sections I plated today came from Petri dishes I set up 48 hours prior to the transfer. They looked like this:

Wait, it's a trap!

Pieces of rhododendron leaves are floating on the water surface, enticing the Phytophthora zoospores to hang out.

Plating fir roots is a time consuming process. It begins with washing the roots to remove any extra soil. The roots are then bathed in a 70% solution of 200 proof ethanol, rinsed, dried, and shallowly embedded in PARP plates. A major reason the process is slow is that contamination must be guarded against. Between steps the tools I’m using need to be sterilized and cooled. (It’s important not to place recently flame sterilized tools in 200 proof ethanol).

Around noon I decided to make a quick trip to Chipotle to purchase and weigh a burrito. I’ve been developing a theory that the weight of a Chipotle burrito can be predicted by the number of patrons in the store (maybe I should be more specific, the number of patrons ordering or in line to order . . . this does not include people eating in restaurant).

When I arrived and saw only three people at the counter ordering I knew things looked promising. And this happened:

610.8 grams of wonderfulness.

That’s the second largest Chipotle burrito I’ve had, and it stops a troubling trend I had been observing in declining weight. So here are my weighed burrito purchases:

Burrito #1: 669.4 grams — Patrons in store: 1
Burrito #2: 588.2 grams — Patrons in store: 6
Burrito #3: 554.6 grams — Patrons in store: 16
Burrito #4: 610.8 grams — Patrons in store: 4

Heed my advice, order your burrito when the store is slow.

I’m not sure about this, but I suspect you might have just read the first blog post in the history of the world to discuss both baiting of Phytophthora and weighing of Chipotle burritos.

Firs, Gravel, Phytophthora, V8, and Rice

In one long day (yesterday) I planted all the firs I’ll be using in my fall experiments in the gravel bed. The gravel bed is temporary housing, I’ll dig them up in early September to plant them at my field experiment sites. Right now I can forget about them for a few months. It’s nice to have that wrapped up. One of the firs was telling me how excited he is to witness PSU football tailgating. I didn’t break it to him that he would only see a game or two before being transplanted.

A Missouri gravel bed of firs near Beaver Stadium.

On Tuesday I picked up fresh cultures of Phytophthora cactorum and P. drechsleri. I made several plate transfers onto V8 agar. Today I transferred parts of those cultures to rice. In two weeks I’ll have about four pounds of rice covered with phytophthora. That rice will be used to inoculate firs in two of my experiments. I’m planning to release the phytophthora (I just typed typed “release the phytophthora” in a “release the Kraken!” fashion) on the firs.

A plant destroyer lurks on V8.

I'm culturing deadly rice (deadly to plants, that is).

It’s been great to see my to do list shrinking. The cost is early mornings, long days at work, and crashing when I get home. I estimate I have another two weeks or so before my schedule regains some sense of normalcy (at least as normal as a schedule can be for a grad student).

Hi! Bye!

Things are a bit hectic right now. This week I have been striving to wrap up my initial round of fir processing. The first level of root plating is done, and right now I am 76% finished with the root inspection, washing, and bagging. My goal is to finish that up by the end of the day Saturday.

Today (actually yesterday, I am writing this around 1:00 AM) I washed roots from my Phytophthora fir experiment. One advantage to spending so many hours in the greenhouse during the past two weeks is that I now barely notice the high temperatures. Today it peaked at 106°F.

During the summer greenhouses get warm.

As I processed the firs today I found myself wishing I had devised a way to measure the foliage color. I noticed the plants that had been inoculated with Phytophthora tended to have lighter green needles.

Canaan firs (left to right): 1) Control; 2) no Phytophthora + 24 hr/week flood; 3) Phytophthora cactorum + 24 hr/week flood; 4) Phytophthora drechsleri + 24 hr/week flood.

When I finally arrived home this evening I discovered the cassette I ordered for my bike arrived. It didn’t fit. It turns out cassettes and freewheels are very different things. I also figured out that removing a freewheel can be very difficult (and quite possibly impossible with the tools I was using). Tomorrow I am going to stop by The Bicycle Shop to see what they charge to replace a freewheel. I also intend to check out a special they are running on a Giant Boulder (that’s a bike, not a big stone). I might end up getting a new bike instead of replacing the freewheel on my old one if the deal is good enough. No word on whether or not they are running a bike version of the cash for clunkers program.

After wrestling with the freewheel to no avail I decided to go grocery shopping. So as midnight came and went I was cruising around a deserted Wegman’s. When a schedule becomes unruly you’ve got to run errands whenever you can. Thank goodness for 24 hour grocery stores.

Now it’s 1:00 in the morning and my plans to start today at 5:00 AM don’t sound so good.

More Apple Baiting

This afternoon I set up a few more apples with with alleged Phytophthora infested roots. Most of the last batch I did developed soft rot symptoms after five days, which is not consistent with Phytophthora infection of apple. The rot should be rather firm. I suspect other pathogens were present either on the root surface or within the root.

This time I used a different procedure to surface sterilize the roots before placing them inside the apples. Hopefully the results are better.

A few of the apples I set up today.