Download Project Summary - Berkeley Cosmology Group

Group Leader: Daniela Goltsman
Katherine Cantarero
Victoria Partida
Jose Villanueva
Microbial Diversity
In our current topics in science class we focused on microorganisms. We
observed microbial diversity that we come in contact with on a daily basis. We learned
how to identify different types of bacteria based on their shape. In this lab, we used
culturing techniques and fluorescence microscopy through a process known as FISH.
FISH stands for fluorescence in situ hybridization. To able to understand what you are
looking at under a microscope you must first understand the phylogeny of life. Before the
discovery of DNA, phylogenies were built using morphological characteristics or
phenotype. Now that scientist have discovered DNA and RNA they are able to classify
different species. More specifically, scientists use the 16S rRNA to differentiate between
different organisms. This sequence is used because it has few changes amongst closely
related organisms. In this lab we were able to see for ourselves the diversity amongst the
microorganisms around us.
In this lab, we performed many different processes. The first process we went
through is known as the inoculation process, which allows us to introduce
microorganisms to an environment. We did this by using gel agarose, which contained
nutrients to allow bacteria, and other microorganisms to grow. We allowed
microorganisms to collect on the gel by leaving the dish out and open over the weekend.
Some of the organisms that were collected were placed on the gel, others were collected.
We obtained samples by leaving out two separate dishes in Jose’s bathroom, and
Katherine’s bedroom. We also collected samples from UC Berkeley’s Strawberry Creek
(Two drops), and from swabbing the Foothill dorm’s rec. room’s keyboard. We then
allowed the microorganisms to form colonies in an incubator at 37 oC.
We observed the many different results in the four different plates. Our first
sample was that of Jose’s bathroom. We observed a fuzzy, purple colony, a gel like white
colored, smooth colony. We also observed a colony with a brown green center, and a
rough outer white rim. We then observed fungus with a web-like look. We also observed
quite a few clear, circular colonies. The most distinct of all of the colonies was the one
tiny red dot colony. One the computer’s keyboard gel we didn’t find as much variety as
we did in the bathroom, but we did observe some things. We noticed fungus, a pink dot,
and little smooth white colonies. Next we observed the colonies on the gel from
Strawberry Creek. We saw that there were white, gel-like, smooth colonies that had
yellow tips. We noticed a fungus that was white and rough. We then observed many
small white and round colonies. Although the creek’s gel was really diverse it had similar
fungi as the other gel plates. Finally we observed the plate that was obtained from
Katherine’s room. We noticed a lot of fungi, a few round white colonies, a few round
yellow colonies, and white blobs that are not circular. These results showed us how
diverse the environment around us is, but how similar things, like the fungi we found,
could still be similar.
The next process we performed was that of isolation. The first step of this process
was to sterilize our environment. We wanted the environment to be sterile, because we
wanted to view the culture as we originally collected. The technique that practiced is
known as the Aseptic Technique, which inquires sterilizing your work bench and all of
your materials. We practiced this technique by spraying our tools with 80% ethanol, and
by lighting a flame that would sterilize the air one meter around. The second step of
isolation was to isolate a colony using a bacteriology loop. We picked one colony of
microorganisms from each plate, and placed each colony on a separate sterile plate. We
then spread the colony, which contained many microorganisms around the media. We
then incubated the plates at 37o for 24 hours. This incubation is needed so that
microorganisms can multiply at favorable conditions. This step is called isolation because
you are taking one colony of microorganisms from a diverse community of
microorganisms, and producing a plate containing only one type of microorganisms.
The step following isolation was fixation of the different colonies. Fixation is the
process in which you immobilize the organisms, or in a simpler form, “killing” the
organisms. Fixation causes the cell to die but the cells membrane will not lyse. The cell
membrane will not lyse or break, because we added the pfa fixative. By killing the cells
and not lysing the membrane you are assured that the DNA and organelles are kept intact.
Each plate contained a wide variety of microorganisms. This is why we had to choose
only four different colonies from each plate to observe, which an overall of 16 colonies
that were chosen is. We had a master mix in four different eppendorf tubes for each
plate. This master mix contained a fixative known as PFA. Next we centrifuged the
tubes to get a pellet that only contained the four different colonies and get rid of the
fixative. Then we incubated the mix at 4 oC to fully fix the cells in each of four different
eppendorf tubes. After that we added PBS and 100% ethanol to create conditions similar
to those inside the cell. At this point we had 4 eppendorf tubes each with four fixed
colonies.
After we fixate the colonies, we added a sample into each well of the slide. The
first well contained microorganisms from the computer’s keyboard. The second well
contained microorganisms from Strawberry Creek. The third well contained
microorganisms from Katherine’s bedroom. Finally the fourth well contained the
microorganisms from Jose’s bathroom. We then let the FISH slide air dry so that the
samples would not mix. So then we put the slide into three different tubes that contained
different percentages of ethanol for three minutes each. Then we allowed to air dry.
We then had to prepare the buffer for the hybridization chamber. The hybridization
chamber was needed so that the buffer in each well would not evaporate. We also added
buffer to each well so our sample would not evaporate. After this step in the process we
added 4 different probes to each well. This is so that our samples could be seen under the
microscope. After this step we put the FISH slide into the hybridization chamber and
incubated at 48o C for two hours. Next we prepared a washing buffer at 48o C. The FISH
slide has to be incubated and washed at the same temperatures so that the cells would not
become shocked and lyse. We washed the slides so that the unnecessary probes would be
washed away. We then finally left the slide to air dry to prepare for visualization under
the microscope.
To conclude this experiment we were able to look at our samples under the
microscope. We did this using different colored fluorescent probes. The different probes
that we coded for universal bacteria, beta proteobacteria, gamma proteobacteria, and
eukaryotes. The probe color for the universal bacteria was green. Then probe color for
the beta proteobacteria was red, but would light up yellow because it would be combined
with universal bacteria, and the beta bacteria. The gamma proteobacteria would be blue,
but would show light blue when combined with the green universal bacteria probe.
Finally the eukaryote’s probe would be the color blue, but would only show as blue,
because it isn’t combined with any other probe. Unfortunately, the camera on the
microscope only takes pictures in black and white. Therefore our project leader had to
take the pictures, and use a software program called Photoshop to color and enhance the
overall quality of our results.
When it came time to visualize the FISH slide we had different results for each
sample. All four wells contained bacteria and this was seen because there were a lot of
green fluorescent shapes. We also saw fungi present in each well except for the well that
contained the pond bacteria. The shape of the fungus was not seen because the probe
only binds to the DNA in the nucleus, so this was the only part of the eukaryote to
fluoresce. The gamma proteobacteria was present in all well but the well for the
keyboard. These specimens fluoresced in light blue. Finally, we also found beta
proteobacteria in the pond and the room. This bacterium was not dominant and it
fluoresced in a yellow color. Overall, these results allowed us to conclude that there is a
diverse community of microorganisms that we come in contact with on a daily basis. We
also learned that that microorganism is an essential part for biology and topics that work
our in that area.