Month: July 2015

There was a lot going on during week 4! One of the main projects we worked on was a bacteria blight assay.

These carrots were infected with the bacteria in the greenhouse so that their growth wouldn’t be influenced by outside factors.

One of the visible effects of the blight is the presence of yellowed, unhealthy leaves…

Additional visible cues of bacterial blight are pockets of bacterial goo…

We chopped off the tops of the carrots and cut up the flowers, stem, and leaves.

We then soaked the chopped carrot medley in phosphate buffer to allow the bacteria to transfer from the plant to the buffer, which we then plated onto petri dishes.

The carrots were then drained from the buffer and dried. The dried leftovers were weighed. Once we get the bacteria information from the petri dishes, we’ll be able to see how much bacteria there is per gram of carrot folliage.

The second project was scoring ergot, which meant counting the number of sclerotia (the infected seed heads) caused by ergot. A couple of weeks ago, I harvested some Kentucky Blue Grass from one of the experimental fields that had been inoculated with the ergot. There were twelve different trials being tested to see which ones would best combat the ergot. These twelve trials were each tested in four different sections of the plot, making a total of forty-eight sampling bags. When picking the blue grass, I took ten handfuls down one side of the row and ten down the other, to make for an even sampling.

First step, dump it out!

Spread out the grass evenly…

Then you randomly choose a seed head without looking.

This seed head is looking healthy!

These two each have multiple sclerotia on them. The sclerotium are the black parts of the seed head.

I tested 100 stalks per bag. For each bag, I wrote down how many stalks were infected and how many seed heads on each stalk were infected. This information will be used to see which trial (type of blue grass) combats ergot the best.

One of the other projects we worked on was continuing the cruciferous plant project. This is what they looked like after planting them…

Tiny forests of micro-mustard, arugula, and broccoli!

We transplanted five to six plants per pot, and six pots per flat.

When we transplanted them, their chance of survival was looking slim. However, they’ve continued to grow stronger and are now looking like healthy little plants again!

 

Extracting DNA might sound like something that you would only see in a Marvel movie for the creation of some amazing, all-powerful Captain Universe. However, even you can do this in the comfort of your own lab! All it takes is some fancy equipment, potentially dangerous chemicals, and some time and well-balanced centrifuging.

Let’s get started. Today, we’re going to be extracting DNA for fusarium PCRs.

Sounds unexciting? Think again. Just look at this beautiful rainbow of potato dry rot samples!

The first thing that we have to do to extract our fusarium DNA is to prepare a tube for it. Into this tube we need to place tiny glass beads to mechanically grind the cell walls and break up the cell tissue.

Now we need to transfer our DNA-containing material into our glass-bead filled tube. A sterilized toothpick works nicely for this job.

In it goes!

Time to bring in the chemicals. A dose of breakage buffer is put into each tube. The purpose of this buffer is to cause the cell to lyse, meaning that it busts open the cell membrane. Consequently, all of the cellular contents are released and merge together, forming a slurry of sloppy, goopy cell components and denatured proteins.

Next, we vortex these bad boys. This is where the beads actually do their mechanical cell-wall-breaking action.

Dangerous chemical #1 alert! Phenol:chloroform:isoamyl is added to separate the mixture into two phases: organic and aqueous. Phenol can cause very serious burns, so it’s important to exercise caution when working with it.

Wear your gloves! Put on those safety glasses! Do your work underneath the hood!

After the phenol has been safely added, a heavy duty centrifuge is put to use. This will spin the tubes around extremely fast and separate the contents into the two phases.

Eight thousand rotations per minute.

It’s like a super-charged tea cup ride.

Our two phases are now  apparent and are separated by the interphase. The DNA has stayed in the aqueous phase (the clear liquid on top) and the other stuff (cellular components, proteins) have traveled to the bottom. The aqueous phase can be pipetted into another tube for the next step.

Dangerous chemical alert #2! Chloroform:isomyl is added and the tube is centrifuged, leaving another layer of clear DNA-containing phase on top that is pipetted into another tube. The purpose of the chloroform is to clean up any of the residual phenols. Chloroform has carcinogenic effects and presents another good opportunity to put those lab safety skills to use.

Sodium acetate is then added, which helps to precipitate the DNA.

Ethanol is the last thing added. DNA is soluble in water, but is insoluble in ethanol; adding ethanol makes the DNA fall out of solution.

Trudging onward! We lug out our trusty centrifuge to use again, this time spinning our solutions at a steady fifteen thousand rotations per minute. This step causes the DNA to gather at the bottom of the tube.

Now we gently pour off most of the remaining liquid, being careful not to dump the DNA pellet at the bottom (which isn’t very visible at this point) into the beaker.

Back into the centrifuge it goes! The rest of the liquid is poured off and a small globule of DNA is the only thing remaining, with some proteins attached. The proteins are what make the DNA visible and colored.

The DNA pellets are then left to dry.

We’re getting close to our final product. The end is in sight!

DNA re-suspension time!

A small amount of Tris-EDTA buffer is added to tube, which makes it easier for the DNA to dissolve. This buffer also contains RNase, which violently chops up any RNA that might have mistakenly found itself welcome in our final DNA product.

A final, twenty-minute rest in a mini dry bath re-suspended our DNA.

Finally, our finished product! It might look unimpressive but just think of all of the genetic information it contains wrapped up in a mere thirty microliters (thirty millionths of a liter).

What do we use the final products for? Fusarium PCRs!

 

 

 

Unfortunately, last week started off with all of the seed-born carrot disease experiments having to be redone. The petri dishes we had previously plated were supposed to have easy-to-count bacterial colonies on them… instead, most of the dishes had surfaces that were completely coated with bacteria that wasn’t the kind we were looking for. This means that something got contaminated in the process of the experiment.

For each treatment, we plated 9 different dilutions: 0 through 8. The 0 trials were the control plates and these did not have any of the phosphate buffer used for dilutions added to them. The other trials all had varying degrees of the buffer added. Because the 0 trial petri dishes did not have nearly as much of a coating of the other bacteria (thought to possibly be xanthomonas), it appeared that one of the main culprits of this problem was the buffer!

Sadly, we had to throw all of the previous work away…   

But I got to wear these super cool safety glasses again while redoing the experiments, so everything turned out alright.

One of the other projects we worked on last week was quantifying numbers of infected carrots at soft-rot carrot plots. We went out to two different plots one morning.

These particular carrot fields were pretty badly affected by the soft rot, the female plants more so than the males.

The affected plants can be identified by wilting flowers, black-ish spots near the base of the plant, and overall brown, unhealthiness that continues up to the top rather than only being located around the base of the plant (which is more likely caused by herbicide).

The carrot, or whatever is left of it, in the affected plants is usually very sad looking. The bacteria eats it away, leaving varying amounts of mushy carrot remnants. The amount remaining depends on how long the plant has been infected.

We went up and down the rows, counting how many affected carrots were in each one.

One last project that was started was planting different mustard seeds. These spicy mustard seeds produce an antimicrobial compound that can be used to prevent unwanted bacteria from growing in soil. After they’re done growing, we will be able to see how well their soil resists bacterial growth.