User:Matias Bifani/Notebook/Biology 210 at AU
Zebra fish Lab 6
This Lab was designed in order to obtain a better understanding of embryonic development through Zebra fishes. Additionally, two groups of fish were classified one as a control and the other containing an ethanol environment. This experiment requires close attention and observation to better comprehend embryonic development through two different environments. It was hypothesized that the fish eggs that were exposed to alcohol will have more deformities with certain characteristics such as; eye size, body size, movement, colour and heart rate then the control group. These traits will be recorded frequently to see the impact of ethanol on the embryonic development of zebra fish.
Prior to observing the zebra fish eggs other zygotes were examined including a chicken, starfish and frog zygotes. Next the zebra fish eggs were examined and certain observations were documented and compared to eggs seen prior in lab. Two groups of zebra fish were separated into a control group and one exposed to diluted ethanol. Both petri dishes received 20 mL of Deerpark water - which will be need to fill up regularly in order to maintain the zygotes environment. Finally a disposable pipet was used in order to transfer the embryos to their new environment.
Data & Observations
It is to premature to distinguish the effects of ethanol of zebra fish, however there are some distinctions between the two groups predominantly concerning the colouration of the embryos. Overall the size of both there bodies and eyes are inconclusive at this stage. Using Zebra fish as a model organism to better understand the effects of alcohol on embryonic development is crucial information and can utilize to further support the effects on Human embryonic development.
The purpose of this lab was to better understand the impotence of invertebrates and study how more simplified systems
developed into complex organisms. In order to do this a Burlese Funnel was constructed from leaves collected in transect 2, which would be used to better determine the invertebrates that live in the Wildlife Habitat.
Materials and Methods
- Using a dissecting microscope observe an annelate (worm)
- Observe the movement of the annelate
- Observe and identify five different invertebrates
- Record characteristics of different invertebrates observed
- Remove 50:50 ethanol/water conical tube from Burlese Funnel
- Remove 15 mL sample from both the bottom and top of conical tube - place into petri dish
- Examine petri dish for any insects using a dissecting microscope
- observe the characteristics of the insects found
Data and Observations
Observation of Annelate
- fairly calm except if aggravated or interrupted
- Dark purple black
- elastic extremely flexible
Invertebrates and Different Sizes
- Spider 4.3 cm
- Centipede 31.3 cm
- Bee 1.5 cm
- Small crab 1.7 cm
- Millipede 34.3 cm
Overall there was very few invertebrates found in transect 2, this is mostly likely due to the cold temperatures and ice that layered the leaves which were utilized in the 500 g Burlese Funnel. However, from the two collected both the Springtail X and Soil Mite were very small in size compared to the invertebrates examined in the lab. Questioning what was found, we considered the a correlation between insect size and their ability to withstand cold environments.
Plantae and Fungi
There were three main objectives in the lab, the primary focus was understanding diversity and characteristics amongst plants, secondly, to better understand what plants subside in transect 2 and lastly interpreting the results of bacteria found in transect 2 by sequencing their genes via PCR.
Materials and Methods
- PCR gel was loaded with 2 DNA samples and a ladder sample - Investigate several different plants in class - Collected 500g of leaves and plant matter from transect 2 - Samples were obeserved
- using a microscope and standard observation
- Burlese funnel was set up on a ring stand
- a screen was placed inside the funnel - a solution of 1:1 Ethanol Water was taped to the bottom of the funnel - Finally 500 g of plant matter was placed in the funnel
Data and Observation
The PCR gel below demonstrates the only one of the two samples ran correctly. The results of the one sample illustrated that the bacteria’s DNA was roughly 1.5 kilobases – 1 kilobase is 1000 base pairs of DNA.
The majority of the plants where smaller then 1 meter, with exception to the first plant which was roughly 1.5 m Tall. Additionally, all plants had a vascular system, except for the second plant – which is moss. All the plants had unique structures that facilitate their overall function. Many of the low plants grow close to the ground in order to expand more rapidly, and or do not have a large enough vascular system to support a taller plant. Despite not finding any seeds the majority of the samples collected from transect 2 reproduced through seeds – which we concluded were monocot- with exception to the first and second transect sample. There were no flowers found in transect 2, this could be due to the whether. Moss uses its spores to reproduces while the first sample used barries. After further investigation it was concluded that the moss was
The varieties of plants collected from transect 2 shared very similar traits. This suggests that certain traits are more advantageous for transect 2. In addition there is a connection between the plants that share common traits, such as reproducing through seeds – At some point they had a common ancestor. This knowledge deepens the understand of the types of Plantae and Fungi living in transect 2. Not only in today’s lab were we able to classify and broaden the plants and fungi in transect 2, but also interpreted the results of the bacteria from the PCR gel. In order to improve the lab, several more trials of the 1.5 kilobase pair bacteria should have been tested. The Burlese funnels were set up with the leaves in order to collect samples of invertebrates. The large majority of invertebrates come from the same phylum, Arthopoda – which includes spiders and many other insects. The collection of insect from the Burlese funnel will be essential in order to determine the types of invertebrates in transect 2.
Unraveling Bacteria of Transect 2 Via Gram stain, PCR DNA Sequencing and Isolating to Anti-Biotic Resistent Bacteria
The lab was preformed in order to identify different bacteria found in transect 2. The challenge was to identify the different bacteria present in the hay infusion sample. In order to characterize and classify the different Archaea, 8 agar plates were inoculated however half the plates were treated with tetracycline- an anti-biotic. This process would assist in identifying the bacteria in addition to preforming a Gram stain and a DNA sequencing using a PCR. A Gram stain was preformed to determine which bacteria contained a layer of peptidoglycan on the bacterial cell wall, this procedure can give further insights on the characteristic and identification of the bacteria. Secondly, a PCR was used to sequence the DNA to help identify the bacteria in transect 2. It was hypothesized that the more time that Hay Infusion simmered there would be a more potent rotten smell, the water will more clear and the leaves will sink to the bottom.
Materials and Methods
- First 8 agar plates were made
- 4 of the plates were treated with tetracycline - 1 week past allowing bacterial growth
- observe the difference in both plates with/without tetracycline
- Additionally count the approximate numbers of colonies in each plate - Factor in the conversion factor in order to determine colonies/mL
- 2 wet mounts were prepared from agar and tetracycline plates respectively
- Observe the difference between colonies found in respective plates - Observe and draw cell found - determine motility of cell
- Prepare a Gram Stain of for each plate
- Once prepared observe slide under both 40x and 100x - 100x must be done under oil immersion - Record - colony description - Cell description - Gram +/Gram -
- PCR for 16s Sequencing
- transfer bacteria to sterile tube and place in a heat block - centrifuge sample - transfer sample to PCR tube with primer - Place sample in a PCR machine to identify 16s PCR reaction
Data and Observations
Observation of Hay Infusion
There was a strong rotten smell, additionally some water that had evaporated making the water thicker and denser. Most plant matter sunk to the bottom of the Hay infusion, and the overall colour was darker.
100 Fold Serial Dilution
This image shows that the number of colonies counted and the amount of nutrients in the agar plates share a strong correlation. The less amount of nutrients that are present the less colonies of bacteria are recorded per mL. However the other 4 plates that were previously treated with tetracycline too displayed a strong correlation between the, abundance and nutrients plus the anti-biotic, and the amount of colonies present.
Traits and Characteristics of Bacteria - Gram Staining
All plates that were not exposed to tetracycline yield Gram positive. The plate the had more food had smaller colonies however more populated and clustered. The agar plate that was less diluted had larger colonies that resembled tubular shaped rods. The agar plates with the anti-biotic showed that the more nutrients and anti-biotic the smaller the colonies, yet the colonies were more disperse with low levels of motility. The less abundant agar and tetracycline plate had larger colonies that were packed closely together, this plate was the only Gram negative observed.
Conclusion and Future Direction
In conclusion, when first smelling the hay infusion our hypothesis was correct the infusion had a stronger more rotten smell, the leaves had simmered to the bottom, yet the water was more murky which was not as predicted. Agar plates were used as cultivate bacterial colonies, some archaea will not be able to go in the agar environment. Archaea's tend to live in more extreme environments as they where first exposed to earth when it under harsher conditions. It could be concluded that through natural selection Archaea geared itself for the earths extreme temperatures and environments therefore not allowing Archaea to grow on what we consider a good medium, such as agar. Plates treated with tetracycline presented bacteria that were both rod shaped and roughly the same size (Traits and Characteristics of Bacteria - Gram Staining). However, overall there was a strong decrease in colonies when comparing the tetracycline inoculated plate to the pure agar and nutrients. This suggest that the anti-biotic does reduces the amount of bacteria growth. In addition it isolates which bacteria become resistance. It has been a common observation that tetracycline can facilitate the growth of anti-biotic resistant bacteria, according to Li Wen in his article, Mechanism of tetracycline resistance by ribosomal protection protein Tet(O). However, Li Wen does explain the mechanism of how tetracycline do kill bacteria. They do so by interrupting the synthesis of proteins, preventing the cell to expand. This outside source helps support the data observed in the lab.
It can be deduced that exposing the bacteria from transect 2 could grow on both agar plates with nutrients in addition to tetracycline. The plates containing the anti-biotic where less populated, and the treated agar plates with less nutrients and tetracycline showed a negative on the Gram Stain - suggesting the the bacteria present does not contain the amino acid peptidogyclan on the cell wall. It was noticed that across all four Gram stains the more populated and condensed the colonies were the smaller the individual colony was. This could be a result of high competition, or a faster way of expansion across medium. Such observation allows us to gather a greater understand of the diversity of bacteria living in transect 2. In order to follow up and fully understand the bacteria living in transect 2, the PCR DNA sequencing results will be available next lab, which will further help to identify the life in transect 2.
Li, W., Atkinson, G., Thakor, N., Allas, Ü, Lu, C., Chan, K., ... Frank, J. (2013). Mechanism of tetracycline resistance by ribosomal protection protein Tet(O). Nature Communications, 1477-1477.
Identifying Algae and Protists 28-01-2015
Purpose: To further develop our understanding of the dichotomous key a sample of transect 2 was used to observe two different niche’s. An experiment was conducted in order to observe 6 wet mounts from 2 different niches of the transects 2 sample. This would allow the lab team to enhance their understating of what types of organism belong to the ecosystem of transect 2. It was hypothesized that organism that belonged to the top section of the sample would have more developed motility then those observed in the bottom niche of the sample.
Materials and Methods:
• First a known organism was observed under a ocular microscope
o A dichotomous key was used to confirm that organism and strength the understanding of the dichotomous key
• 6 wet mounts were collected from transect 2 sample
o The 2 samples from the top and bottom of the sample first need to diluted
o 3 samples were collected by a pipette from both the bottom and top niches of the sample respectively
o Before looking at the sample you have to dilute the sample
• Therefore dilute the original sample 4 times using sterile broth
• Add 1 drop of paralytic agent to each slide the slow down the organisms motility
• Once all 6 wet mounts from the 2 separate niches observe the organism under and ocular microscope
• Find one organism in each wet mount and identify it using the dichotomous key
o Once identified draw and record organism characteristics
• Secondly, plate 6 agar plates with the sample
o Make sure three of the plates have an antibiotic
1.) 3 organism from the top niche of sample
2.) 3 organism from the bottom niche of sample
Conclusion and future directions:
In conclusion after observing the organism all three organism found in the top niche had means of motility. However it is true that organism found at the bottom of the niche too have cilia and flagella. Therefore there is no clear determent between organisms found in the two different niches. In order to further improve the results more sample trails can take place to better isolate what kinds of algea and protest are found between the two different niches.
Great job, very complete! ML
26th January 2015 – Lab Entry 1
Introduction & Purpose:
Charles Darwin developed the Theory of Natural Selection, which is natures way to isolate and pass on traits that are more suitable for a certain environment and ecosystem. There are three factors that are necessary to achieve natural selection, variability amongst species, heritability, differential capacity for survival and reproduction. In order to track evolution through natural selection 3 different organisms were observed from the Volvocine line: Chlamydomonas, Gonium and Volvox. Certain evolutionary specializations such as number of cells, colony size, motility, specialization of cell and method of sexual reproduction where observed to differentiate the different characteristics of the different green algae, chlorophyta. In order to familiarize ourselves with classifying traits a dichotomous key was used to determine different traits and organisms. A sample was later collected from transect 2 in order to classify and observe the different organism of that location. The dichotomous key will be a necessary tool to be used in order to classify and determine what organism is being observed. Such observations will be needed to determine the ecology and the biodiversity of a designated transect.
Materials and Methods:
In order to determine the different characterizes of the organism in the Volvocine line, three wet slides were created which consisted of Chlamydomonas, Gonium, and Volvox. A paralyzing agent was added to reduce motility, which allowed for observations under a microscope with an ocular lense. The dichotomous key was used to determine different evolutionary specialization and to clarify what organism it is. In order to determine the biodiversity of the transect 2 a Hay Infusion Culture. 10.7 grams of the transect sample was added to a 500 mL solution of Deer Park Water. Once the sample was added to the Deer Park Water Solution roughly 0.1 grams of dried milk was added and mix for 10 seconds. When mixing the ensure the jar is closed, however once mixed remove the lid to allow organism to breathe (Make ATP!). Finally label the jar to ensure a consistent sample, and clean up the lab station. Next week, a sample collected will be used to determine the biodiversity in the transect, this will be achieved by creating a wet slide, with a paralyzing agent, and using the dichotomous key to help classify the organism found.
Data and Observations:
Characteristic Chlamydomnas Gonium Volvox Number of Cell 2 4 8 Colony Size (µm) 17 45 45 Cell Specialization No Yes Yes Mechanisms of Motility Flagella Enivornment Cilia and Flagella Isogamous or Oogamous Both Oogamous Oogamous
Therefore, the more evolved organism of the Volvocine line have larger colonies. In addition you can see the familiar traits that are in all three organism such as reproduction.
Where is your transect information? Make sure you follow the information in the red boxes. The information that you do have is very complete though. ML