User:Zachary Thorogood/Notebook/Biology 210 at AU

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Lab 6.

Objectives:

  • Learn the stages of embryonic development.
  • Compare embryonic development in different organisms.
  • Set up an experiment to study how environmental conditions affect embryonic development.

Procedure I: Starfish (Asterias) Development:

  1. Observe Several Factors:
    • Relative Egg Size.
    • Amount of Yolk.
    • Distribution of Yolk.
    • Blastulation.
    • Gastrulation.
    • Larval Stage.
    • Environment of Development.
    • Fertilisation.
    • Mechanism of Waste Disposal.
    • Physical Protection.
    • Parental Care.
  1. Record results.

Procedure II: Frog Development:

  1. Observe Several Factors:
    • Relative Egg Size.
    • Amount of Yolk.
    • Distribution of Yolk.
    • Blastulation.
    • Gastrulation.
    • Larval Stage.
    • Environment of Development.
    • Fertilisation.
    • Mechanism of Waste Disposal.
    • Physical Protection.
    • Parental Care.
  1. Record results.

Procedure III: Chick Development:

  1. Observe Several Factors:
    • Relative Egg Size.
    • Amount of Yolk.
    • Distribution of Yolk.
    • Blastulation.
    • Gastrulation.
    • Larval Stage.
    • Environment of Development.
    • Fertilisation.
    • Mechanism of Waste Disposal.
    • Physical Protection.
    • Parental Care.
  1. Record results.


Table I: Comparison of Embryological Features of Developing Starfish, Frog, and Chick.
Feature Starfish Frog Chick
Relative Egg Size Small. Larger. Very Large.
Amount of Yolk Small Amount. More Yolk. Large Amount.
Distribution of Yolk Isolecithal. Telolecithal with animal and vegetal poles. Meroblastic cleavage because of so much yolk.
Blastulation Holoblasatic division to morula stage and then blastula. Holoblastic but uneven division and asymmetrical blastula with two poles. Meroblastic division to form Blastodisc; develops over the yolk.
Gastrulation Develops ectoderm, endoderm, and mesoderm. 3 germ layers with blastopore formation and then neural tube. 3 germ layers develop from blastodisc to primitive streak.
Larval Stage Organogenesis to form motile bipinnaria. Organogenesis to motile tadpoles. Organogenesis to nonmotile embryo.


Table II: Comparison of Ecological Aspects in Sea Stars, Frogs, and Chicks Development.
Feature Starfish Frog Chick
Environment of Development Water. Water. Egg.
Fertilisation External. External. Internal before shell forms.
Mechanism of Waste Disposal Water Environment. Water Environment. Allantois.
Physical Protection Motile. Motile. Egg shell and membranes.
Parental Care None. None. Mother keeps eggs warm.

Procedure IV: Zebrafish Experiment Procedure:

  1. After reading and analysing a published paper about the effect on Zebrafish development - create a hypothesis, prediction, and experimental plan.
  2. Observe the Zebrafish embryos carefully and determine their developmental stage.
  3. Set up the control group and the test group in covered in petri dishes.
  4. Use 20mLs of Deerpark Water and 20 healthy translucent embryos per dish. Use a dropper to transfer the eggs to the dishes with the appropriate water. If another variable is being tested, then every time the water is change that variable must also be included.
  5. Organise the observation schedule and procedure.
  6. When embryos are 4 - 5 days old, remove 10mLs of water with any empty egg cases and add 25mLs fresh water.
  7. Save any dead embryos in paraformaldehyde that will be provided.
  8. One week after the experiment has begun, remove 5mLs of water with any egg cases and add 5mLs of fresh water or test solution. Preserve 1 - 3 embryos from the control and the experimental groups in paraformaldehyde.
  9. Between 1 week and 2 weeks incubation time, remove 5mLs of water and add 10mLs of water.
  10. Feed two drops of paramecium starting after one week.
  11. Make final observations and measurements at two weeks.
Table III: Zebrafish Larvae Observation Results
Day Control 0.03% Salt Solution 0.3% Salt Solution 3% Salt Solution
Monday, 24th Feb. 2014 19 - 21 Alive; 2 - 3 Hatched + Dead. 1 - 2 Hatched + Dead.
Tuesday, 25th Feb. 2014 4 Unhatched + Dead; 5 Hatched + Dead; 38 - 40 Hatched + Alive. 2 Unhatched + Dead; 4 Hatched + Dead; 20 - 21 Hatched + Alive. 1 Unhatched + Dead; 1 - 2 Hatched+Dead; 28 - 30 Hatched + Alive. 25 Hatched + Dead.
Thursday, 27th Feb. 2014 30 - 40 Hatched + Alive. 25 - 30 Hatched + Alive. 8 Hatched + Dead; 18 Hatched + Alive. 23 Dead.
Tuesday, 4th March. 2014 3 Dead. 8 Dead. All Dead. All Dead.

Additional Notes:

  • On Tuesday, 25th Feb. 2014 - New test subjects were used since past subjects died on Monday, 24th Feb. 2014 due to high salt solution concentrations. New test subjects were previously fertilised on Wednesday, 19th Feb. 2014 and placed into testing conditions on Friday, 21st Feb. 2014.
  • As the experiment continued, movement of Zebrafish decreased dramatically to most of them being unable to move unless gently shaken.



Lab 5.

Objectives:

  • To understand the importance of Invertebrates.
  • To learn how simple systems (including specialised cells and overall body plan) evolved into more complex systems.

Procedure I: Observing Acoelomates, Pseudocoelomates, and Coelomates:

  1. Observe the acoelomate, Planaria, with the dissecting scope and a cross sectional slide of Planaria.
  2. Record your results.
  3. Observe the nematodes and a cross sectional slide of their pseudocoelomate structure.
  4. Record your results.
  5. Observe the coelomate, Annelida.
  6. Record your results.
  7. Describe the movements of the three types of worms and how the movement relates to their body structure.

Acoelomate – Planaria:

  • It moves by using a film of mucus to glide over surfaces.
  • Tripoblast – lacks a body cavity, with a single-opening digestive tract.

Pseudocoelomate – Nematodes:

  • It moves by a process of muscle contractions.
  • Smooth, unsegmented bodies, lacks cilia.

Coelomate – Annelida:

  • It moves by using peristalsis – or creating ripples that pass along the body.
  • Tripoblast – segmented bodies.

Procedure II: Analyzing the Invertebrates Collected with the Berlese Funnel:

  1. Break down the Berlese setupd and transfer the preservative solution to a Petri dish.
  2. Examine the contents under a dissecting microscope.
  3. Try to identify the invertebrates by using a dichotomous key.
  4. Record your results.

Conclusions:

Invertebrates Discovered: There was three arthropods found - none of the invertebrates had wings, rounded bodies, and several pairs of legs. None of the arthropods could be classified with true certainty, but two of the invertebrates looked closely like fleas or lice.

Table IV: Arthropod Identification Results
Feature Number of Wings Number of Antennae Number of Legs Number of Body Segments Class
Arthropod 1 2 Wings. 2 Antennae. 6 Legs. 3 Body Segments. Insect.
Arthropod 2 No Wings. 2 Antennae. Many Legs. Many Body Segments. Centipede.
Arthropod 3 No wings. 2 Antennae. 10 Legs. One Body Segment. Crustacean.
Arthropod 4 No Wings. No Antennae. 8 Legs. 2 Body Segments. Arachnid.
Arthropod 5 No wings. 2 Antennae. Many Legs. Many Body Segments. Millipede.



Lab 4.

Objectives:

  • To understand the characteristics and diversity of plants.
  • To appreciate the function and importance of fungi.

Procedure I: Collecting Five Plant Samples From The Transect:

  1. Bring three bags and proceed to the transect.
  2. Obtain 500g of leaf litter sample from a site at the transect - leaf litter samples include the crumbly top layer of the soil and plan matter above the soil.
  3. Obtain five representative samples from five plants. Try to aim for the greatest diversity.
  4. Record the data.

Procedure II: Plant Vascularzation:

  1. Obtain a sample of moss, Mnium, and compare the sample to the stem of the angiosperm, lily.
  2. Examine the cross section slide of the lily stem. FInd the xylem and phloem layers.

Procedure III: Plant Specialization:

  1. Examine the leaves of the moss and try to identify specialised appendages.

Procedure IV: Plant Reproduction:

  1. Examine the moss, Polytrichum, and try to identify the male and female gametophytes and the sporophyte.

Procedure V: Observing Fungi:

  1. Obtain a sample of the fungi, Rhizopus stolonifer, and observe under a microscope.

Procedure VI: Setting Up the Berlese Funnel to Collect Invertebrates:

  1. Pour 25mL of the 50:50 ethanol:water solution into the flask of or bottle.
  2. Fit a piece of the screening material into the bottom of the funnel.
  3. Place the funnel into the neck of the square-sided bottle.
  4. Carefully put the leaf litter sample in the top of the funnel.
  5. 5. Place a lighted 40 watt lamp above the funnel with the bulb about 1-2 inches from the top of the leaf litter.
  6. Leave the lighted setup on the lab bench for one week.



Lab 3.

Objectives:

  • To understand the characteristics of bacteria.
  • To observe antibiotic resistances.
  • To understand how DNA sequences are used to identify species.

Procedure I: Quantifying and Observing Microorganisms:

  1. Make one more check on your Hay Infusion Culture and describe any changes.
  2. Count the total number of colonies on a plate.
  3. Record the data.

Procedure II: Antibiotic Resistance:

  1. Observe the difference between the nutrient plate vs. the nutrient + tetracycline.
  2. Record the data.

Procedure III: Bacteria Cell Morphology Observations:

  1. Choose four colonies, out of the eight, that best represent bacterial growth and microorganisms.
  2. Try and get two colonies from the nutrient agar and two from the nutrient + tetracycline plate.
  3. Prepare a wet mount and gram stain for each colony.
  4. Record the data.

Procedure IV: Stat PCR Preparation for DNA Sequence Identification:

  1. Select one plate from the two colonies that has the best characterisation.
  2. Isolate DNA from bacteria in the colonies and use two primer sequences (27F and 519R) to amplify the 16S rRNA gene.
  3. Transfer a single colony of bacteria to 100ul of water in a sterile tube.
  4. Incubate at 100C for ten minutes and centrifuge.
  5. Use 5ul of the supernatant in the PCR reaction.


Data:

Hay Infusion - Lots of evaporation occurred. Plant matter sunk to the bottom of the jar and the water has turned to mirky/amberish colour.

Table I: Bacteria Cell Morphology Observations.
Colony Nutrient with/without Tetracycline Colony Description Cell Description Gram Positive or Negative
2 Nutrient Mainly small sizes clumped together to form a spread out form. Variation in colour from dull orange to white/grey. Small rod and circular cells, motile. Some Bacilli arrangement. One clear Staphyloccocus. NEGATIVE
4 Nutrient Blocks of colours mainly white in colour that form longer, but lesser farms. Size ranges from small to large colonies. Small "black" circular cells, slow moving, difficult to make definitive conclusions on arrangement of cells. NEGATIVE
T3 Nutrient + Tetracycline Bright orange colonies, mainly medium sized cells, some fungi growth present. Small "black" circular cells, slow moving, difficult to make definitive conclusions on arrangement of cells. POSITIVE


Table II: 100-fold Serial Dilutions Results.
Dilution Agar Colonies Counted Conversion Factor Colonies/mL
10-3 Nutrient 300 x10^3 300000
10-5 Nutrient 50 x10^5 5000000
10-7 Nutrient 6 x10^7 60000000
10-9 Nutrient 1 x10^9 1000000000
10-3 Nutrient + Tet 90 x10^3 90000
10-5 Nutrient + Tet 0 x10^5 0
10-7 Nutrient + Tet 0 x10^7 0



Lab 2.

Objectives:

  • To understand how to use a dichotomous key.
  • To understand the characteristics of Algae and Protists.

Procedure:

  1. Obtain a dichotomous key and make a wet mount from a sample of random organisms.
  2. Add Protoslo to increase chances of finding an organism.
  3. Record observations.
  4. Using the hay infusion, that was created from the previous lab, take random samples from varying points to see which organisms inhabit the hay infusion.
  5. After gathering the sample, create a wet mount and add Protoslo to increase chances of finding any organisms.
  6. Record observations.
  7. Obtain four 10ml test tubes that contain sterile broth and label them 2,4,6,8.
  8. Obtain eight agar plates - four nutrient agar plates and four agar + tetracycline plates.
  9. Mix the hay infusion carefully, yet enough to create an adequate mixture.
  10. Obtain a micropipette that is set to100 microlitres.
  11. Take 100 microlitres of the mixed hay infusion and place into the 10ml test tube labelled 2.
  12. Take 100 microlitres of the the mixture in test tube 2 and place into test tube 4.
  13. Take 100 microlitres of the the mixture in test tube 4 and place into test tube 6.
  14. Take 100 microlitres of the the mixture in test tube 6 and place into test tube 8.
  15. Take 100 microlitres of the the mixture in test tube 2 and place into both agar plates.
  16. Take 100 microlitres of the the mixture in test tube 4 and place into both agar plates.
  17. Take 100 microlitres of the the mixture in test tube 6 and place into both agar plates.
  18. Take 100 microlitres of the the mixture in test tube 8 and place into both agar plates.

Data:

Organisms with Dichotomous Key: Very few organisms were found and those that were we could not definitively classify.

Hay Infusion Observations: Water is amber brown with plant and soil matter primarily located at the bottom of the infusion.

  • Niche 1 in Hay Infusion: Plant and soil matter located at the bottom.
  • Niche 2 in Hay Infusion: Plant and soil matter located in the middle/top.



Lab 1.

Objectives:

  • To understand natural selection.
  • To understand the biotic and abiotic characteristics of a niche.

Procedure 1: The Volvicine Line.

  1. Prepare a slide of living Chlamydomonas and examine it microscopically, adding protoslo will slow motile creatures down and make them easier to observe.
  2. Observe the living culture of Gonium on a slide. It is a progressively more complex species consisting of a colony of 4, 8, 16, or 32 cells. They are held together in a gelatinous matrix and each cell can form a new colony.
  3. Observe the living culture of Volvox on a slide. It represents the peak of evolutionary complexity in this line of green algae consisting of thousands of spiked cells making up a spherical Volvox colony.

Procedure 2: Defining a Niche at AU.

  1. Set the 20x20 feet dimensions of the transect (either 1, 2, 3, 4, or 5) with four popsicle sticks.
  2. Describe the general characteristics of the transect: Location, Topography, etc.
  3. List the abiotic and biotic components of the transect.
  4. Use the sterile 50ml conical tube to take a soil and ground vegetation sample. Make it is representative of the ground and what is on the surface of the ground.

Procedure 3: Make a Hay Infusion Culture.

  1. Weigh 10 to 12 grams of the soil/ground sample and place in the plastic jar with 500ml of deerpark water.
  2. Add 0.1 grams of dried mil and gently mix it for about 10 seconds.
  3. Take the top off and place the jar in a safe place in the lab.
  4. Label the jar so the group can find it.

Data:

Table I: Biotic and Abiotic Observations in the Transect
Biotic Observations Abiotic Observations
Two Squirrels. Two Metal Light Poles.
A Bird. Loose Garbage.
Shrub/Trees. Sidewalk.
Ground Cover Plants. Mulch.
Tall Grass. Soil.

Hay Infusion Observations:

Mirky dark water with transect components mixed all around. Mass of the overall hay infusion is 11.03 grams.

Very good start. Don't rewrite protocol try to put into own words. Could include some more detail. SK

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