BES Summer Internship

Week 10 brought this internship to a close. This week, we got to work on some sunflowers that had been growing outside.

First, we made a spore solution using spores that had been collected from a petri plate on which they were growing. We also had some diluted potassium chloride to spray on the sunflowers that weren’t getting treated with the spore solution.

 

All of the potassium chloride treatments got covered up so that they wouldn’t get any of the spore solution on them.

Spore solution…

Direct hit!

After applying this treatment, we were going to wait and see if the spore treated plants developed a pinkish residue that had been found on some other sunflowers, to see if those spores caused that problem.

 

This internship was such a great experience and I am very thankful that I got this opportunity to work with and learn so much from everybody!

 

During this internship, we extracted a lot of DNA from various samples. In week 9, I was able to take some pictures of how we obtained those samples.

 

The first step is to put a large cube of the agar into a flask containing a broth…

Then the flasks are put onto a shaker that spins the broth and agar around. We left them like this for about a week.

 

 

We waited until enough growth had occurred to harvest it…

Then we strained the flasks, collecting the tissue in autoclaved filter paper.

We then pressed the excess water out of the tissue. It kind of looked like (not appetizing) cheese!

It got put into tubes that were then sealed with kim wipes and parafilm.

 

Into the freezer they went! Afterwards they were lyophilized and then used for DNA extractions.

Week 8 was another busy week! It started out with a visit from a group of middle schoolers who were at a camp at the experiment station for the week. We did DNA extractions along with running some gels. They thought it was pretty cool and it was fun to get to help teach other people the skills I’ve learned over the summer.

A few weeks ago, we picked some garlic that had come from a soft rot field. This week, we weighed out the bulb heads and counted how many would count as “marketable” to see which treatment worked best at fighting the soft rot.

Unfortunately, not many of the bulbs were ranked as marketable. But I did find one that looked like a heart!

We also inoculated some Yukon Gold potatoes a couple weeks ago with dry rot. We did this by cutting a small core out of one end of the potato and then used dry rot grown on agar plugs to fill in the hole. Parafilm, a stretchy, breathable band- aid like material, was wrapped around the wounds and then we let them sit in a slightly humid environment for a little while. After seeing some signs of dry (and soft) rot, we decided to try cutting them open to examine the inside.

 

Here’s a top view of the cored sections where the potatoes were inoculated-

Potato surgery in progress!

We didn’t see quite as much dry rot as we expected. Only one trial really showed growth of the dry rot outside of the cored area. The potato in the below picture in the upper left hand corner actually turned out to be suffering from soft rot. We threw the soft rot potatoes away because we are focusing on the dry rot growth. The soft rot potatoes also smelled pretty bad!

A new project that we started this week was planting a brassica plot.

I weighed out certain amounts of seeds such as different kinds of mustards, broccoli, and arugula. Then I mixed those seeds with sand that had been inoculated with verticillium wilt.

Now we’re going to see how the mustard, broccoli, and arugula combat the verticillium wilt.

We also found a frog!  He’ll be keeping an eye on the plant growth for us.

 

With the verticillium project going on, we’re using tons of petri plates and slants. These plates and slants are filled with mediums that have different levels of selectivity- the more selective medium prevents less things from growing on it than does a less selective medium.

The mediums are made out of a variety of different ingredients. They’re gel-like in substance and contain agar, which is kind of like a gelatin that comes from algae.

This day, we needed to make some more slants and plates. All of the recipes are in this handy dandy book.

The slants we made had CPA or Czapek-Dox Agar. This recipe was just for the broth, but we added agar.

And the plates we made were 1% Potato Dextrose Agar.

First I made the PDA. This recipe is pretty simple and only has a couple ingredients. First is water!

500 mL makes quite a few small sized plates.

Next is the potato dextrose and agar. The potato dextrose smells a little bit like instant mashed potatoes!

In they go…

And then the same general instructions follow for the CPA.

For the slants, we fill them about 1/3 of the way full. We don’t pour the plates yet.

After pouring the slants, we autoclave both them and the un-poured plate medium. The autoclave is like a huge pressure cooker and sterilizes anything that goes into it.

After the autoclave, we tip the slants sideways so that they melted medium gels tilted.  

The plates are made by simply pouring the medium into the empty petri dishes and left to cool. We do this under the hood, which helps to keep everything sterile.

From here, we have lots of nice, sterile plates and slants we can use for future use!

 

 

Week 6 was another busy week! We started out with spore counting.

This is the slide that we used to put the solution containing the spores in to. The silver notches are where you pipette the spores into and then they spread across the surface where there is a tiny grid etched in it.

After placing the spores on to the special slide, we headed over to the microscope.

Here’s a view inside the microscope! The very small grid is seen here. The smaller squares are the ones that we counted the spores in.

Here you can see the actual spores. We used a clicker to count the spores in each square and counted 16 squares at a time. These 16 squares are seen contained inside the triple white lines.

Next, we finished one of our experimental carrot plot projects where the  carrots were being measured for amounts of soft rot. We went through and counted the number of healthy versus diseased plants in each plot and collected the data to see which treatments worked best.

There were lots of diseased carrots!

We also measured and flagged some plots for future use. One of these plots is for more sclerotia measurement and another one is for mustard growth.

We used this tool, which allows you to see the view from left, right, and center, all at once through the three windows. This makes squaring up the corners a lot easier than using the Pythagorean Theorem and running around trying to make everything match up!

I also picked garlic that had been growing in another experimental plot and chopped off the tops, saving the bulbs. We will then weigh the bulbs and see which plot produced the most.

Beautiful blue skies!

 

During week 5, we got to take a whole day to spend in the fields! The first place we went was to a potato field where we talked with some farmers who were having problems with their field. One of the things that is great about this internship is seeing how the research is being used in real world applications. Being able to meet the farmers and listen to them and the researchers communicate is really neat.

Next, we visited two different peppermint fields. In these fields, we counted the number of plants that were infected with verticillium wilt.

The fields were both about 35 acres.

We divided each field into four sections and then walked six diagonal lines in each section to most accurately view and see all the plants.

The mint fields looked innocent from the outside but were actually quite jungle-like once we got moving through them. In some places the plants were knee high; in others, waist high!

Everything smelled very minty fresh. My shoes smelled like peppermint for at least a week afterwards.

At first, identifying the plants with verticillium wilt seemed like trying to find a needle in a haystack! There was so much mint to look through.

After a while, identification got easier. The plants with verticillium wilt were often crunchy and crispy looking like this…

Yellowed leaves were also a giveaway.

In addition to yellow, the plants also had a purple hue on the leaves.

Some of the transects had barely any verticillium wilt and others had quite a bit.

Some just had a lot of ladybugs.

Soil samples had previously been taken from these same fields so the next step will be to look at how those match up with the number of affected plants in each transect.

Peppermint fields forever!

 

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.

 

Another project that I was able to work on during my first week was PCR using DNA from different samples of ergot, a fungi that grows on plants such as blue grass and wheat. If ergot is ingested it can lead to vasoconstriction and can also cause hallucinations.

To perform the PCR, we had to combine primers, water, and master mix into each capsule along with the different DNAs. The master mix is a bright green color because it contains ingredients that help it sink into the gel later in the PCR. When pipetting all of the primers, master mix, and DNA together, you have to be very careful to not contaminate any of the capsules and to not mix the DNAs together. It can be tricky to remember what you put where with so many different tiny tubes!

After all of the solutions were combined, the capsules were centrifuged and then put into the PCR machine. This machine changes the temperature for specific amounts of time so that the primers and master mix can work to replicate certain sections of the DNA over and over. After the PCR machine was done, we created a gel using agarose. We put combs into the gel to make tiny wells. When the gel had firmed up, it was placed into the electrophoresis machine. Each DNA solution had to be carefully transferred into the wells of the gel using a pipette.The voltage of the electrophoresis machine was turned to 120 V. DNA has a negative charge so it travels towards the positively charged end of the machine. The agarose gel created a matrix for the DNA to move through which made the smaller pieces move farther than the larger pieces.

The sheet of agarose was carefully placed into a GelDoc-It Imager.The window of the imager allows you to see the gel under UV light.
The imager also allows you to record a computer image of the picture to examine for later use.