User:Student 59/Notebook/Biology 210 at AU

Caroline Sell

Bio 210 - 007

---

---

Lab 3: 01-28-16

Before starting today's procedures, we took one last look at the Hay Infusion Culture. As of today, it has been sitting for 2 weeks. There were no changes in smell but the appearance did change. All the organisms we saw growing at each layer were nearly gone. There was a lot of dirt that had sunk to the bottom, and the top layer was extremely reduced. We observed that 50% of the water had evaporated. We hypothesize that there was a change in appearance because there were no more nutrients left in the culture and everything died. However, we did see that there was growth on our agar plates because there were nutrients present allowing bacteria to cultivate.

Image of Hay Infusion culture after 2 weeks, part 1

Image of Hay Infusion culture after 2 weeks, part 2

Procedure 1: Quantifying and Observing Microorganisms

We used colony morphology to help determine the type of bacteria growing on our plates. We counted the colonies on each plate, and made sure we divided by 100 to keep the units the same (colonies/um) since we made the cultures with 100 μL.

Table1: 100-fold serial dilutions results

According to this table, there were no results for the 10^-5 tet, 10^-7 with and without tet, and 10^9 with and without tet plates.

Procedure 2: Antibiotic Resistance

Based on the table above and the images of the plates below, we saw that for the previously mentioned plates, there was no growth at all. The only plates we could compare tet/no tet were the 10^-3 plates and we saw that the growth looked slightly different. The colonies on the plate with tet were larger, more spread out, and brighter in color whereas the colonies on the plate without tet were smaller and covered more of the plate. Neither of these was quite enough to be a lawn. The differences in colony type could indicate that the antibiotic did not allow for as many smaller colonies to grow as we saw with the plate without tet. As seen in the table above, there were significantly fewer colonies on the plate with tet than on the plate without tet. There appeared to be presence of some fungi on each of the plates where there was growth.

Top: 10^-3 without, Bottom: 10^-3 with tet

Top: 10^-5 without, Bottomr: 10^-5 with tet (condensation present)

Top: 10^-7 without (condensation present), Bottom: 10^-7 with tet

Top: 10^-9 without (condensation present), Bottom: 10^-9 with tet

Tetracycline is a broad-spectrum antibiotic that inhibits the growth of

Procedure 3, part 1: Bacteria Cell Morphology Observations

To observe the bacteria from our sample plates, we used a toothpick to pick up a colony of bacteria from 4 plates (10^-3 with tet, 10^-3 without, 10^-5 with tet, 10^-5 without tet) and put them on slides with a drop of water. We placed a cover slip on top and used a compound microscope to take a closer look at the shape and mobility of the bacteria. Below are the images we collected, all at 40x magnification:

plate 10^-3 without, shape: coccus (sphere), motility: individual cells moving individually diagonally from top right to bottom left

plate 10^-3 with tet, shape: bacillus (rod), motility: individual cells moving back and forth, not changing their original location much, just shifting back and forth, up and down

plate 10^-5 without, shape: bacillus (rod), motility: individual cells moving back and forth, not changing their original location much, just shifting back and forth, up and down

place 10^-5 with tet, no bacteria present

Procedure 3, part 2: Gram Stain

To perform the gram stain, we had to first make a wet mount by sterilizing an inoculation loop

Image 1: Gram Stain Image 2: Gram Stain

Procedure 4: PCR setup for 16S Amplification

We selected to compare the 10^-3 without and 10^-3 with tet samples for our PCR test.

---

Lab 2: 01-21-16

With the Hay Infusion Culture we made last week in lab, we made wet mounts for microscopic observation of the culture. The Hay Infusion Culture consisted of 10-12 grams of soil/ground vegetation from the sample we took last week, 0.1 grams of dried milk, and then everything was mixed together. The top of the jar was left open for one week.

First, we observed the Hay Infusion Culture in the jar. There was no particular smell. We did notice a bit of mold on the top of the culture.

Image of Hay Infusion Culture after 1 week, notice the different layers

Image of top of Hay Infusion Culture after 1 week

We noticed some plant matter (algae) near the top of the culture and this would be because of the need to perform photosynthesis. Along with the algae at the top level we also saw Paramecium, which would need the nutrients to survive.

Below are images of the organisms found at each level (top, middle, and bottom) along with their size and name.

Image of Diatom, top level, brownish color, 100 μL

Image of Paramecium, Top level, colorless and motile, 10 μL

Image of Diatom, middle level, brownish with patterned grooves and rows, 36 μL

Image of Diatom, bottom level, brown in color, 150 μL

Image of Hydrodictyon, bottom level, grass green color, non motile algae, rod shaped cells joined in a net-like conformation, 42.5 um

. . .

Paramecium: As per the Freeman text, paramecium are protists that are motile. They are a single cell, part of the Alveota lineage, and have 1 macro and 1 micro nuclei. They can reproduce asexually by binary fission or sexually with conjugation. In order for them to survive, they must consume the nutrients surrounding them in their environment, which is why we found them with the diatoms at the top of our culture.

If the Hay Infusion "grew" for another two months, we would expect more growth of algae such as diatoms. We already saw these on each level of our culture. However, there was only 0.1 grams of dried milk and at some point the nutrients would run out and nothing else would be able to grow in the culture.

. . .

We also prepared and plated serial dilutions using our Hay Infusion Culture.

Image of process used to prepare agar plates through serial dilutions

CLS

---

Lab 1: 01-14-16, Examining Biological Life at AU

Purpose

The purpose of this lab was to take a closer look at the biological life around us here at AU. While we study biology, it is important to understand the biological life we interact with each day. This semester, each group will examine a select 20x20 transect located on campus. We had transect #2 which is located by the Amphitheater and Hughes/McDowell Hall. There is a creek, many rocks, bushes, and trees. Below are images and a diagram of the transect. For this week's activity, we collected samples of all elements found in the transect to make a Hay Infusion Culture in lab. We also took notes to describe the biotic and abiotic components of the transect.

Materials and Methods

• 1 Gallon Ziplock Bag
• Latex Gloves
• Flashlight (*transect observed after dark)
• Camera/camera phone
• Glass Jar (w/ 1L capacity) with lid
• 500ml of purified water
• 0.1 gram of dried milk (*sustenance for living organisms in hay infusion culture)
• Sharpie
• Painter's tape or labels

We observed our transect by collecting aout 10-12 grams of material from the transect

Data and Observations

Image of Hay Infusion Sample

Image of Map of Transect

Image of Transect part 1

Image of Transect part 2

Image of Transect part 3

Biotic: bush, trees, moss in water

Abiotic: rocks, sticks, dead leaves

Conclusions and Future Directions

CLS

.