L16. Epidermis cells

On Monday 2nd of March we did two experiments using the microscope, and now i'm going to explain the first one. The objective of this experiment was to identify the shape of epidermis cells, and to identify and explore the part of the stoma and see how it changes its shape when we add salt water.The pores open to facilitate uptake of carbon dioxide and close to limit the loss of water

MATERIALS

-Slide
-Cover slip 
-Tap water
-10% salt water
-Forceps
-Dropeper -Scissors
-Needle
-Leek

PROCEDURE

1-First we cut the stalk of the leek and pulled out the transparent part of the epidermis using forceps.
2-Then we placed the peel into the slide containing a drop of tap water (so the cells don't die) .
3-Next we took a cover slip and placed it gently on the peel with the aid of a needle.
4-We viewed it in the microscope and took pictures of it.
5-Then we prepared a 10% salt solution and put the solution with a dropper on the left part of the slide (so it touched the cover slip) and placed a piece of cellulose paper in the opposite side of the cover slip to let the dissolution go through the sample.
6-Finally we looked through the microscope once more and took more pictures.


Results and observations:  
When we first saw the cell we noticed the characteristic shape of a plant cell, a geometric one, a squareand the cell wall. Then we looked closer at it and saw the stomas: they were open. Stoma opens when the guard cells are turgid, when the water potential of the cells adjacent to the guard cells are higher than that in the cell sap of the guard cells and the water molecules from the adjacent cells move into the guard cells by osmosis. The opening of the stoma is an advantage because it allows gaseous exchange to take place.
Then we added salt water and took a second look. Now the stomas were closed because the adjacent cells were hypertonic and the guard cells hypotonic so the water molecules moved out of the guard cells into the adjacent cells by osmosis. When this happens, the guard cells become plasmolysed which in turn causes the stoma to close.

QUESTIONS

1- What is the major function of a cell membrane?
The membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. Also it protects the cell from its surroundings.

2- What is the major function of the cell wall?
It surrounds the cell membrane and provides structural support and protection to it. Also it acts as a filtering mechanism and as a pressure vessel, preventing over-expansion when water enters the cell.

3-How does salt affect the cells shapes? And the stomes?
I explained it earlier.

 

L13. ANIMAL CELLS vs PLANT CELLS

Last Monday we compared animal and plant cells with the mycroscope. The objectives were to identify the major components of cells, differentiate between animal and plant celss and to measure dimensons of the entire cell and the nucleus.

First we peeled off a leaf from an onion. dyed it with safranin stain (red) and then viewed it in the mycroscope.

Then we did some calculations to know the real size of the cell and its nucleous.

After that, we extracted a cell from our cheeks. 

L12. DNA Extraction

On Monday 26th of January we did a new experiment about DNA extaction. The objective of this experiment was to study the DNA structure and to understand the process of extracting DNA from a tissue.

MATERIALS

- 600 ml Beaker
- 10 ml graduated cylinder
- Small funnel
- Glass stirring rod
- 10 mL pipet
-Safety goggles
-Cheesecloth
-Automatic pipet
-Kiwi
-Pineapple juice
-Distilled water
-90 % ethanol (ice cold)
- 8 ml DNA buffer (25 ml dish soap, 7 g NaCl, 450 ml tap water)

PROCEDURE

First of all we prepared the buffer in a 600 ml beaker. We put 450 mL of tap water, 25 ml of dish soap and 7g of NaCl and stirred the mixture careful because we don't want any foam or bubbles to form! 

1-We peeled the kiwi and chopped it to small pieces. We placed them in one 600 ml beaker and smashed them with the pestle to become a juice puree. 
2- We added 8 ml of the buffer to the mortar.
3- We mashed the kiwi puree carefully for 1 minute without creating many bubbles.
4- Then we filtered the mixture: we put the funnel on top of the graduated cylinder. We placed the cheesecloth on top of the funnel and added all the contents of the mortar carefully on top of the cheesecloth to fill the graduated cylinder. The juice drained through the cheesecloth but the chunks of kiwi didn't pass through.
6- We added the pineapple juice to the green juice (4 ml because we had 20 ml of green mixture DNA solution). This helped to obtain a purer solution of DNA because pineapple juice contains an enzyme that breaksdown proteins.
7- Next, we tilted the graduated cylinder and poured in an equal amout of ice-cold ethanol with an automatic pipet. We put the ethanol through the sides of the graduated cylinder to form a clear layer on top of the DNA solution.
8- We placed the graduated cylinder to eye level and using the stirring rod we stirred only the ethanol and DNA came up. 

Results and observations:
The DNA looks like long, small, white and thin fibers.  

QUESTIONS
1- What did the DNA look like?
 The DNA looks like long, small, white and thin fibers.  

2-Why do you mash the cells? Where it is located inside the cells?
Because you want to libreate the DNA that is located inside the nucleus.

3- Explain what is the function of every compound in the buffer
The salt breaks the nucleus and the cell and the soap takes away the proteins.

4-DNA is soluble in water, but not in ethanol. What does this fact has to do with the method of extraction?  
This means that we can only see the DNA in the part of the ethanol because if it touches the water it will dissolve.

On Monday 2nd of February we repeated this experiment but we extracted the DNA from our own cells from our mouths. We did the exact same procedure but instead of mashing the kiwi we took mineral water with salt and we rinsed our mouth with it. At the end we observed the DNA in a microscope. 

L.11 Cytochrome C Comparison Lab

PROTEINS AND EVOLUTION

Genes are made of DNA and are inherited from parent to offspring. Some DNA sequences code for mRNA which, in turn, codes for the amino acid sequence of proteins. Over time, random mutations in the DNA sequence occur. As a result, the amino acid sequence of Cytochrome C also changes. Cells without usable Cytochrome C are unlikely to survive. The cytochrome C is a small protein found loosely associated with the inner membrane of the mitochondrion. It is found in eucariotic cells and has an hemeprotein. It is essential to the electrone transport chain and it is involved in using energy in the cell (ATP).

The purpose of this practice is to compare the relatedness between organisms by examining the amino acid sequence in the protein.

METHOD

First, we compared the amino acid sequence of Cytochrome C in various organisms:
-horse, donkey, whale (mammals)
-penguin, chicken (birds)
-snake (reptile)
-moth (insect)
-wheat (plant)
-yeast (fungi)

1- We marked the amino acids which were different.
2-Then we counted and recorded the total number of differences.
3- We shared the data with the rest of the class to complete Table 1.


After that, we made a cladogram (branching tree)
1- The two most closely related species had the fewest differences in amino acid sequence. We placed the two most closely related species on the two shortest branches of the tree.
2-Then we placed the next two closest species on the next shortest branches.
3- And we continued until all the species had been placed.

RESULTS AND OBSERVATIONS

CONCLUSIONS

There are 0 differences between chickens and turkeys in their Cytochrome C amino acid sequence.

We think that the horse and the zebra have 1 or 2 differences, like the donkey and zebra.
To make this prediction we used this information: if they can reproduce and if the offspring will be fertile or not, we compared organs, compared embrios...

More closely related organisms have more similar Cytochrome C because evolutionarily, it hasn't been that long since they separated. If the species are close, it means that less time has passed since they separated (there are less accumulated mutations). They have a common ancestor.

Other data, including genes, suggests that fungi are more closely related to animals than plants. But Cytochrome C data suggests that fungi, plants and animals are equally distantly related because if there are more than 40 genes, there are too many mutations to see it clearly.

L9. Protein identification

On december 22nd we did an experiment about proteins. The objectives are to identify the peptide bonds and to compare the protein concentration in different foods.

Biuret's test
The Biuret's test is a chemical test used for detecting the presence of peptide bonds (the bonds that link together amino acids that form chains --> proteins). A peptide bond can be broken by hydrolysis. The intensity of the colour of the Biuret's test is directly proportional to the protein concentration. The solution to be tested in treated with a strong base followed by a few drop sof copper II sulphate. If the solution turns purple protein is present. Only peptides with a chain of at least 3 amino acids give a significant measurable colour shift with these reagents (polypeptides).


MATERIALS

-250 ml beakers
-Test tube rack
-6 test tubes
-6 x 10 ml pipet
-Mortar
-Glass marking pen
-Gloves
-Goggles
-Milk
-Rice milk
-Egg (white and yolk)
-Yogurt
-Potato
-Distilled water
-NaOH 20%
-10 drops of CuSO4

PROCEDURE

First of all we had to dilute the protein:
1- We added 100 ml of distilled water to each 250 mL beaker, and with the glass marking pen we labeled them as M (milk), R (rice milk), EW (egg white, EY (egg yolk), Y (yogurt) and P (potato).
2- Then we added 10 ml of a dispersion of each food in the belonging beakers, we separated the egg white and yolk in two different beakers and mashed the potato.

Then we prepared the samples:
3- We cleaned and dried the test tubes and labeled them like the beakers. Each group used the dispersions from the same beakers. Then we added 2 mL of every food dilution of the beaker to each corresponding test tube.
4- After that we added 2 mL of 20%NaOH dissolution to each test tube, Andrea did this because NaOH is caustic so she had to put gloves and goggles on.
5-We shook gently and added 5 drops of CuSO4 in each test tube. Then we allowed the mixture to stand for 5 minutes.
6-Finally we saw the colour change (to pink or purple) amd we compared the test tubes.

Results and observations:
Milk - positive
Rice milk - negative
Egg white - positive
Egg yolk - negative
Yogurt - positive (it has less water, the protein is more concentrated)
Potato - positive, but our teacher said that it had to be negative because it had starch.

We saw that all the foods of animal origin gave a positive result, they all have proteins. Rice milk is negative because it has starch like the potato (but strangely the potato was positive). Also, we ordered the food from more concentration of protein to less according to the colour: 1- egg white, 2- yogurt and potato, 3- milk.

QUESTIONS

1- Which food has proteins?
Eggwhite, milk yogurt and potato.

2- Which food has more proteins? Why?
Eggwhite because it's animal food and it's rich in proteins. Then the yogurt and potato and finally the milk.

3-Do you find any difference between rice milk and cow milk?
Rice milk doesn't have proteins however cow milk does as i had said before.

4- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk because the protein is more concentrated than in the milk, it has less water.

L8. Saponification

On the 1st of december we decided to experiment the saponofication, we wanted to make soap. Saponification is a reaction that produces soap when you mix fats and a strong base (NaOH).

MATERIALS

-32 gr NaOH
-90 ml H2O
-270ml of olive oil
-safety goggles
-2 beakers
-gloves
-balance
-stirring rod
-spatula
-heating plate

PROCEDURE


First we put on our safety goggles and gloves to protect ourselves. Then we mixed in a 600 ml beaker the 32 grams NaOh with the 90 ml of water, slowly, we noticed that the beaker heated up, it is a endotermic reaction! After that we slowly started to pour the olive oil in the beaker, at the same time mixing the mixture with the stirring rod. We mixed it for 20 minutes, also we heated the beaker on the heating plate to accelerate the reaction. Then we put a couple of drops of aroma and finally we put the soap mixture in a mold.

Results and observations: We will let the soap mixture sit for a couple of days and then we will obtain hard soap. The reaction of saponifiaction:

First by hydrolisis we obtained glicerine and oleic acid, then the oleid acid joined the NaOH and soap and water were formed.

L7. Lipids properties

On december 1st we did a new interesting experiment related with lipids. These compounds are characterized as natural substances that do not mix with water but dissolve in organic solvents, C, H and O are the principal elements of lipids. The objectives of this experiments are to test the solubility of lipids, indetify lipids in liquids compunds and to understand what is an emulsion and the effect of detergents. We can divide this experiment in three.

MATERIALS

-Test tube rack
-250 mL beaker
-Water
-6 test tubes
-Cellulose paper
-Dropper
-Scissors
-Glass rod

-Olive oil
-Soap
-Milk with different fat content (full-cream, semi-skimmed, and skimmed milk)
-Petroleum ether
-Ethanol
-Sudan III

SOLUBILITY OF SOME LIPIDS
PROCEDURE

 1-First we cleaned and dried three test tubes, we labeled them as W (water), E (ethanol, and PE (ether).
2- Then we added 3 drops of oleic acid to the three test tubes.
3- We added 1 ml of water in the first test tube (W).
4- 1 ml of ethanol in the second test tube (E).
5- 1ml of petroleum ether in the thirs test tube (PE).
6- Finally we shaked each test tube and recorded solubility and observations in our worksheet.

Results and observations: We saw that in the test tube W the oleic acid didn't dissolve, in the E test tube micelles had formed and in the last PE test tube it dissolved completely because ether is an organic dissolvent.

LIPIDS IDENTIFICATION:
A. TRANLUSCENT MARK:

1- Our teacher gave us two pieces of cellulose paper.
2- In the first paper we put one drop of water, we saw a translucent spot and then we waited a little bit to see what was happening.
3- Then we put a drop of olive oil on the second piece of cellulose paper and we saw a translucent spor also. We waited to see what was happening.
4- Finally we wrote the results in our worksheet.

Results and observations:  The drop of water of the first piece of paper dried after a while and didn't leave any marks but the drop of oil was still visible when dried, it was translucid.

B. SUDAN III dye

1-We took the W test tube of the first experiment and added 2 drops of Sudan III.
2- Then we prepared three test tubes with full-cream, semi-skimmed and skimmed milk and also put 2 drops of Sudan III in each test tube to stain it.

Results and observations: The water had to turn black if there were fatty acid in the solution but in our experiment it didn't work and nothing happened.

PERMANENT EMULSION:

1- We took a 250 ml beaker and put 100 ml of water there.
2- Then we added 1 ml of olive oil and with a glass rodd we stirred the mixture vigorously and let it stand for a few minutes.
3- After taking note of what was happening we added 2 drops of soap and stirred the mixture again, and waited for a little bit to notice the difference.

Results and observations: First when we added oil to the water a layer of oil formed in the top part of the water. When we added soap microdrops appeared, little micelles. Soap doesn't dissolve the lipid, it just wraps it so the oil layer can't be formed.

QUESTIONS:
1-From your observations, which compunds can dissolve lipids?
Organic solvents can dissolve lipids, for example in our case ether dissolved them.

2-Do the oil and water mix? No
What can you conclude about the polarity of oil if you know water is polar?
I can say that lipids tend to be non-polar because they are made of longs chains of hydrocarbons with relatively little oxygen (hydrocabron chain), and this fatty acid tail is hydrophobic.Water molecules are strongly attracted to each other, this is the same for oil, because they are more attracted to their own molecules they just don't mix together. They separate and the oil floats above the water because it has a lower density.

3- Why is olive oil liquid at room temperature? And why not the lard?
Olive oil is liquid at room temperature because it is unsarurated (the chain bends and it only bonds on one part, it's easier to break). Lard isn't liquid because it's saturated (so it's harder to break the bonds)

4-Why does a lipid leave a translucent spot on paper?
In their liquid state oils and fats easily penetrate into the pores of dry substances. The lipids soak into the paper fibres, but the lipid spot evaporates much more slowly than water (because the forces that hold the lipid molecules together are stronger than those in water molecules)

5-  Which type of milk contains more lipids? Why?
The full-cream milk contains more lipids because it has more fat (fatty acids).

6- Did the oil and water mix when you added soap? 
Yes. Detergent is attracted to both water and oil helping them all join together and form something called an emulsion.

7-What did the soap do to the fat? 
Soap on one end it is hydrophillic (that is polar) and on the other end it is hydrophobic (non polar). So, the polar end of the soap molecule will attract the water molecules while the non-polar end of the molecule attracts the oil molecules.
Soap doesn't dissolve the lipid, it just wraps it so the oil layer can't be formed.

8- Can you think about process and locations were compounds like the soap would be important to an animal? 
Soap makes insoluble particles become soluble which allows water to rinse the particles away. This means that while oil doesn’t naturally mix with water, soap can suspend oil/dirt in such a way that it can be removed. They are useful in bile acids.