User:Marshal G. MacCartney/Notebook/Biology 210 at AU

Zebrafish Development: Effects of Rhodamine 6-G within embryo and larva development, 2015-02-18 through 2015-03-04

This experiment was done to observe, identify, and record the effects of Rhodamine 6-G (R 6-G) on the growth and development of Zebrafish embryos and larvae. There has been no documentation of the effects of this experiment, of the complete exposure of Rhodamine 6-G on freshwater fish, so it is being used as a baseline for the lab and for the directors of the lab. Furthermore, after doing moderate research of this chemical, it was hypothesized that the addition of R 6-G to water will have adverse effects on vertebrate development. If Zebrafish embryos and larvae are exposed to Rhodamine 6-G then their embryological development will be stunted in comparison to a control group of distilled water.

Set up control group environment with 20 mLs of distilled Deerpark water, then placed 20 healthy translucent embryos within the petri dish. For the variable control group three different petri dishes were made. The first was made by taking 200 μL of R 6-G and 18.8 mLs of Deerpark distilled water and 20 healthy translucent embryos all being combined in the petri dish together the same day as the control group. The second was made exactly like the first except only 11 embryos were added to the petri dish and it was made three days after the experiment was started. This was due to an unforeseen mishap that resulted in the death of 11 embryos in the first test dish that was caused by protists attaching and eating "bad eggs". These eggs were selected by the lab group but were faulty eggs before selection and should not be weighted when viewing results. The last test group was created on day 13 of the experiment by taking four of the larva 2 from each of the experiment groups and adding it to a petri dish of pure Rhodamine 6-G. On day 4-5 the water in the control and R 6-G/water in the first two experiment dishes were changed by removing 10 mLs and adding 25 mLs of water or R 6-G/water respectively. All dead eggs and larva were removed when found and identified. Day 7 removed 5 mLs of liquid or each petri dish and added mLs of the proper liquid to each dish (Deerpark water to control and Rhodamine 6-G/water mixture to both experiment groups). One drop of paramecium was added to each of the three petri dishes. The liquid changing process mirrored that of day 7 for the rest of the experiment. Observations on heart rate, overall development, eye size, movement v.s. control, dead/alive, total length, fin development, and tail length were all recorded throughout the experiment process.

Day 3

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	1 living	N/A	N/A	N/A	N/A	N/A	N/A
	19 dead
Test	9 living embryos, all in 20 somite stage	N/A	N/A	N/A	N/A	N/A	N/A
	11 dead

Day 6

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	20 living	325 µm	250 µm	50 µm	No visible fin development	144 beats per minute	Darker coloration
	15 hatched						Quick startle time
	5 in 20 somite stage
Test 1	8 living, all hatched	362.5 µm	262.5 µm	75 µm	Visible beginnings of pectoral fin development	120 beats per minute	Able to rotate eyes
	1 dead						Spin in rapid, stationary circles
							Lighter coloration
Test 2	11 living	325 µm	250 µm	25 µm	No visible fin development	132 beats per minute	Darker coloration
	9 hatched						Quick startle time
	2 in 20 smite stage

Day 8

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	19 living, all hatched	3,500 µm = 3.5 mm	2750 µm = 2.75 mm	150 µm	Visible pectoral fin development	Not visible	Darker coloration
					Beginning dorsal fin development		Quick startle time
					Fins more developed than in tests		Visible gills
Test 1	7 living, all hatched	2875 µm = 2.875 mm	2500µm = 2.5 mm	175 µm	Visible pectoral fin development	Not visible	Jerks around non-stop with thrashing tail
					Noticeably underdeveloped, smaller than control		Swims into walls of Petri dish
Test 2	11 living, all hatched	4250 µm = 4.25 mm	3500 µm = 3.5 mm	375 µm	Visible pectoral fin development	Not visible	Jerks around non-stop with thrashing tail
					Noticeably underdeveloped, smaller than control		Swims into walls of Petri dish

Day 10

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	20 living, all hatched	3,500 µm = 3.5 mm	2250 µm = 2.25 mm	325 µm	Visible pectoral fin development	144 beats per minute	Darker coloration
							Quick startle time
							Visible gills
Test 1	8 living, all hatched	3250 µm = 3.25 mm	2750 µm = 2.75 mm	175 µm	Visible pectoral fin development	Not visible	Moves more quickly
	1 dead						Jerks around non-stop with thrashing tail
							Appears Rhodamine may be in the blood stream
Test 2	11 living, all hatched	3375 µm = 3.375 mm	2500 µm = 2.5 mm	200 µm	Visible pectoral fin development	222 beats per minute	Moves more quickly
							Jerks around non-stop with thrashing tail
							Appears Rhodamine may be in the blood stream

Day 13

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	19 living, all hatched	4375 µm = 4.375 mm	3000 µm = 3 mm	625 µm	Visible pectoral and dorsal fin development	Not visible	Swim away from pipette
					Fins more developed than in tests		Eyes more developed, can differentiate white of eye and eyeball
					Developed tail fin		Visible gills, flattened head sides
Test 1	6 living, all hatched	4375 µm = 4.375 mm	3100 µm = 3.1 mm	250 µm	Visible pectoral fin development	Not visible	Not a lot of movement
	1 dead				Beginning dorsal fin development		Sudden spasms with tail thrashing
					Developed tail fin		Swims into walls of Petri dish
					Noticeably underdeveloped, smaller than control		Crooked tail development
Test 2	11 living, all hatched	4000 µm = 4 mm	3000 µm = 3 mm	300 µm	Visible pectoral fin development	Not visible	Sudden spasms with tail thrashing
					Beginning dorsal fin development		Swims into walls of Petri dish
					Beginning tail fin development		Crooked tail development
					Noticeably underdeveloped, smaller than control		Very light colored, almost transparent

Day 15

'	Number Living/Dead:	Length:	Tail Length:	Eye Diameter:	Fin Development:	Heartbeat:	Other Observations:
Control	19 living, all hatched	3635 µm = 3.625 mm	2500 µm = 2.5 mm	275 µm	Visible pectoral, dorsal, and tail fin development	Not visible	Fins held close to body
					Fins more developed than in tests
Test 1	All 6 dead	4000 µm = 4 mm	3125µm = 3.125 mm	275 µm	Pectoral, dorsal, and tail fins are deteriorating/crumpled	Not visible	Protists still alive
							Stuff coming out of eyes
							Crooked tail
Test 2	10 living, all hatched	3875 µm = 3.875 mm	2625 µm = 2.625 mm	300 µm	Visible pectoral, dorsal, and tail fin development	Not visible	Jittery movement
	1 dead				Noticeably underdeveloped, smaller than control, fins appear crumpled/deformed		Fins held close to body
							Crooked tail
Test 3	All 4 dead	N/A – too deformed to measure	N/A – too deformed to measure	N/A – too deformed to measure	Crumpled/deformed completely	Not visible	Dyed a vivid pink color
					Not possible to identify what are fins		Eyes different sizes
							Crooked tails

In order to observe, record, and obtain information on the chemical, Rhodamine 6-G, effects on freshwater fish embryos and larvae development, Zebrafish were used as a test subject and a two week trial was composed. When looking at the results it is clear that Rhodamine 6-G in diluted amounts cause developmental issues in the larvae, not initially but when exposure time is around 13 days signs of developmental stress can be observed from physical traits. Furthermore, when the Zebrafish were exposed to pure Rhodamine 6-G with no other source of liquid in the petri dish like in Test 3 the larvae did not last longer than two days, which proves that fish this size cannot survive on Rhodamine 6-G alone. There has been no other studies or research performed that could be found on complete fish exposure to this chemical. If another experiment were to be performed the use on multiple dilution levels with Rhodamine 6-G should be view first. Once the level of toxicity is established then another experiment should be performed to test which stage in development for freshwater fish is the most safe to expose Rhodamine 6-G to their environment.

Vertebrate Analysis and Food Web from Group 4 Transect, 2015-02-11

16S Sequence Analysis from Group 4 Transect, results received 2015-02-18

Observe, Identify, and Understand Invertebrates from Group 4 Transect, 2015-02-11

This study was performed in order to comprehend the importance of invertebrates, and to learn the evolution between simple systems to more advanced systems. In addition, invertebrates were to be observed and recorded that were specifically found within the transect. From the purpose of this study it can be acknowledged that there will be an abundance and variety of life that will be present within this niche.

The Berlese Funnel was set up with a plastic funnel full of transect leaf litter attached to a conical tube containing ethanol, a light was placed above the contraption and was left to have all invertebrates to fall into the conical tube. It was taken down after one week of being set up and the contents within the conical tube were poured into two separate petri dishes. The dishes were examined using a dissecting microscope and the different invertebrates were observed, identified, and recorded.

All observations were recorded in the table below:

Organism	Length in mm	Number in sample	Description of Organism	Location
Thros	~2mm	3	winged, antenna	top
Soilmite	~1mm	1	antenna, tail-like structure, dark color	top
Springtail	~1mm	6	8 legs, brownish	top
Termite	~1.5	5	large lower body, antenna, 6 legs, tanish	bottom

                                                (Figure 1: Burlese Funnel set-up)

The movement of a Nematode (roundworm) is through pseudocoelom, a planaria moves by using acoelomate, and plathelminthes uses acoelomate as well to move. In regards to the observed invertebrates within the transect Burlese Funnel, the size of the organisms ranged anywhere from about 3mm to about 1 mm. The largest of the organisms was the Thros and the smallest of the invertebrates was a tie between soilmites and springtails. The most common invertebrate within the leaf litter were soilmites. The data solidly supports the hypothesis showing a large number of different invertebrates found within the leaf litter representing the transect as whole. If repeated, the time of the collection of leaf litter should be conducted when the temperatures are warm and with no snow or ice on the ground because that is when this type of ecosystem is thriving and most abundant of plants and animals.

Observe, Identify, and Understand Plantae and Fungi from Group 4 Transect, 2015-02-04

This study was performed to view plant and fungi life growing or dead within the transect area. In regards to plants, it was necessary to observe the differences of characteristics that make plants so varied. In regards to fungi, it was necessary to acknowledge the function and understand the importance this group has on ecosystems and life in general. From the purpose of the study it can be expressed that there will be a variety of diverse plants and fungi taken from transect group 4 due to its location within a community garden.

Obtained three Ziplock bags. Took samples of leaf litter, plant matter and soil placed into one bag. Took representative samples of five different plants (Brussels sprout, Shelf mushroom, weed, kale leaf, and clover) without damaging the plant too much and placed into a second bag. Took sample of dormant shrubs, scattered pine needles and put into the third bag. Returned to lab. Observed all of the five samples and filled out chart provided by the lab manual. Set up a Berlese funnel with the remaining leaf litter.

Transect Sample Plants	Location and # in transect	Description (size and shape)	Vascularization	Specialized Stuctures	Mechanisms of Reproduction
#1	Northwest, Brussels sprout vegetation box	Brussels srpout, green/brown, ~3.0 cm in diameter, round w/ short stem	Vascular xylem and phloem	Oravy, ovule	Angiosperm (seeds)
#2	Northeast, on the side of the lettuce vegetation box	Shelf mushroom, orange/brown, ~3 cm in diameter, half circle shape	Hyphae	Rhizoid, basidomycota	sexual basido spores
#3	Inbetween the northwestern vegetation boxes	Weed, tan/brown, ~7 cm long, ~0.75 cm long needles,	Stem and needles, xylem and phloem	No stomata, needle like structures	spores on needles
#4	Southwest, Kale vegetstion box	Kale leaf, green/leafy/ tough, ~6 cm-by-~3 cm	Dicot, bundles in rings	Guard cells, Cuticle, stomata, cell walls, chlorophyll	Angiosperm (seeds)
#5	Inbetween the northwestern vegetation boxes	Clover/weed, green/flimsy, long/stringy, ~19 cm long from root to tip	xylem and phloem, long roots	Guard cells, stomata, chlorophyll	Angiosperm (seeds)

                                                         (Figure 1: Brussels Sprout)

                                                         (Figure 2: Shelf Mushroom)

                                                         (Figure 3: Weed)

                                                         (Figure 4: Kale Leaf)

                                                         (Figure 5: 3 Leaf Clover)

The leaves taken from the transect were most brownish/grey and were dead. The leaves came from the two living plants within the transect, Brussels sprout and Kale. The pine needles collected were most likely blown into the transect from the surrounding area. There were not tangible seeds that could be brought back to the lab. However, the Brussels sprout plant is an angiosperm that could have flowers when in blooming season. Furthermore, spores were identified on plant number 3 which was examined under a microscope to prove that spores were present. Fungi sporangia are small round structures that grow upward. They are important because they contain cells used for fungus reproduction called spores. The proposed hypothesis can be confirmed because there was a variety of plants that could be collected from the transect; however, in regards to fungi only one sample could be found thus providing evidence that there is not an abundance of difference types of fungi.

-M.M.

Microbiology: Identifying Bacteria from Group 4 Culture Plates, 2015-01-28

This experiment was performed to understand the basic differences in bacteria, view bacteria that are resistant to antibiotics and understand the procedures and reasons to due so. When looking at the basic differences it consists of classifying the bacteria according to cell morphology which entails size, shape and other various physical qualities. The bacteria can be categorized into six prokaryotic sub categories that are depend upon morphology as well nutritional requirements and lastly their DNA. It is proposed, if there are resistant stands of bacteria within the Hay Infusion Culture that were grown on the plates then, there will be less colonies formed than on the plates with out the antibiotics.

The Hay Infusion Culture was inspected and observed. Both sets of culture plates were brought to the table and separated according to whether they contained tetracycline or not. A rough count was taken for all of the colonies grown on each plate, the number was recorded. Samples were taken from both normal nutrient agar plates and the plates with nutrient agar + tetracycline from the 10^3 and 10^9 plates and made into wet mounts there were viewed and studied to determine morphology. The use of the 100X lens with oil was needed in order to observe the samples. Furthermore, four gran stain slides two from the nutrient agar plates and two from the nutrient agar + tetracycline plates from the 10^3 and 10^9 plates. Went through the gram staining procedure, found on page 30 of the lab manual. Observed the stained slides and recorded all observations.

All data observed was recorded and formatted in two separate tables

(Table 1: 100-fold Serial Dilutions Results)

Dilution	Agar Type	Colonies Counted ~	Conversion Factor	Colonies/mL
10^-3	nutrient	500	x10^3	5,000,000
10^-5	nutrient	300	x10^5	300,000,000
10^-7	nutrient	400	x10^7	40,000,000,000
10^-9	nutrient	67	x10^9	670,000,000,000
10^-3	nutrient + tet	175	x10^3	1,750,000
10^-5	nutrient + tet	292	x10^5	292,0,000
10^-7	nutrient + tet	264	x10^7	26,400,000,000
10^-9	nutrient + tet	30	x10^9	300,000,000,000

(Table 2: Bacteria Characterization)

Colony Label	Plate Type	Colony Description	Cell Description	Gram + or Gram -
10^-3	nutrient agar	2mm diameter, irregular shape, opaque white, raised, powdery	No motion, shape- rod(single), arrangement- individual	Negative
10^-9	nurtient agar	4mm diameter, circular shape, orange, raised, smooth/glistening	No motion, shape- rod(chain), arrangement- individual	Positive
10^-3	nutrient agar + tet	4mm diameter, circular shape, yellow/orange, raised, smooth/glistening	No motion, shape- rod(single), arrangement- individual	Positive
10^-9	nutrient agar + tet	4mm diameter, circular shape, yellow/orange, raised, smooth/glistening	No motion, shape- rod(chain), arrangement- rows	Negative

I do not believe there are any Archaea species bacteria in the samples due to the rather un-extreme the environment the transect location is in. When observing the Hay Infusion Culture the smell is about the same is foulness but there is a lot more decay and scum growth. These changes may be due to the microorganisms living in the culture. If the culture is left untouched then, the organisms living in it will continue to live and go though their life processes which will lead to the complete decay of most objects which will increase the foulness of the smell. Based on the data recorded and observed with the plates, the wet mount slides and the gram stain slides it is inferred that there are antibiotic-resistant bacteria that has grown on the nutrient agar + tetracycline plates and is alive and thriving. When looking just at the plates there is no apparent difference with the majority of the colonies in both sets of plates. This indicates that the bacteria on the plates are roughly the same type however, there is just a mutation within the population that allowed for growth on the plates with the tetracycline. There is about half the amount of bacteria in the agar + tetracycline plates when comparing them to the agar plates. There are quite a few different species that seem to be unaffected by the tetracycline but the exact number is unknown. Tetracycline works by inhibiting the production of proteins. Specifically, it does not allow transfer-RNA from binding to the (A) site on a ribosome. It affects both gram positive and gram negative bacteria, parasitic protozoans, chlamydiae, rickettsiae, and some mycroplasams. (Chopra, 2001). The hypothesis discussed in the beginning of the notebook was proven true, there are less colonies of bacteria on the tet. plates than the normal agar plates.

Chopra, Ian, and Marilyn Roberts. "Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance." Microbiology and Molecular Biology Reviews. American Society for Microbiology, June 2001. Web. 03 Feb. 2015.

-M.M.

Identifying Protists and Algae from the Group 4 Hay Infusion Culture, 2015-01-21

Within this Hay Infusion Culture the 500 mLs of water in the jaw acts as its own ecosystem. Inside this small, simple ecosystem are different niches that organisms can live in. These organisms can vary upon location in the jar as well as depths all depending on their life necessities and which area they are best adapted for. In this lab protists and algae will try to be observed as well as any other organisms that are competing for the same resources as well as utilizing all biotic and abiotic factors. With quite a bit of diversity within the culture it is predicted that there will be organisms that are different from the two sample depths that were taken due to competition.

The culture was brought to the lab table without disturbing or mixing its contents. The smell was observed as well as its appearance, all observations were recorded and documented. Two samples were taken from the culture, one from the top of the waters surface and the second from the bottom mucky area. Wet mounts were made from the separate niches and put under a compound microscope to observe what organisms were present using a dichotomous key to help separate the observations. The organisms observed were measured, recorded and drawn.

The initial observations are as followed: smell- bad, foul, moist, decaying; appearance- murky, bottom layer is soft, wet, lose soil, top (surface)layer filled with scum or mold; middle(water) layer is brownish in color and not transparent; objects in suspension- pine needle, shriveled Brussels sprout, leaf speckled with white mold. The first sample was taken from on top of the floating decomposing leaf, and the second was taken from the bottom amidst the soil and muck in the center of the jar. Organisms might differ from there locations because near the leaf there may be decomposers or organisms that feed on decomposers and near the bottom where the human eye can only see wet soil there may be nutrients or a food source for a different type of organism to survive. After observing the wet mounts six organisms were studied all of which were protozoans. From the bottom sample wet mount a motile- Paramecium Aurelia ~130μm, a non-motile- Gonium colony ~90μm in diameter and a motile- Colpidium ~60μm were all observed and recorded. From the top sample wet mount a motile- Pelomyxa ~2mm, a motile- Paramecium ~140μm, and a non-motile- Pandorina colony ~212μm were all observed and recorded. According to page two of the Freeman text the Paramecium Aurelia found from the bottom layer wet mount meets all the needs of life. In the energy department a Paramecium engulfs smaller organisms to obtain the necessary energy to survive. A Paramecium is a unicellular organism that is protected by a cell membrane. Paramecium Aurelia has genetic info and can horizontally transfer some of its DNA to other members of its species. In regards to replication it can sexually or asexually reproduce and is the result of evolution.

                          (Figure 1; Paramecium Aurelia found on both the top and bottom Hay Infusion samples)

                          (Figure 2: Gonium colony found on the bottom layer of the Hay Infusion {LEFT})
                          (Figure 3: Pelomyxa found on the top layer of the Hay Infusion {RIGHT})

                          (Figure 4: Colpidium found on the bottom layer of the Hay Infusion)

                          (Figure 5: Pandorina colony found on the top layer of the Hay Infusion)

This Hay Infusion Culture seems to not have been contaminated to the best of knowledge and is thriving with various types of life at all depths. The data collected supports that there are different organisms at various depths but what is also true is that there is the same type of organism that can survive at both the surface and the bottom and that is the Paramecium. It was quite difficult to find and identify the organisms and if replicated it may be best to use the "slowmo" oil. Furthermore more than one wet mount was made for each layer in order to find and identify the organisms. It would be interesting to see if the Hay Infusion Culture could survive on its own for two months. In my opinion I think it would survive but the quality and the amount of life would significantly decrease. Furthermore, carrying capacity in regards to resources available would be a huge selective pressure that would kill off portions of the population within each niche and could affect the overall ecosystem that is the Hay Infusion Culture.

-M.M.

American University Transect: Group 4, 2015-01-14

The amount of organisms through out the world keep increasing due to new organisms being discovered everyday. These organisms can live in a variety of places. At American University it will be observed what organisms are present in the soil all across campus. Due to the location where this sample was taken it is predicted that there will be a decent amount of life such as bacteria, protists, plants, and animals living together in this one area.

Went to the group 4 transect site to examine the biotic and abiotic factors of the niche. A 20meter-by-20meter square was observed and from that the soil samples were taken from all over the site and combined in a 50 mL sterile conical tube. Returned to the lab with the soil sample and made Hay Infusion Culture. Comprised of 11 grams of the soil sample, 500 mLs of deer park water, 0.1 grams of dried milk in a plastic jar. Mixed ingredients for 10 seconds and left out in the lab with the lid removed.

The transect where group 4 took the soil samples was northwest of the soccer field in a campus garden that is gated off from the rest of the of the normal grass by a 10 foot high plastic gate. Inside the transect area there are four separate wooden boxes that vegetation could be grown in. At the time Brussels sprouts and Kale were being grown in two separate boxes of the four that were there. Soil samples were taken from each of the boxes as well as from two other sections of the transect outside the boxes. In addition to the soil that was collected rabbit feces, a Brussels sprout, Kale leaf, snow, a pine needle, a dead leaf and grass was included. All things went normal when making the Hay Infusion Culture. Abiotic factors include: direct sunlight, frozen soil, frost/ice on plants, four wooden vegetation boxes, plastic fencing, plastic irrigation hoses. Biotic factors include: grass, decomposing plant matter, animal traffic(feces), kale, Brussels sprouts, clover.

This lab was merely the process of obtaining the soil sample and setting up the Hay Infusion Culture. From just inspecting it with the naked eye there is a lot of diversity in the soil sample and should provide a positive environment to allow living organisms to survive.

-M.M.

1/28/2015 Figured out -M.M.