User:Christina Hwang/Notebook/Biology 210 at AU

March 18, 2015

In this lab, presentations regarding the transect lab report were given.

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March 4, 2015

As done in the previous lab, the PCR results of the bacterial DNA was acquired and analyzed the strain through the NCBI Blast website, and found that the bacterial strain for MB3, could possibly be Variovorax sp. S15 partial 16S rRNA gene, strain S15. Whereas, the MB4 strain that underwent the PCR machine had the results of Pseudomonas sp. YP19 16S ribosomal RNA gene, partial sequence.

Zebrafish Procedure

After the first initial series of observations for the first week, many of the zebrafish embryo for the control experiment died, and were missing, or improperly collected to begin with; therefore, a second set of embryos for the control were collected. For each visit of the zebrafish, a couple of fish were fixed each time, depending on how many were remaining.

Date	Number of Living	Number of Dead	Stage of Development	Color	Eye Coloration	Movement	Size
2/25/15 Control	8	0	Juvenille	Clear, slightly yellow, black speckles	black	agile	5 mm
2/25/15 Control 2	16	3	Juvenille	Clear, slightly yellow, black speckles	black	agile	5 mm
2/25/15 Experimental	19	1	Juvenille	Clear, slightly yellow, black speckles	blackish brown	agile	5 mm
2/27/15 Control	8	0	Juvenille	Clear, slightly yellow, black speckles	black	agile	5 mm
2/27/15 Control 2	18	1	Juvenille	Clear, slightly yellow, black speckles	black	agile	5 mm
2/27/15 Experimental	18	0	Juvenille	Clear, slightly yellow, black speckles	black	agile	5 mm
3/2/15 Control	7	1	Juvenille	slightly yellow, black speckles	black with pupils	agile, but freeze in light	5 mm
3/2/15 Experimental	11	7	Juvenille	slightly yellow, black speckles	black with pupils	agile, but freeze in light	5 mm
3/2/15 control 2	0	gone missing
3/4/15 control	2	5	Juvenille	slightly yellow, black speckles	black with pupils	agile, but freeze in light	6 mm
3/4/15 Experimental	0			not fluorescent

• https://instagram.com/p/z0xgkOAkV3/
• https://instagram.com/p/z0xcSsgkVu/

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February 25, 2014

Procedure I: Starfish (Asterias) Development

In this procedure, the embryos of starfish (Asterias) were observed under a microscope and was characterized by its features.

Feature	Starfish
Relative Egg Size	~23µm
Fertilization	External
Amount of Yolk (Holo/Mero)	Holoblastic
Distribution of Yolk	Isolecithal
Blastulation	yes
Gastrulation	Endoderm, Ectoderm, and mesoderm
Larval Stage	Yes
Waste Disposal	Digestive Tract
Protection	Hyaline layer

Procedure II: Frog Development

In this procedure, the embryos of frogs (tadpoles) were observed under a microscope and was characterized by its features.

Feature	Frog
Relative Egg Size	~1650µm
Fertilization	External
Amount of Yolk (Holo/Mero)	Holoblastic
Distribution of Yolk	Telolecithal
Blastulation	yes
Gastrulation	Endoderm, Ectoderm, and mesoderm
Larval Stage	Yes, tadpole
Waste Disposal	Digestive Tract
Protection	Rubbery shell

Procedure III: Chick Development

In this procedure, the embryos of chicks were observed under a microscope and was characterized by its features.

Feature	Chick
Relative Egg Size	~38100µm
Fertilization	External
Amount of Yolk (Holo/Mero)	Holoblastic
Distribution of Yolk	Telolecithal
Blastulation	yes
Gastrulation	Endoderm, Ectoderm, and mesoderm
Larval Stage	No
Waste Disposal	Digestive Tract
Protection	Hard shell

Procedure IV: Human Development

In this procedure, the embryos of chicks were observed under a microscope and was characterized by its features.

Feature	Human
Relative Egg Size	~150µm
Fertilization	Internal
Amount of Yolk (Holo/Mero)	Holoblastic
Distribution of Yolk	Isolecithal
Blastulation	yes
Gastrulation	Endoderm, Ectoderm, and mesoderm
Larval Stage	No
Waste Disposal	Umbilical Cord
Protection	Amniotic Sac

Embryogenesis Experiment with Zebrafish Larvae

For this experiment, my lab partner and I read a published paper on the effects of caffeine on Zebrafish embryo and presented our reading in class. Then we conducted our own experiment with a chemical called Rhodamine 6-G, an orange fluorescent chemical, and sought to determine what the effects this chemical would do on Zebrafish embryos compared to that of Zebrafish embryos that were just in distilled water. For this experiment, twenty embryos for the experimental dish was collected and twenty embryos for the control dish was collected, creating a total of forty zebrafish embryos to be tested. The first few days of observations were collected and compiled into a table.

Date	Number Living	Number Dead	Stage of Development	Color	Eye Coloration	Movement	Size
2/18/15 (experimental)	20	0	17-20 Somites	clear	N/A	N/A	1 mm
2/18/15 (control)	20	0	17-20 Somites	clear	N/A	N/A	1 mm
2/20/15 (experimental)	20	0	31 Somites	clear/black spots	black/clearish	N/A	1 mm
2/20/15 (control)	8	7	31 Somites	clear/black spots	blackish/clearish	N/A	1 mm
2/20/15 (control 2)	20	0	31 Somites	clear/black spots, less speckling	blackish/clearish	N/A	1 mm
2/23/15 (experimental)	19 hatched, 1 in egg	0	60 Hours	slightly yellow, black speckling	black	fast, agile, freeze in light	4 mm
2/23/15 (control)	8 hatched	0	60 Hours	clear with black	black	fast, agile, freeze in light	4 mm
2/23/15 (control 2)	20, 1 fixed, 4 misshapen	0	60 Hours	clear with black	black with some brown	fast, 4 spinning more than spinning, freeze with light	4 mm

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February 18, 2015

In this lab, we learned about the different types of invertebrates.

Procedure I: Acoelomates, Psudocoelomates, and Coelomates

During this procedure, different types of invertebrates were observed under a dissecting microscope. The first one that was observed was the Flatworm, also named Platyhelminthes, which had a fluid motion and moved rather slowly. The flatworm, because it is an unsegmented and soft-bodied invertebrate, moves in a fluid motion because it lives in water. Because of their soft unsegmented bodies, their movements flow more, rather than that of a segmented invertebrate. The second one that was observed was a Rhabditis, which had a spirally movement, like streams. The Rhabditis is a nematode, with a pseudocoelom, with an incomplete body cavity, resulting in the spiraling movements. The third invertebrate that was observed was an earthworm, which was rather large, and was segmented, and moved rather slowly. Because of its segmented body shape, the way the earthworm moved was by scrunching up the segments then inch forward and expand their segments.

Procedure II: Arthropods

The arthropods provided are being observed and characterized from a dichotomous key.

'	Arthropod
1	Bumble Bee
2	Arthropoda-centipede
3	psudeoscorpian-crab from reef tank
4	arthropoda arachnida araneae-spider
5	Diplopoda-millipede

Procedure III: Analyzing the Invertebrates Collected with the Berlese Funnel

In this procedure, the invertebrates found in our transects, collected in the leaf litter, and collected from the Berlese Funnel are being observed and characterized through a dichotomous key.

Organism (phylum and class)	Length in mm	Number in sample	Description of Organism
Arthropoda Arachnida/tick	1 mm	1	Brown, with about 6 legs, with a clearish/white color along its lower back
Isopoda/Pillbug or Sowbug	2-3mm	1	white, has more than 6 legs, looks like it has segments on its back
Siphonaptera/Flea	2mm	1	Striped with black and grey color, almost clearish, has a long pointed end, looks kind of hairy
Collembola/Springtail	1mm	6	Clear color, small, looks segmented, has antennae
Hymenoptera/Wingless Wasp	2mm	2	Looks like a small and wingless wasp, colored dark brown/black and light yellow

• http://instagram.com/p/zO4QMSgkcV/
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• http://instagram.com/p/zO4cekAkcj/
• http://instagram.com/p/zO4z5-gkdD/

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February 11, 2015

The four different PCR samples from last week's lab, resulted in all four samples on a similar place on the ladder, with the first sample at three areas on the ladder. http://instagram.com/p/y8S5q6gkRJ/

Procedure I: Collecting five plant samples from the transect

In today's lab, we studied the difference between plants, land plants and aquatic plants, and vascular plants and non-vascular plants. In this lab, five plant samples were collected to identify and characterized. In this lab, transect 4, which is the farmland was visited to collect five plant samples. These samples include leafs from a lettuce plant, brussel sprout plant, a piece of a kale leaf, a leaf from a bush, and some clover samples. The lettuce sample was taken from the lettuce garden, which is a box designated for the lettuce plants to grow. The brussel sprout sample was taken from the brussel sprout garden. The kale sample was taken from the garden growing the kale plant. The leaf was taken from a bush next to the gate, which is also next to the area where the leaf litter sample was taken from. The clover sample was taken from next to the lettuce garden. For the leaf litter sample, the leaf litter included pine needles and dead leaves in an area where there was moisture in the ground.

Transect 4: Plant Samples

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Procedure II: Plant Vascularization

Plant vascularization was studied in this procedure and was used to characterize the vascularization of the plant samples that were collected from the transect. It was seen that all the leaves were vascular plants.

Procedure III: Presence of Specialized Structures

Within this procedure, the plant samples that were collected from the transect where studied, their shape, size, and cluster arrangement. All plant samples collected were of leafs, which include the lettuce, kale, brussel sprout, bush and the clovers. These leaves were observed to see if they had any specialized structures. A specialized structure that was noticeable under the microscope was the stomata. However, for the kale sample, the stomata were not very visible. The brussel sprout sample had the same result, where the stomata were not visible; therefore, it was concluded that both the kale and brussel sprout samples did not contain specialized structures. This resulted with the leaf found on the bush, next to the gate and the leaf litter, where under a microscope, stomata were not visible. The spinach leaf sample; however, had a different result, where it did contain visible stomata, as well as the clover, that was growing next to the spinach garden. This then allowed for the conclusion that the spinach and clovers did contain specialized structures.

Procedure IV: Mechanisms of Plant Reproduction

Plant reproduction was a subject of which, we learned the difference between a dicot and a monocot, which was also used to characterize the five plant samples.

Transect Sample Plants	Location and # in transect	Description	Vascularization	Specialized Structures	Mechanisms of Reproduction
1	Kale Garden #4	Green, small, very leavy, fairly sturdy	yes	dicot, no noticeable stomata	flowering
2	Brussel Sprout Garden #4	Green, roundish leaf with stems, thick	yes	dicot, no noticeable stomata	flowering
3	Spinach Garden #4	Green, leafy, fairly flimsy and soft	yes	dicot, visible stomata	flowering
4	Bush near the gates #4	purplish, darkish green, with a hint of red. Fairly rough, with spiked edges	yes	dicot, no noticeable stomata	flowering
5	next to the spinach box garden, located in the middle of transect #4	Green, small, in pairs of 3, leaves are round	yes	monocot, visible stomata	flowering

This table summarizes all the results concluded from the previous procedures.

Plant Samples

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• http://instagram.com/p/y8SlI4AkQx/

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February 4, 2015

Procedure I: Quantifying and Observing Microorganisms

In this lab, we used the bacteria that was grown in the agar plates and observed the bacteria and the difference between the bacteria growing in the agar plate with nutrient and agar with the tetracycline. In each agar plate, the number of colonies grown in each agar plate was recorded, some were observed to have to many to count, which is what is defined as a lawn, others were approximated to a certain number, because although they were many colonies and not everything could have been specifically counted, it would not be considered as a lawn. Other agar plates, for both nutrient and tetracycline, the agar plates with dilutions were observed to have less bacteria growth, compared to the agar plates that have been less diluted or the plates that have not been diluted.

Table 1: 100-fold Serial Dilutions Results

Dilution	Agar Type	Colonies Counted	Conversion Factor	Colonies/mL
10^-3	nutrient	lawn	x 10^3	lawn
10^-5	nutrient	480	x 10^5	4.80x10^7
10^-7	nutrient	17	x 10^7	1.7x10^8
10^-9	nutrient	1	x 10^9	1x10^9
10^-3	nutrient+tet	370	x 10^3	3.70x10^5
10^-5	nutrient+tet	14	x 10^5	1.4x10^6
10^-7	nutrient+tet	1	x 10^7	1x10^7
10^-9	nutrient+tet	0	x 10^9	0

Pictures of Agar Plates

Agar Plate with nutrient + tet

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Agar Plate with nutrient

• First agar plate (10^-3) picture is missing
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Procedure II: Antibiotic Resistance

The observations of this experiment then concluded that tetracycline, an antibiotic, does affect bacterial growth. After observing the agar plates of the nutrient and nutrient+tetracycline, and seeing that the agar plates, even diluted, with just nutrient, were able to develop a larger growth of bacteria. For example, the agar plate with the 10^-3 nutrient had a lawn, which occurs when the bacterial growth forms colonies too numerous to count, while the agar plate with tetracycline had a noticeable difference in bacterial growth, where the 10^-3 agar plate of nutrient+tet had a countable number of colonies. Although bacteria was still able to grow on the agar plate that contained tetracycline, it did affect the total number of bacterial growth that could have occurred, had it not been for the tetracycline on the agar plates. Some bacteria are affected by the antibiotic, tetracycline, and others are not; for example, chlamydiae, is a type of bacteria that has been showing activity with tetracycline (Chopra and Roberts; 2001).

References: Chopra, I. and Roberts, M., "Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance". Microbiology and Molecular Biology Reviews. June 2001. 65: 232-260.

Procedure III: Bacteria Cell Morphology Observations

In order to observe the types of bacteria that have grown in the agar plates, slides were prepared and observed under a microscope at 40x and 100x using immersion oil at 100x. Two samples each from the nutrient agar plates and from the nutrient + tet agar plates were collected and observed. Within these samples, the types of bacteria observed were that of bacillus and coccus.

Gram Stain Procedure

First, in order to make a gram stain of a bacteria of the two samples, a loop must be sterilized in alcohol and the alcohol must be burned off with fire. Then the loop must gather a sample, which has been first observed and the observations of the colony collected, and mixed into a drop of water on a sterile glass slide. Then the water on the slide must be dried by passing it over the flame until the water dries off the slide. Next, the bacteria must be stained with crystal violet and must sit for a minute, then washed off with water. This procedure is repeated, except instead of using crystal violet, Gram's iodine is used. After using Gram's iodine, the slide must be de-colorized with 95% alcohol for ten to twenty seconds. Then the slide must be smeared with a safranin stain and then after 20-30 seconds, the stain must be rinsed off with water. The excess water is then blotted off with provided Kimwipes and set to dry. After these steps, the slide is then observed under 40x and 100x with immersion oil. These steps are repeated for the remaining bacterial slides.

Table 2: Bacteria Characterization

Colony Label	Plate Type	Colony Description	Cell Description	Gram + or Gram -
1N	10^-5 no tet	raised, golden yellow, undulate	budding	negative
2N	10^-7 no tet	filamentous, flat, white-ish	bacillus, streptobacilli	positive
1T	10^-5 tet	curied, convex, yellow, butter color	coccus, singular	negative
2T	10^-7 tet	umbonate, greenish black, round	streptobacilli, club rod, diplococci	positive

Procedure 4: Set up PCR for 16S sequencing

For the four bacteria samples, in order to isolate the DNA of the bacteria, the PCR is used to amplify the 16S gene. In order to do so, a single colony of bacteria from the selected agar plates must be collected and transferred to a 100μL of water in a sterile plastic tube. Then the plastic tube is incubated at 100°C for ten minutes in order to denature the template strands of DNA. After the ten minutes are over, the sample must centrifuged in 20 μL of a primer mixture for five minutes in order to separate the supernatant and the pellet. After the two separate, 5μL of the supernatant is transferred to a 16S PCR reaction and placed into the PCR machine. These steps are then repeated for the other three samples of the agar plates selected.

Hay Infusion Observation

• The smell is not as pungent as it previously was; however, the smell is still pretty awful. Smells like something rotting.
• Volume of the environment reduced by half. Half of the water in the jar has been dehydrated.
• The presence of mold is not as great as it was in the previous week.
• It seems as though everything had settled to the bottom of the jar.
• The remaining liquid felt like water through gloves, but at the same time, had a kind of slippery feeling to it.

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January 28, 2015

Procedure I: How to Use a Dichotomous Key

In this lab of "Identifying Algae and Protists", we observed and identified different types of protists by using a dichotomous key. This was done by making a wet mount of known protists. After doing so, I observed the wet mount under a microscope and identified a single organism using the dichotomous key. Using the dichotomous key, my lab partner and I made the observation that the known organism was a Paramecium multimicronucleatum. We were able to get to this conclusion because the organism was colorless, trumpet-shaped, a fast swimmer, had a cilia, and was about 250μm, which allowed us to narrow down the selection to the Paramecium multimicronucleatum. Another organism that was observed was an organism named by Colpidium, which was identified by its clear, colorless nature, small body, oval shape, and fast swimming, which was also between 50-60μm.

Procedure II: Hay Infusion Culture Observations

Within the hay infusion culture created in the last lab, an ecosystem developed. Within this ecosystem, I gathered a sample from the top layer, middle layer, and the bottom layer of the ecosystem, made wet mounts for each of the layers, and observed them under 4x, 10x, and 40x.

• http://instagram.com/p/yYa2ivAkW2/
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Top Layer Within the top layer, two types of organisms were observed and identified using the dichotomous key. 1. The first organism was colorless. Its body was covered in cilia, as well as being oval shaped (almost spherical). It was small, a fast swimmer, and it had a small mouth. It was about 52.5μm. The organism was identified to be a Colpidium

• http://instagram.com/p/yYUDx6gkca/

2. The second organism was long and stringy. The color was not clear; however, it did not clearly have any color. The organism was about 1100μm. The organism was identified as a Spirostomum.

• http://instagram.com/p/yYVl9Zgker/

Middle Layer Within the middle layer, two types of organisms were observed and identified using the dichotomous key. 1. This organism was found next to the pine needles, and was colorless and stick-like. This organism was approximately 1000μm and was identified to be a Spirostomum.

• http://instagram.com/p/yYWGRogkfl/

2. This organism was colorless and about 52.5μm. This organism was identified to be a Stentor.

• http://instagram.com/p/yYWvveAkQe/

Bottom Layer 1. This organism was black, long, and a stick-like figure. This organism was 550μm and was identified to be a Spirostomum.

• http://instagram.com/p/yYXRM3gkRL/

2. This organism was black, but basically colorless and was approximately 52.5μm. This organism was identified to be a Stentor.

• http://instagram.com/p/yYYIU7gkSj/

Observations: Within all three layers of the ecosystem of the hay infusion culture, it was observed that the Spirostomum was present.

Procedure III: Preparing and Plating Serial Dilutions

"Tube Dilution"

Tubes	100µL: 10mL
1	10^-2
2	10^-4
3	10^6
4	10^8

"Plates (Agar Nutrient with and without Tetracycline"

Nutrient	Tetracycline
1 - 10^3	1 - 10^3
2 - 10^5	2 - 10^5
3 - 10^7	3 - 10^7
4 - 10^9	4 - 10^9

This is great! Very well documented and you walked me through the steps very well. In addition, I knew exactly what you were looking for in the pictures with your descriptions. Keep up this excellent work! ML

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January 21, 2015

Today in lab, we studied the structure of the Volvocine line, which included the Chlamydomanonas cell, Gonium cell, and the Volvox cell. The characteristics of these cells were observed using an optical microscope to view the differences in these cells and noticed varying characteristics within the members of the Volvocine Line.

Procedure I: 'Observations'

Characteristics	Chlamydomonas	Gonium	Volvox
Number of Cells	1	32	too many to count
Colony Size (µm)	2.5 µm	50 µm	340 µm
Specialization of Cells	no specialty	beginning to specialize	specialization beginning
Mechanisms of Motility	flagellum, very fast	driven by water current	not motive
Isogamous or Oogamous	isogamous	oogamous	oogamous

Procedure II: Observing a Niche at AU

The transect that my group was assigned was transect 4, which is a controlled area (a community vegetable garden). In this transect, samples of soil and leaves of different areas of the transect were collected into a sterile 50 mL tube. In this environment, both abiotic and biotic factors were observed.

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Abiotic	Biotic
snow	brussel sprouts
rocks	clover
plastic irrigation sprinkler	wood dome
plastic label	pine needles
paint	bushes

After these observations, back in the lab, my group made a Hay Infusion Culture by placing 10-12 grams of the collected samples into a jar, pouring in 50 mL of water into the jar with the samples, and placing in 0.2 grams of dried milk for food. The jar was then shaken slightly and left for a week with the lid removed.

Great job! Thanks for inserting the links to your pictures and for including the information about the volvocine line. Very complete. ML

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January 14, 2015

I'm just trying to get a hang of this. I hope it works.