David Zepeda-Campos
Katia Garcia
Hector Ibarra
Ashleigh Richelle
Topics in Science: Presentation #2
Microbial Diversity
Have you ever wondered what kinds of things live in your refrigerator or
bathroom? What about in creeks or the inside of your mouth? In our group, we were able
to answer some of those questions. During our group sessions, we collected samples from
these places, incubated them, prepared them and viewed them under the light microscope,
and finally we used the Fluorescence In Situ Hybridization method (FISH), to identify the
organisms present. The key microorganisms we viewed under the microscope with and
without the FISH method were fungi and bacteria. We were able to isolate these
microorganisms from the sample we had collected, and the outcome of the work we had
done was really interesting and amazing to look at.
Our first day, we were each handed a Petri dish plate with nutrient rich-medium in
it and were told that our task was to find a place where we could leave it open or swab in
order to find out what kind of microorganisms grew there. We each chose different
places. David and Hector decided to leave it in a refrigerator, Katia left hers in the girls’
bathroom while Ashleigh used swabs from dirt water, her mouth and a garbage can. After
completing that task, we incubated the plates at 37C for 2 days. On Tuesday, we found
that we are surrounded with more bacteria than we thought. Ashleigh even got some
fungi to grow in hers. Once we had observed them with our naked eye, we used a
dissection microscope. This allowed us to see colony morphology and see variety of
shapes and colors, such as blob-shaped colonies to circular-shaped colonies, as well as
colors like whites, yellows and browns. Another characteristic that was evident our first
day investigating was the horrible stench being emitted by the various bacteria. We were
also able to identify hyphae which are the filaments from fungi. To see the cell
morphology we each took our sample, placed it on a slide and used the light microscope.
This microscope was connected to a computer. It was able to take pictures and project a
bigger image on the computer screen. When we were looking at the image projected by
the microscope on the computer, we were able to see that the bacteria were still alive!
Some of them were moving across and others were just moving in place. At this point we
were able to categorize the bacteria by their shape including circular-shaped bacteria
called cocci and rod-shaped bacteria called bacilli
The FISH protocol is an essential method in microbiology for narrowing down
and identifying microorganisms that may be present in an environment setting. The base
of this method all starts off with a molecule called 16S rRNA. The ribosomal RNA
(rRNA) is present in all organisms and it is involved in the synthesis of proteins. This
specific type of ribosomal RNA is given its name (16S) due to its density after
centrifugation. Such variety of the 16S rRNA in organisms is due to the evolution and
natural selection of the organisms. While all organisms contain this rRNA, there are a
variety of sequences among the population. This procedure, however, is not limited to
prokaryotic cells. It can also be used for eukaryotic cells; the difference though is that the
probe used to identify the eukaryotic cells would be targeting 23S rRNA. This method
works by designing 16S rRNA probes and adding them to a sample of bacteria. These
probes are designed to target a specific sequence in a prokaryotic cell and “hybridize” or
fuse to it. Probes can be designed to identify short regions in a sequence ranging from
twelve to twenty-five nucleotides in length. Once the protocol is done, scientists can
assess such characteristics like phylogenetic identity, morphology, number, and spatial
arrangements.
For the FISH process, we first had to fix the sample on a slide. We used a specific
kind of slide which was actually separated into eight sections so we would have enough
room to place all our samples together, but apart from each other at the same time. The
samples that we each used were from the colonies of bacteria that we obtained from the
different locations we chose. We scooped up small portions of the various colonies with a
loop tool, placed the samples in eppendorf tubes and added 500ul of fixative (PFA 4%).
After incubating the samples for about two hours at four degrees C, we centrifuged the
tubes, and added one volume of PBS ph 7.0, plus one volume of 100% ethanol. After we
had fixated the solution, we went into the actual F.I.S.H. procedure. The first things we
had to do were turn on a Bunsen burner, so to kill off any and all unnecessary bacteria
floating in the air. Then we put on our gloves. After putting 5ul of each sample on four
of the wells (one per person) of the FISH slide and letting it air dry, we had to make the
hybridization buffer in a 2ml eppendorf tube. We did this by adding 898ul of miliQ
water, 700ul of formamide, 360ul of NaCl 5m, 40ul of buffer Tris-HCl ph 8.0, and 2ul of
10% SDS. We then dehydrated the slide in 50%, 80%, and 90% ethanol solution for three
minutes each. After letting it dry again from the dehydration process, we prepared the
hybridization chamber by placing a kimwipe in a 50ml tube and adding 1.8ul of
hybridization buffer followed by 8ul of the buffer to each well and 1ul of each probe as
well. We then placed the slide in the hybridization chamber and incubated it again at 46
degrees C for 2 hours. After that, we rinsed the slide with washing buffer which consisted
of the same thing the hybridization buffer has. We rinsed the slide one last time with the
washing buffer in order to get rid of any leftover probes. After the slide dried, we saw it
through a light microscope. The FISH protocol detects the different organisms by lighting
them up a different color. There are 4 probes that hybridize, or attach, to the bacteria and
make them light up. The probe for eukaryotes is green, the probe for general bacteria is
blue, the probe for Gama probacteria is green, and the probe for firmicutes is red.
Sometimes some microorganisms will hybridize with two probes, so there will be more
combinations of colors that can result from this process. If an organism attaches to a blue
and a green probe, then the organism will show as light blue. If an organism attaches to a
red and a green probe, then it will show up as yellow. If an organism attaches to a blue
and a red probe, then it will show up as purple. And finally, if an organism attaches to a
red, blue, and green, then it will show up as white. Our results ultimately showed that we
had almost every kind of the organisms we went looking for. Practically every color on
the chart was evident in our visualization of the bacteria samples.
From collecting the samples to preparing them and viewing them under the light
microscope, the overall experience of finding out was a great one. We were able to see
that sometimes the places we would love to keep really clean, like our mouths and
refrigerators, can’t always be kept clean. There will always be something living in or
around us other than humans and that is what makes life interesting and diverse in many
ways.