User:Valerie Moon/Notebook/Biology 210 at AU

February 5, 2014: Week 4 V.M. – Edited February 26, 2014

Introduction – Characteristics, diversity, and importance of plants & fungi

In today’s lab we focused on the plant and fungi specimens in our transect. After collecting our samples we notes distinguishing characteristics. We paid particular attention to the vascularization of the plants we collected, as well as specialization and the manner of reproduction the plants use. All of these characteristics make the plants specific to our transect able to thrive in the ecosystem. In addition, after collecting all of our data, we will begin to see the role that plants and fungi may play in our small transect.

Purpose

The purpose of today’s lab is further familiarize ourselves with the living components of our transect. Because our transect is very small, I'm not sure how much plant life will be present, but I expect to see at the minimum, grass and rose bushes, but we will see what else there is.

Materials and Methods

Part One Materials:

1.) 2 bags

2.) Petri dish to use as a shovel

Part One Methods:

1.) Bring two empty bags to the transect (make sure there are no holes)

2.) In the first bag, collect about 500g of leaf litter sample (dead leaves, soft soil)

3.) In the second bag, take samples of 5 plants in the transect. In this bag, also put any seeds, pine cones, etc.

4.) Take photos of plants if necessary

5.) Identify the plants in the transect using a guide

6.) Describe the vascularization of the plants present

7.) Observe the arrangement, and other characteristics of the leaves collected from the transect

8.) Identify and observe the seeds from the transect

9.) Use a dissecting microscope to make any further observations about the plant samples

10.) Observe the fungi previously collected on the agar plates. Look at these samples under a dissecting microscope.

11.) Draw and describe the fungi

Part Two Materials:

1.) 25 ml of 50:50 ethanol/water solution

2.) small bottle

3.) screening material

4.) funnel

5.) tape

6.) lamp

7.) stand with a clamp

Part Two Methods:

1.) Pour 25 ml of 50:50 ethanol/water solution into the small bottle. There should be a little bit of room left at the top of the bottle.

2.) Tape a piece of screening material to the bottom of the funnel

3.) Place the small bottle on the stand. Clamp the funnel so that it rest slightly above the opening of the bottle.

4.) Pour the leaf litter into the funnel, and fill the funnel to the top. (The leaves should be placed at the bottom of the funnel, covering the screen).

5.) Place a light over the funnel and then cover everything with foil.

Data and Observations

We collected 5 samples of plants from our transect. Below is a photo of our 5 collected plants, and their names:

Sample 1: Leaf from a black oak tree

Sample 2: Leaf and seeds from a rose bush

Sample 3: A clump of grass

Sample 4: Clover

Sample 5: Moss

Below is a table that we created, depicting the vascularization, specialization, and reproduction/type of seed.

We observed a sample of fungi in a petri dish under a dissecting microscope. We determined the sample was, indeed, fungi because we could observe the black sporangia made of hyphae. Also, on the underside of the petri dish, the rhizoids are visible, which are hyphae that have grown into the agar like roots.

Below is a drawing of the fungi we observed: The black-bread mold (Rhizopus stolonifer)

January 29, 2014 V.M. - Edited February 16, 2014

Introduction - Characteristics of Bacteria and Antibiotic Resistance

In today's lab we once again observed our hay culture infusion and made note of any differences. Then, we then moved on to observe the petri dishes we setup last week, and we observed the differences/morphology between the plates with/without antibiotic. Next, to further observe cell morphology we observed four samples of agar/antibiotic and agar plates. Finally, we prepared gram stains of the four samples.

Purpose

The purpose of conducting these different procedures in today's lab is to familiarize ourselves with the characteristics of bacteria, and observe antibiotic resistance, using samples from our transect.

Materials and Methods

Part One Materials:

1.) Hay Culture Infusion

2.) Last week's prepared petri dishes

Part One Procedure:

1.) Record observation about Hay Culture Infusion. Note any changes in appearance, smell, etc.

2.) Count the number of colonies on each agar plate

Part Two Materials:

1.) Last week's prepared petri dishes

Part Two Procedure:

1.) Observe distinguishing characteristics between colony types on plates with and without the antibiotic. Think about what this indicates.

2.) Observe/determine what is the effect of tetracycline on the total number of bacteria and fungi? How many species of bacteria are unaffected by this antibiotic?

3.) Research how tetracycline works, and research which bacteria are sensitive to it.

Part Three Materials:

1.) Sterilized loop

2.) Last week's prepared petri dishes

3.) Slides

4.) Cover slips

5.) Microscope

6.) Bunsen burner

7.) Wax pencil

8.) Staining tray

9.) Crystal violet

10.)Wash bottle filled with water

11.) Gram's iodine

12.) 95% alcohol

13.) Safranin stain

14.) Paper towel

15.) Oil

Part Three Procedure:

1.) Make a wet mount for two samples from the nutrient agar plates, and two from the nutrient agar plus tetracycline plates

-Sterilize a loop over a flame

-Use the loop to scrape up a tiny amount of growth from the agar plate

-Mix the sample into a drop of water on a slide

-Cover with a coverslip

-Observe under a microscope (first observe at 10x then at 40x)

-Determine observed cell shapes and whether they are motile

-Draw each organism observed

2.) Gram stain the four preps

-Label the slides with a sharpie, and use a wax pencil to circle on the underside of the slide where the specimen is.

-Pass each slide through a flame a few times with the bacterial smear slide up.

-Place the slides on a staining tray, and cover the slides with crystal violet for 1 minute.

-Rinse the stain off using a wash bottle.

-Cover the slide with Gram's iodine mordant for 1 minute. Then rinse gently.

-To decolorize, flood the slide with 95% alcohol for approximately 20 seconds. Then rinse gently.

-Use a paper towel to blot excess water, then allow the slides to air dry.

-Observe the slide at 40X

-Write down observations and draw sketches

3.) Prepare PCR for DNA sequence identification next week

-Choose two samples, one from plate with tetracycline, and one without the antibiotic.

-Mix a small amount of the colony in 100 microliters of sterile distilled water.

-Heat the tube of sample and water in a heating block for 10 minutes.

-Spin the sample for 1 minutes at 13.6K rpm ion microcentrifuge.

-Add 23 microliters of mastermix with forward and reverse primers to the PCR bead in the small tube. Mix well and carefully.

-Add 2 microliters of the bacteria DNA sample (out of the microcentrifuge) to the small tube with PCR bead.

-Place this tube into the PCR machine after mixing the tube carefully.

Data and Observations

Observations of Hay Culture Infusion

-Water level receded

-Water seems to have become clearer - you can look through the top of the water and see the bottom of the jar/the bottom layer of sediment

-Less of an odor (only very slightly)

-I suspect that the water level receded due to evaporation, and perhaps the culture became clearer because the sediment all fell to the bottom of the jar.

Observations of Petri Dishes

We observed that there was much more bacterial growth on the agar plus tetracycline plates, perhaps this is because the bacteria from our hay culture infusion happen to have antibiotic resistance. We also observed that there was fungi present on the the 10^-9 plate (without Tetracycline) and the 10^-7 and 10^-5 plates with Tetracycline. Also, all colonies appeared circular, but the two variations we found appeared to be either orange or beige in color.

We made note of the petri dishes that housed bacterial colonies, and were surprised that not all of the petri dishes had bacteria. We expected the more diluted samples to have less bacteria and the less diluted samples to have more colonies, but our observations did not reflect this prediction, perhaps because of an error in how we spread the samples on the agar, or how we conducted our dilutions. The following table represents our observations of the colonies present.

Comparing the 10^-3 plate and the T10^-3 plates, both plates have growth from the orange and beige types of bacteria. However, on the T10^-3 plate, the two types of bacteria seemed to thrive more and grew to become larger. However, in comparing the two types of bacteria on the T10^-3 plate, the orange bacteria colonies seemed to thrive more than the beige and became larger. In addition, all colonies, both orange and beige appear to be circular. Below are photos taken of the two plates in comparison. Therefore, we found that our two different types of bacteria colonies are unaffected by Tetracycline.

We made the following table about our three samples of gram stains to describe the colonies, and whether they are gram positive or negative.

We made the following diagrams of our colonies.

Conclusion

In this lab, we found that not only bacteria colonies formed on some of our agar plates (the normal agar plates, and the ones mixed with Tetracycline) but also fungi formed on some of these plates. We observed the colony morphology, and used a gram stain to determine which were gram positive and which were gram negative, and found that out of our three samples, the two from normal agar plates were gram positive, and the sample from the agar plate with tetracycline was gram negative. After doing some research into how Tetracycline functions, I found that this antibiotic inhibits the bacteria's ability to produce essential proteins to its survival. Therefore, this antibiotic prevents the bacteria from spreading. In next week's lab we will conduct gel electrophoresis on our PCR products.

Information about Tetracycline taken from http://www.netdoctor.co.uk/sex-and-relationships/medicines/tetracycline-tablets.html

2/6/14, lab 1 notes

Great work! Some notes:

-Make sure each lab entry is signed with initials

-Make sure photos are included by Sunday

-Start working on building a map of your transect to detail your land and where your samples are taken from. We will talk about this more Wednesday

Great job! AP

January 22, 2014 V.M.- Edited February 9, 2014

Introduction - Hay Culture Infusion and Preparing Serial Dilutions

In today's lab we observed the Hay Infusion cultures that we set up in last weeks lab, in order to explore possible organisms in the different niches of our ecosystem. After observing out Hay Culture Infusion, we setup 100-fold dilutions of the culture spread these on agar petri dishes. We hope that next week when we observe our petri dishes that there will be prokaryotic organism and maybe some fungi present.

Purpose

The purpose of setting up and observing the Hay Culture Infusion this week is to observe the carrying capacity and the organisms present in the different niches of our ecosystem. The second step of today's lab was to set up agar petri dishes with dilutions of culture in the hopes that next week we will observe prokaryotic organisms and fungi. We hypothesize that next week we will see life in our petri dishes and we predict that we will see the most dense amount of prokaryotic/fungal growth on the less diluted petri dishes we created.

Materials and Methods

Part One Materials:

1.) Paper

2.) Writing utensil

3.) The Hay Culture Infusion

4.) Dropper

5.) Cover slide

6.) Slide

7.) Dichotomous key

8.) Microscope

Part One Procedure:

1.) Carefully bring the Hay Culture Infusion to station

2.) Make observations about the culture (i.e. smell, appearance, visible life)

3.) Take a few samples from the culture from different niches

4.) Note where the samples were extracted from in the culture

5.) Use dropper to place a small sample of the culture onto a slide, then cover with a coverslip

6.) Repeat step 5 for samples from at least 2 separate niches

7.) Observe samples under a microscope

8.) Draw pictures of observed organisms

9.) Using dichotomous key, attempt to identify the organisms

10.) Attempt to make other characterizations about the observed organisms

11.) Measure/record size of the organisms

Part Two Materials:"

1.) Four tubes of 10 ml sterile broth (make sure they're labeled 2, 4, 6, 8)

2.) Micropipeter set at 100 microliters (and tips for micropipeter)

3.) Four nutrient agar petri dishes

4.) Three agar plus tetracycline petri dishes

5.) Hay Infusion Culture

6.) Paper

7.) Writing utensil

Part Two Procedure:"

1.) Label the petri dishes with group initials, T (pm the tetracycline dishes), and the correct dilution on each plate

2.) Swirl the Hay Infusion Culture

3.) Take 100 microliters from the culture and aseptically add this sample to the 10 ml of broth in tube labeled 2 (for a 1:11 dilution)

4.) Swirl the tube well

5.) Take 100 micro liters of broth from tube 2 and add this to tube 4 (dilution of 1:10,000). Repeat this step twice again for tube 6 and tube 8

6.) Take 100 micro liters from tube 2 and aspectically spread onto the agar plate labeled 10^-3, and tetracycline agar plate also labeled 10^-3

7.) Take 100 micro liters from tube 4 and aspectically spread onto the agar plate labeled 10^-5, and tetracycline agar plate also labeled 10^-5

8.) Take 100 micro liters from tube 6 and aspectically spread onto the agar plate labeled 10^-7, and tetracycline agar plate also labeled 10^-7

9.) Take 100 micro liters from tube 8 and aspectically spread onto the agar plate labeled 10^-9

10.) Leave the petri dishes for a week to incubate at room temperature

Data and Observations

Hay Culture Infusion Observations:

-Murky water

-Dirt has settled on bottom

-Grass resting in middle

-White specks on top

-Layer of gray film on surface of water

-Tiny piece of fuzzy mold on piece of grass sticking out of the water

-Distinct and unpleasant odor

Samples - Extracted from Hay Infusion Culture:

We extracted two samples for observation. The first sample was extracted from the top of the water where we observed white speckles that had developed. The second sample, we extracted from the very bottom of the jar where the dirt and other vegetation lay. We hoped that these two different areas would house organisms from different niches. The water at the top could possibly house organisms that require more light, while the organisms at the bottom may not require light but may require nutrients from the vegetation.

Top of culture:

Organism 1 observed at 10x

-7 ocular spaces = 70 micrometers

-Seems to glide/spin and does not change shape

Organism 2 observed at 10x

-6 ocular spaces = 60 micrometers

-Moving very little

Organism 3 observed at 10x

-3 ocular spaces = 30 micrometers

-Very active and bouncing around

Bottom of culture:

Organism 4 observed at 10x

-2 ocular spaces = 20 micrometers

-Very agile, bouncing around

I did not come across as many organisms as I was expecting to find, and some of the organisms seemed to resemble one another, although they differed in size. I’m not sure if they are different organisms because of the differences in size, or if they are the same organisms at different maturity levels, and that is why they differ in size. (Ex: organisms 1, 3, and 4). This factor made it difficult to identify the organisms with the dichotomous key, and I did not come to conclusion about the organisms' identities.

The majority of organism I observed were motile, meaning that they were able to meet the needs of life by searching for nutrients, moving to shelter when in need, and moving to/from away whatever necessary.

Thoughts about Hay Culture Infusion:

Because we were observing organisms of different niches, it is important to think about the selective pressures that could possibly affect the composition of our sample. I believe that the selective pressures of our sample could include food availability, and availability of light. In addition, if I were to observe the hay infusion culture in two months, I would expect to see more mold growth. I would also expect to see more organism activity as the current organisms reproduced.

Conclusion

In today's lab we observed a number of different organisms taken from our Hay Culture Infusion. I did not seem to notice any distinguishing characteristics of the organisms from the different niches (samples from the top/bottom of the jar). However, we were able to observe the organisms we did find, and observe that there were all motile. In next week's lab we will observe the petri dishes that we set up today, and I expect to see prokaryotic growth, and hopefully some fungi as well.

Figures

Figure 1:

January 15, 2014: Lab week one V.M.

Introduction - First observations of transect

In this study we will be observing a transect of land on the American University campus in order to better understand how all the biotic and abiotic elements coexist and create a functioning ecosystem. Although the dimensions of our transect is 20 by 20 feet, this bit of land is still considered its own ecosystem, and over the coming weeks we will take the time to understand what we can about this small ecosystem.

Purpose

The purpose of today's lab is to become better acquainted with our small ecosystem and to observe the abiotic and biotic elements, as well as take notice of anything of interest. At this point, we do not have any hypothesis or predictions of interest, although we are familiar with the AU campus, we have an idea of what biotic and abiotic characteristics will be present.

Materials and Methods

Materials:

1.) paper

2.) writing utensil

3.) sterile 50 ml conical tube

4.) 500 ml of deerpark water

5.) .1g of dried milk, and a jar.

Procedure:

1.) Ensure that the transect's borders are marked with 4 popsicle sticks.

2.) Record the general characteristics of the transect, including the location, topography, and anything else important.

3.) Record the biotic and abiotic components.

4.) Use the 50 ml conical tube to collect a sample of the transect. We selected a sample including both soil and vegetation.

5.) With this sample, create a hay infusion culture. Begin by weighing 10-12 g of the sample.

6.) Place the 10-12 g of the transect sample into the plastic jar with the 500 ml of deerpark water.

7.) Add .1 g of dried milk into the jar and mix everything together gently for about 10 seconds.

8.) Label the jar with group members' names.

9.) Leave the jar in the back of the lab room in a safe spot.

Data and Observations

Location: Center of quad (center of footpath through the middle of the quad, and part of the adjacent lawn)

Topography/general characteristics: The transect includes grass, concrete, soil, stone, and some vegetation. It is completely flat, level, open and not protected by any trees or other forms of shelter.

Abiotic characteristics include: concrete, stone, soil

Biotic characteristics include: rose bushes, grass, unidentified green vegetation

Conclusion

All of our observations of the characteristics of our transect indicate that our ecosystem is somewhat diverse, regardless of its size. We were able to visibly see a number of biotic and abiotic characteristics, but with further research I feel confident that we will also discover a number of organisms that are not visible to the human eye. After setting up our hay infusion this week, the following week we may be able to draw some conclusions about our transect from observing the hay infusion culture, and the week after that we should be able to observe some samples of the hay infusion culture under a microscope and observe any organisms present.

January 22, 2014 Entered username and text successfully V.M.Excellent! MB