Cassandra Bivens

Honors Experience Journals

17SS

Undergraduate Pre-Professional Research

January 18, 2017

I have now been back in the lab for two weeks after going home
for Christmas break. There hasnt been much going on lately, since
several of the studies in the lab are wrapping up for now. Jennas study,
AD-2, studies the effects of antidepressants on the developing
adolescent brain, and it requires me and the other undergraduate
students to dose rats with antidepressant (or saline, for control
groups). This is the study I work on the mostI do something related
to AD-2 every day that I go into the lab, which is about three to four
days a week. Jenna often has me help out other studies that focus on
different drugs by running behavioral tests for them, such as the
Cincinnati Water Maze and the Morris Water Maze.
While I have been doing the Morris Water Maze since last
semester, I just learned how to run the Cincinnati Water Maze this last
week. Essentially, each rat gets five minutes to swim through a
complicated maze in total darkness, while we sit outside of the room
and watch them on a camera, keeping track of how many mistakes
they make and how long it takes them to reach the finish. Out of the
two water mazes Ive learned how to do, I definitely like Morris better!
It takes much less time and seems more accurate than Cincinnati (or
Cincy, as everyone calls it), since Morris tracks the rats with the
attached computer, as opposed to Cincy, which needs the human eye
to track the rats. I also learned a new dosing technique this last week.
Emily, the graduate student who also taught me how to run Cincy, is
running a study that tests the effects of pesticides on fetal and young
rats, and her study requires the dose of drug to be administered
directly into the stomach. The technique she taught me is called
dosage by gavage. Essentially, you fill a syringe with the drug and
attach a long, hollow, needle-like instrument with a rounded tip. You
then stick it down the baby rats throat until it reaches the stomach,
then dispense the drug and remove the needle. The process seems
almost like intubation, but the needle goes down the throat and into
the stomach rather than into the lungs. This is especially difficult to
perform on very small baby rats, so you have to be very careful not to
puncture anything internally. Overall, I have already learned a lot of
new lab skills in the brief time I have been back.

February 1, 2017
Since several studies have slowed down lately and are no longer
doing behavior, I havent been doing too much in the lab for the past
couple of weeks. Im still coming in several days a week, but with less
behavior to do, I havent been there for as long. Jennas study, AD-2,
does not do behavioral testing, since that was done in the preceding
study, AD-1. I have been doing a small amount of behavior for other
studies, as well as dosing for AD-2 and Emilys study, which uses the
gavage method of dosing. Jenna has said that she would like to get the
undergraduate students started on doing some benchwork in the lab,
which would entail working with the tissues collected from the rats
rather than the rats themselves. Tomorrow Im schedule to learn how
to do protein assays with Jenna in the lab, so hopefully that all goes
well. I would really like to learn more techniques that can be used in
the actual laboratory.

February 15, 2017

In the last couple of weeks, I have been doing more work in the
laboratory in addition to my work with dosing the rats and performing
occasional behavioral tests. Jenna had me start learning how to do
protein assays and Western blotting, both of which are important
common lab techniques. Protein assays involve a lot of pipetting, which
I have done in many of my academic labs. However, in a research lab,
accuracy and precision is much more important, and it must be
statistically confirmed that there has been nearly no error in the
lengthy process before the results can be considered significant. After
my first protein assay was finished, we ran it through a machine that
determined how much error was present. The machine indicated that
there was a significant amount of error, meaning that the assay I had
done was not going to be able to be used in the study. While this was
certainly disappointing, Jenna informed me that she didnt expect me
to have usable results on the first try and that it would take a while to
get used to such a precise process.
Additionally, Jenna began to show me how to perform the
Western blotting procedure. It was supposed to be a two-day process;
however, it needed to be two consecutive days, and I started it on a
Tuesday and am not available on Thrusdays due to classes. Because of
this, Jenna finished the second day of it for me. However, it seems as
though the lab does not have the necessary funds to order more of a
specific chemical that the blot requires, so it looks like we will no
longer be using that technique in the lab. However, Jenna assured me
that she is absolutely looking into the countless other laboratory
techniques we can use on our samples of rat brain tissue.

March 1, 2017
The last two weeks have been really difficult, and I have barely
been at my lab. Last week, I got very sick very quickly with what
turned out to be severe strep. I had to go to the emergency room due
to my throat swelling shut and be given a heavy dose of antibiotics. I
wasnt able to leave my bed for two days, so I missed a lot of classes
and some work. Just a couple of days after going to the ER, I went to
the doctor since I still seemed to be pretty sick. It turned out that on
top of the strep, I also had the flu, as well as a sinus and ear infection
all of this while still being on antibiotics for strep! Ive felt terrible for
the past two weeks and have gone to class most days, but I have only
been at the lab once or twice. Jenna has been very understanding of
everything. It seems like I have just run myself down this semesterI
am taking 18 credit hours (including the notoriously awful organic
chemistry), working at the lab 12-15 hours a week, and volunteering,
in addition to trying to stay active and go about normal life activities.
Although I dont feel 100% myself yet, I am so glad to be feeling better
after how awful Ive felt recently! I am very glad that there hasnt been
much for me to do in the lab, since I have had to call off several days
due to being sick, and I would have felt terrible leaving other people to
do all of my work. The couple of times I have been in the lab, I have
only had to dose or do brief behavioral tests, since some studies still
have not started back up yet.

March 15, 2017

The undergraduates in the lab are on spring break this week, so
there isnt too much going on at the moment. I am home for break, but
I have worked in the lab the last couple of weeks. I have been doing
the standard dosing for AD-2, as well as dosing for Emilys study and
behavior for whichever studies need help. It looks like Jenna is planning
on having more for us to do in the lab with new laboratory techniques
when I return from break.

March 29, 2017

Since returning from break, I have been working with a Cryostat
machine as well as doing the usual AD-2 dosing and dosing for Emilys
study. The Cryostat is not located in our lab, but another lab is allowing
us to use theirs. Essentially, it cryogenically freezes and slices up rat
brains into pieces too thin for any human to cut them themselves, so
that they can be mounted onto microscope slides and examined. Each
slice is 20 micrometers thickthats 20/1000 of a millimeter. The slices
are so thin that they often roll up and have to be flattened out with the
help of tiny brushes that look like very thin, precise paintbrushes. Each
slice has to be cut, flattened out, and mounted on a slide individually,
so the process takes several hours to get through an entire rat brain,
even though the brains are each no bigger than the pad of my thumb.
Were supposed to collect and mount between 120 and 150 slices from
each brain, so that we can run tests on them and observe them
through a microscope at a later date. After mounting the slices, we put
them on a hot plate for about 10 seconds so that the proteins in the
brain tissues can properly anneal to the slides. The brains must be
frozen before slicing so that they can be cut more easilyif they were
left at room temperature, they would be too gooey to obtain uniform
slices from.
The Cryostat machine seems fairly easy to operate, but it will
definitely take some getting used to. There are several settings that
have to be set very specifically, or else it will become very difficult or
nearly impossible to obtain the slices we need. The region of the brain
that should be most visible in the slices is the hippocampal region,
which looks sort of like the M in a McDonalds sign. If you hold the
microscope slides up to the light, this region becomes very clear.
Although we have to make sure that we obtain the best possible slices,
they dont have to always be perfect; as long as the hippocampus and
the area beneath it are intact, we should be able to obtain the
information we need. I am so happy and interested to be learning new
laboratory techniques and procedures so that I can feel like Im doing
more to contribute to the study and the scientific community!

April 12, 2017

Over the last couple of weeks, I have been doing a lot of work
with the Cryostat as well as a new procedure called cresyl violet
staining. The Cryostat can be very frustrating, because something as
small as the machine being even a single degree warmer or cooler
than usual can make the slices very difficult to obtain. This is a
challenge for me, because I am very much of a perfectionist and like
everything to be as ideal as it can be. So it will definitely take some
getting used to, since working with brain tissues on such a microscopic
level is bound to go wrong at some point, if not frequently! Jenna has
been very patient and understanding, especially with the whole
learning curve that comes with using the machine and finding the right
settings. She has spoken to many people who are very experienced
with using the Cryostat, and it seems that they have all had their fair
share of mishaps! Hopefully this will be beneficial with helping me to
accept that not everything I do can always be perfectlearning how to
make mistakes and grow from them is an important life skill in any
profession.
The cresyl violet staining is my favorite laboratory technique Ive
learned so far. After we obtain the brain slices from the Cryostat and
mount them, we freeze them until they are used for cresyl violet
staining. Before they are stained, the slices are pretty much
transparent on the slides, due to their being so thinly sliced. After
removing them from the freezer, we fix them by soaking them in a
toxic chemical called PFA, which stands for paraformaldehyde. This
helps to ensure that the slices are properly stuck to the slides and will
not slide off. After theyre fixed, the slides are soaked, four slides at a
time, in various chemicals for five minutes at a time. First, they are
soaked in a chemical called xylene, which is also toxic. This is all done
in a chemical hood, so that the fumes being breathed in are kept to a
minimum. Then, they are transferred to a second container of xylene
for the next five minutes. After that, they are soaked in two separate
containers of 100% ethanol, one container of 95% ethanol, and one
container of 70% ethanol, each for five minutes. They are then briefly
dipped in de-ionized water to rinse them before transferring them to
the container of cresyl violet staining solution for 30 minutes. The
cresyl violet solution is a thick, dark purple, gooey liquid. After 30
minutes, they are removed and go back through the various containers
of liquids in the opposite order they did beforefive minutes each in
de-ionized water, 70% ethanol, 95% ethanol, and two containers of
100% ethanol. This time, however, they are not soaked in xylene. Once
the slides are removed from the last container of ethanol, they are
allowed to dry. I usually go up to the animal behavior suite and dose
during this time. When I come back down, the slides are dry and ready
to have cover slips mounted onto them; this is done with a clear, glue-
like substance. After the staining process, the tissue is a dark, pretty
purple color, and the individual parts of the brain visible within the
slice are outlined much more clearly than before so that they can be
easily observed under a microscope.
While I have absolutely enjoyed working with the rats throughout
the year, doing dosing and behavior with them, it is pretty cool to be
using laboratory techniques that are used in biomedical laboratories all
over the world. It makes me feel like Im really contributing to the
overall purpose of the study. Its also interesting to be using some of
the techniques that I have previously heard or read about in some of
my classes, since actually doing them myself makes them seem much
more real.

April 26, 2017

Today was my last day in the lab this year, which is a very
strange feeling. My last final was today, so Ill be leaving to go home
for the summer tomorrow morning. In the last couple of weeks, Ive
been doing a lot more with the Cryostat and cresyl violet staining.
Jenna has been letting me do most of it on my own, since Im pretty
much finished with learning how to do it. Everything has been going
really well, and between myself and the other undergraduate students,
were collecting plenty of slides to stain and examine under a
microscope and observe any possible physical structural effects of
antidepressants on the brain, in comparison to those treated with the
saline control. This entire year has taught me a lot about what being a
part of the scientific community entails, including being a part of the
biomedical research community, which is a different entity in itself.
While it is no longer my goal to attend medical school, a decision that I
made this year, I have really enjoyed my time working in the lab.
Additionally, the experience I have gained from working with laboratory
animals will help me when it comes to applying to veterinary school,
since any sort of experience handling and dosing animals is important
in a competitive applicant.
Becoming a part of the scientific community was one of the main
goals I set for myself this year, and I would say that I certainly
achieved that. When I look back at my first few weeks in the lab,
everything was so new and shocking to me. Now, I feel very
comfortable doing everything I have learned and am constantly looking
to learn more and contribute as much as I possibly can to the study,
even though it may not necessarily directly relate to what I plan to do
for a career anymore. However, it has introduced me to several options
in the veterinary world that I never previously knew existedeach
hospital that uses animals for its research must have a practicing
veterinarian and veterinary services staff to ensure the humane
treatment of the animals. Additionally, Jenna has been very
understanding of my decision to switch from pre-med to pre-vet, and
she has even offered to connect me with one of the hospital vets when
I return to the lab in the fall, so that I can potentially shadow him when
Im not busy working in the lab.
I would say that this year has allowed me to grow as a person
and in a professional sense. I have been exposed to many situations
where people much older and more educated than me treated me as
an equal, and it was nice to be taken seriously. I feel that many people
do not take pre-professional students seriously until they have shown
that they are dedicated to doing what it takes to get ahead and make
themselves competitive applicants, and that is definitely an area I have
grown in this year. I have also been allowed to make mistakes and
learn from them, which has been really beneficial to me personally,
since I have always been very afraid of messing up or being less than
perfect. My experience throughout the last semester and year has
challenged me in so many new ways, and it has really helped me on
my way to becoming who I want to be. I am very excited to be
returning to the lab and continuing the study in the fall!