User:Sydney Marshall/Notebook/Biology 210 at AU

March 9, 2014

Lab 6: Embryology & Development

Introduction
This lab is entitled Embryology and Development. The purpose of this lab is to identify the different stages of embryonic development and compare embryonic development between three different organism, specifically a frog, starfish, and chick.

Materials and Methods
Procedure I: Starfish Development

• Stages of starfish development were viewed.
• A starfish zygote was compared to an unfertilized sea star egg.

Procedure II: Frog Development

• Prepared slides of early and late cleavage of frogs were examined.
• Live tadpoles were examined.

Procedure III: Chick Development

• 72 hour chick embryos were observed using a dissecting scope.
• Parts of the fertilized egg were identified.

Observations and Data
Table 1: Comparison of Embryonical Features of a Developing Sea Star, Frog, and Chick

Feature	Starfish	Frog	Chick
Egg Size	microscopic	tiny	200 ocular spaces at 4x
Amount of yolk	holoblastic	holoblastic	meroblastic
Distribution of Yolk	isolecithal	telolecithal	partal meroblastic
Blastulation	holoblastic	holoblastic	meroblastic
Gastrulation	ectoderm, endoderm, mesoderm	open cavity, neurulation	primitive streak
Larval Stage	motile	tadpole	enclosed in egg

Table 2: Comparison of Ecological Aspects in Sea Star, Frog, and Chick Development

Feature	Starfish	Frog	Chick
Environment of development	aquatic	freshwater	egg shell
Fertilization	external	external	internal
Mechanism of waste disposal	diffusion to environment	environment	extra embryonic membranes (allentois)
Physical Protection	none	none	shell protection
Parental Care	none	none	parent

Conclusion and Future Plans
The purpose of this lab was to gain a better understanding of the embryonic development of the starfish , frog and chick. This was done by identifying their embryological features and differences in ecological development. This experiment was important because it is going to help set up an experiment with zebrafish. The experiment is going determine how an environmental condition, an independent variable, is going to affect zebrafish embryonic development, the dependent variable .

March 9, 2014

Lab 5: Invertebrates and the Importance of their Systems

Introduction
This lab is entitled Invertebrates and the Importance of their Systems. The purpose of this lab is to understand why invertebrates are important and to observe the evolution of their systems, specifically their body plans.

Materials and Methods
Procedure I: Observing Acoelomates, Pseudocoelomates, and Coelomates

• With the dissecting scope, Planaria, an acoelomate was observed, how its movements related to its structure were noted.
• Under a microscope, the digestive system of an acoelomate was observed.
• The above steps were repeated with a nematode, a pseudocoelomate and Annelida, a coelomate.

Procedure II: Analyzing Invertebrates from Transect 2 using Berlese Funnel

• A preservative solution was analyzed after breaking down Berlese funnel.
• Invertebrates were to be identified, however none were found in the transect.
• Instead, five leaf-litter organism were identified.

Procedure III: Vertebrates and Niches

• Vertebrates that could inhabit the transect were identified.
• A food web was constructed of biotic and abiotic components that could benefit from transect.

Observations and Data
Procedure I: Observing Acoelomates, Pseudocoelomates, and Coelomates

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  Figure 1: Planaria move by gliding across a surface.

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  Figure 2: Nematodes have 4 muscles within their structure that contract and expand when moving across a surface.

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  Figure 3: Coelomates expand and contract near their thorax area but are able to utilize their entire body muscles for movement.

Procedure II: Analyzing Invertebrates from Transect 2 using Berlese Funnel
Table 1: Leaf-Litter Organism Descriptions

Kind of Organism	Length in mm	Brief Description of Organism
Black Housefly	13 mm	black with brown tint, small wings, oval shaped body
Fruit fly	4 mm	very small, reddish brown, round body, three antennae
Black Fly	17 mm	large oval body,opaque black color
Millipede	25 mm	reddish brown, long, flatter body, many legs
Fly	20 mm	reddish brown, flat body, three antennae, large wings

• The size range of the organism measures are between 4mm – 25mm. The largest organism was the fly which was 20 mm long, but larger in girth than the millipede. The smallest organism was the fruit fly which was 4mm. The most common organisms that were found in the leaf litter were different types of flies.
  

Procedure III: Vertebrates and Niches

• Five Organism That May Inhabit the Transect
  • Red Shouldered Hawk
  • Great Crested Flycatcher
  • Great Blue Heron
  • Kingfisher (Setters, 2010)
  • American red squirrel (Rubin, 2012)
    

Figure 4: Food Web of Observed Organism in Transect


Many different species of birds inhabit the transect, especially in the pine tree, such as the ones listed above. Squirrels benefit from pine cones as a source of food. Squirrels are a source of food for the larger birds that inhabit the pine tree. In the web, humans can benefit from pine trees by using them as lumber for construction or decoration for the Christmas holiday. Bacteria inhabit dead leaves while fungi are decomposers in the soil.

Conclusion and Future Plans
The purpose of this lab was to gain a better understanding of invertebrates of a niche, and this was fulfilled through observing different worm body plans and how this affected their type of movement. Since vertebrates were unable to be found in our transect we observed leaf litter organisms instead. Observing leaf-litter organism allowed us to understand what types of vertebrates are present in other niches. Since we were not able to identify any invertebrates in our transect, in the future we could possible test whether or not our transect is uninhabitable for invertebrates.

References
Setters, Loret. Pine trees are for the birds. 2010. Beautiful Wildlife Garden. (9 March 2014) <http://www.beautifulwildlifegarden.com/pines-trees-are-for-the-birds.html>
Rubin, Catherine. Tamiasciurus hudsonicus. 2012. Animal Diversity Web. (09 March 2014) <http://animaldiversity.ummz.umich.edu/accounts/Tamiasciurus_hudsonicus/>

February 24, 2014

Lab 4: Observing Plantae and Fungi from Transects

Introduction
This lab is entitled Observing Plantae and Fungi from Transects. The purpose of this lab is to view the diversity of different plant types and any possible fungi that grew as a result of the previous lab. Specifically, five different leaf types were taken from Transect 2 and fully described in a table based on their vascularization, specialization and reproductive organs. After the descriptions were noted, the last procedure was done as a precursor for next week's lab.

Materials and Methods
Procedure I: Collecting five plans samples from Transect 2

• Use three bags to obtain the following samples from the transect: leaf litter sample, 5 representative plants, any flowering or seeding plants.
  

Procedure II: Plant Vascularization

• Observe moss and lily and note heights.
• Describe the vascularization of plants found from the transect.
  

Procedure III: Plant Specialization

• Observe moss leaves.
• Describe the shape, size and composition of the leaves found at the transect.
  

Procedure IV: Plant Reproduction

• Observe reproductive parts of moss and lily.
• Identify any seeds in transect as monocot or dicot.
• Check for proof of spores or flowers.
  

Procedure V: Observing Fungi

• Observe black bread mold under a dissecting microscope.
  

Procedure VI: Berlese Funnel setup for collecting invertebrates

• Pour 25 mL of 50:50 ethanol/water solution into a bottle.
• Place screen at the bottom of the funnel.
• Carefully add leaf litter to funnel.
• Place under lighting and cover everything with foil.
• Leave for next week’s lab.
  

Observations and Data
Table 1: Descriptions of 5 transect plants

Transect 2	Location and number in transect	Description, shape, size, composition	Vascularization	Leaves and Special Characteristics	Seeds, evidence of flowers or other reproductive parts
Pine Tree	Sprouting from ground	Tall tree, typical tree	gymnosperm	Sharp leaves on ends, long, thin	cones
Holly Tree	Sprouting from ground	Shorter tree than pine	dicot	Sharp pricks around sides of leaf,	evidence of berry production
Vine	Covering entire Transect	Plant	monocot	leaves grow along stem	none
Oak Tree	Sprouting from ground	Shading entire transect	dicot	a little thicker than sycamore leaf	none
Sycamore Tree	Sprouting from ground	Large round tree	dicot	very thin and flimsy	none

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  Figure 1: Tree From Transect

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  Figure 2: Vine Branch

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  Figure 3: Sycamore Leaf

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  Figure 4: Pine Branch

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  Figure 5: Oak Leaf

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  Figure 6: Holly Branch

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  Figure 7: Black Bread Mold

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  Figure 8: Moss Plant

Conclusion and Future Plans
There is much plant diversity in Transect 2. The significance of botanical diversity in a niche is important for other organisms to colonize. Fungi sporangia are the reproductive parts in fungi. This is important because it allows fungi to reproduce asexually. In the future, invertebrates located in the transect are to be observed. It is predicted that there will be presence of an invertebrate in the next lab because of the diversity of the transect.

February 16, 2014

Lab 3: Understanding The Characteristics Of Bacteria By Observing Antibiotic Resistance And Understanding How DNA Sequences Are Used To Identify Species

Introduction
This lab is titled Understanding The Characteristics Of Bacteria By Observing Antibiotic Resistance And Understanding How DNA Sequences Are Used To Identify Species. The purpose of this lab is to identify the morphology of the bacterial colonies and how these are affected by the antibiotic tetracycline. In order to understand the characteristics of bacteria colonies, wet mounts were prepared to be observed under a microscope, gram-staining of bacteria was done, and PCR preparation of DNA was done, which was a precursor next week’s lab which to purify these products of the PCR reaction. The hypothesis for today’s experiment was that the tetracycline will inhibit the growth or colonies.

Materials and Methods
Procedure I: Observing and Counting Colony Growth on Nutrient Agar and Nutrient Agar + Tetracycline Plates

• Observe changes (such as smell or appearance) in the Hay Infusion Culture.
• Count the amount of colonies on each bacterial plate, record in Table 1 and multiply by their conversion factors to accurately determine how many colonies there are per mL.

Procedure II: Antibiotic Resistance

• Note differences between colonies in nutrient agar and nutrient agar + tetracycline plates
• Infer the effects of tetracycline on total number of bacteria and fungi.
• Determine if there are any species of bacteria that were unaffected by tetracycline.

Procedure III: Observations of Bacterial Cell Morphology and Gram-Staining

• Observe a prepared slide using 100x oil immersion and its three stained areas containing samples of bacillus, coccus, and spirillum.
• Take 4 samples from 4 seemingly distinct colonies from both the nutrient agar plates and nutrient agar + tetracycline plates.
• Prepare 2 wet mounts for each bacteria sample by putting a drop of water on a slide and scraping the bacteria on the sample and covering one group with a cover slip and another without.
  • Examine the covered slide under a microscope and identify the bacteria.
  • Gram Staining
    • Heat the slips on a Bunsen burner three times.
    • Cover the slips with crystal violet for 1 minute then rinse off with water.
    • Cover slips with Gram’s iodine for 1 minute then rinse off with water.
    • Immerse slips with 95% alcohol for 10-20 seconds and rinse gently with water.
    • Cover slips with safranin stain for 20-30 seconds and rinse off with water.
    • Dry slips with a paper towel and air dry.
    • Observe slips under a microscope and record observations.

Procedure IV: PCR Preparation for DNA sequence identification

• Transfer on colony of bacteria into a 100uL of water in a sterile tube.
• Incubate at 100 degrees Celsius and place in a centrifuge.
  • Place 5uL of the supernatant of the centrifuged liquid in the PCR reaction.

Observations and Data
Procedure I: Description of Hay Infusion Culture

• Stronger smell
• Loss of water

Figure 1: Bacterial Colonies in Plates before Observation

Table 1: 100-fold Serial Dilutions

Dilution (Plate Label)	Agar	Colonies Counted	Conversion Factor	Colonies/mL
10^-3	nutrient	5000+	X10^3	5000000
10^-5	nutrient	240	X10^5	24000000
10^-7	nutrient	129	X10^7	1290000000
10^-9	nutrient	17	X10^9	1.7 X 10^10
10^-3	nutrient + tet	960	X10^3	960000
10^-5	nutrient + tet	268	X10^5	26800000
10^-7	nutrient + tet	29	X10^7	2900000000

Procedure 2-3:
Description of Colonies

• Tetracycline plates
  • orange color
  • Bigger
  • uniform in size
• Nutrient Agar
  • Combination of cream and orange colored colonies
  • less uniform in size

Figure 2: Sketches of Bacteria Cells

Table 2:Description of Sampled Bacteria Colonies

Colony Label	Tet or NA	Colony Description, color, shape, texture, etc.	Cell Description, motility, shape, arrangement	Gram pos or neg
-5	NA	light yellow, smooth, small, clumped together	grouped together, circular, blue	negative
-7	Tet	orange, circular, smooth like saturated fat	rod and cocci observed, grouped together	negative
-9	NA	white, yellow, round	motile, not grouped together, round	negative

Conclusion
Tetracycline causes colonies to be less but more uniform in size and shape. There was an increase in orange colored colonies compared to nutrient agar places. It indicated that the colonies were responsive to the added tetracycline in the sense that their ability to reproduce was inhibited, which confirms my hypothesis. Bacterial species that tetracycline is unresponsive to are those that have developed evolutionary resistance to it. Since tetracycline treats many gram-negative and gram-positive bacteria, it is rare to fine species that are not responsive. For the next lab, we will determine if our bacteria contains DNA running the PCR products on an agarose gel.

February, 9, 2014

Lab 2: Identifying Algae and Protists from a Hay Infusion Culture using a Dichotomous Key and Preparing and Plating Serial Dilutions

Introduction

The title of this lab is Identifying Algae and Protists from a Hay Infusion Culture using a Dichotomous Key and Preparing and Plating Serial Dilutions. The purpose of this lab was to understand the characteristics of Algae and Protists and utilize a dichotomous key to identify the species of such organisms. This procedure helped in determining the organisms observed from different niches in our hay infusion culture. The main hypothesis for this experiment is as follows: *If our transect consisted of a large variety of botanical the hay infusion culture is going to have a large variety of organisms.

Materials and Methods

Procedure I: Using a Dichotomous Key to Identify Organisms in Hay Culture Observations

• Carefully, bring the jar to your workstation and note its appearance and smell.
• Take 1-drop samples from two major niches in the jar (middle and bottom) using a transfer pipette and note specific locations of the samples.
• Observe the organisms on a microscope and sketch detailed pictures of what is seen in the slides while noting its size in micrometers.
• Characterize the different organism by determine the mobility, photosynthesizing ability, and similarities with the dichotomous key.

Procedure II: Using a Hay Infusion Culture to Prepare and Plate Serial Dilutions

• Take 4 tubes of 10mL sterile broth and label them 2, 4, 6, and 8.
• Obtain four nutiret agar plates and four agar plus tetracycline plates and label them 10-3, 10-5, 10-7, and 10-9.
• Using a micropipeter, obtain 100 microliters of hay infusion culture and place it in the tube 2 to make a 10^-2 dilution. Mix well.
• Obtain 100 microliters of the mix in tube 2, and place it in tube 4 to make a 10^-4 dilution. Mix well.
• Repeat for tubes 6 and 8 to make 10^-6 and 10^-8 dilutions, respectively.
• Take 100 microliters of mixed liquid in the 10^-2 tube, and carefully spread it on the surface of the nutrient agar plate labeled 10^-3.
• Repeat step 5 using tube 4 on the 10^-5 plate, tube 6 on the 10^-7 plate, and tube 8 on the 10^-9 plates.
• Incubate for a week at room temperature.

Observations and Data

Procedure I: Using a Dichotomous Key to Identify Organisms in Hay Culture Observations
Description of Hay Infusion Culture

• Smell: strong and musty
• Appearance: dirty, murky, moldy, has a layer of film on the surface of water

Figure 1: Two organisms found in middle and bottom of hay infusion culture, respectively.

Table 1: Descriptions of the two organisms found in hay infusion culture

'	(left)	(right)
Type of organism	Bacteria	Algae
Shape	Bacilli	Oval
Color	Black	Green
Size	2 micrometers	2 micrometers
Photosynthesic capabilities?	No	Yes

Figure 2: Nutrient Agar Plate Set-Up

Conclusion

In this lab, we were supposed to determine whether the diversity of botanical life corresponded to the amount to life in our hay infusion culture. I determined that since I was only able to identify two organism that my hypothesis was proved false. I interestingly found bacteria in addition to algae and thought it met all the needs of life because it is unicellular, acquires and uses energy by decomposition of organisms, processes and transfers genetic material by means of conjugation, capable of replication through colonies that will be observed next week, and is a product of evolution because it can evolve by being resistant to certain antibiotic strains.

If the hay infusion culture were to be observed for another few months, I would predict that more organisms could be easily identified. I managed to find bacteria as one of my organisms which is usually very tiny, and given time, more could have been found in the culture. Also, more mold could grow on the sides of the culture. Some selective pressures that affected the compositions of my samples were that my transect was in a very shady area surrounded by large trees. Therefore, there is a lot of moisture held in this area giving rise to diverse populations of algae. Even though I was only able to find one algae sample, it is possible that there were more types in other niches in my culture.

January 30, 2014

Lab 1: Observing the Evolutionary Specializations of Cells in the Volvocine Line and Determining the Characteristics of a Niche on Campus

Introduction

The title of this lab is Observing the Evolutionary Specializations of Cells in the Volvocine Line and Determining the Characteristics of a Niche on Campus. The purpose of this lab was to determine the evolutionary changes of cells within the Volvocine line, specifically from the Chlamydomonas, Gonium, and Volvox. The second procedure was to take a sample of our transect #2 in order to make a hay infusion culture for studying bacteria in the following week. The two hypotheses for each procedure this experiment are as follows:

• If the origin of the Volvocine line is Chlamydomonas, then the evolutionary progress of the following cells should exhibit more complexity.
• If the transect is in an area not frequently disturbed by human presence, then there will be a wide range of organisms to observe.

Materials and Methods

Procedure I: Observing the Evolutionary Changes in Cells within the Volvocine Line

• Prepare slides of Chlamydomonas, Gonium, and Volvox using a light microscope.
• Analyze the evolutionary specializations such as number of cells, colony size, relations of structure to function, and reproductive specializations.

Procedure II: Observing Characteristics of a Transect on Campus

• Go to a 20x20 foot transect near campus and describe general characteristics such as location and land profile.
• Determine the biotic and abiotic components of of the transect.
• Take a soil sample from the transect that is an accurate representation of the transect as a whole.
• Create a hay infusion culture using the retrieved soil sample.
  • Put sample in a jar with 500ml of deerpark water.
  • Add .1 grams of dried milk in the jar and label it for next week's use.

Observations and Data

Procedure I: Observing the Evolutionary Changes in Cells within the Volvocine Line

Table 1: Evolutionary Specialization of Members of the Volvocine

Characteristic	'	Chlamydomonas	Gonium	Volvox
Number of Cells		13	14	10000+
Colony Size		1μm	4μm	5μm
Describe any functional specialization of cells		motility (flagella)	form colonies with similar cells	motile flagellate cells, form spherical colonies made of glycoproteins
Describe any reproductive specialization (isogamy vs oogamy)		isogamy of motile cells fusing together to make a gamete	isogamy - cells can function as gametes and fuse together to make a zygote	oogamy - female is nonmotile and males are motile

Figure 1: Sketches of Three Organisms within the Volvocine Line

Figure 1: Sketches of Three Organisms within the Volvocine Line

Procedure II: Observing Characteristics of a Transect on Campus
General Characteristics:

• Located Behind Cassell Hall and in front of Wesley Theological Seminary
• Shaded under flowering and non-flowering organisms
• Abiotic components:
  • Holly
  • Ivy
  • American Sycamore
  • Pine Tree
• Biotic components:
  • Aluminum Foil
  • Soil

Conclusions

Relating back to the purpose of the lab, our group was to determine whether there was significant evoltionionary change in the original Chlamydomonas algae based on its predecessors, the Gonium and Volvox. According to Table 1, the number in cells in each slide increased, as the Chlamydomonas algae evolved over time. Also, the colony size increased from 1um to 5um. The most significant differences were the structures, starting as individual motile cells to spherical motile colonies made from glycoproteins. In addition, the reproductive specialization changed from isogamy, the fusion of cells to make gametes to oogamy, with the female gamete being nonmotile and the male sperm motile. Therefore, the original hypothesis is confirmed by this increase in complex progression over time. A sample sketch of what was seen in the slide can be found in Figure 1.

Based on the transect observations, it was to be determined whether or not a lack of human presence within the transect indicated a large amount of organisms. Our group found that there were a variety of biotic factors found such as Ivy, holly, pine, and sycamore. Only one unique abiotic component, other than soil, was found, which was aluminum foil, and I assumed that because of this, this transect was not commonly disturbed by human company. Although this information is not enough to determine whether the transect is a place containing a large variety of organisms, I conclude that my data does not support my hypothesis because visually, our group could not find organisms other than botanical organisms . However, I believe that in next week’s lab, I will be able to look at a microscopic view of the organisms that the transect can contain, possibly supporting or refuting my hypothesis. Because of this lack of information in the experiment, I would maintain my hypothesis for the next experiment to determine whether or not there is a large variety of bacteria and protists, In addition, it was hard to see the transect in the dark, therefore had there been an adequate amount of light, there would have been other organisms that our group could have observed.

Excellent Lab 1 entry. Very thorough, well explained and organized. SK

January 22, 2014

Test.