Activitat enzimàtica CATALASA

Introducció: 
Valorarem de forma quantitativa l'activitat enzimàtica de la Catalasa del fetge de pollastre.La pràctica la dividirem en dos parts: -en la 1era observarem la diferència d'activitat de la catalasa en diferents                                               teixits animals i vegetals.
                                          -en la 2ona veurem la influència de determinants factors en l'act.enzimàtica

La catalasa és un enzim present en els peroxisomes de les cèl·lules animals i vegetals que s'encarrega d'eliminar l'aigua oxigenada que es forma en algunes reaccions del metabolisme. La reacció química és la següent:          

PART 1- Quin teixit presenta més activitat de la catalasa?

Materials:

• Patata
• Tomaquet
• Pastanaga
• Fetge
• Cor
• Aigua oxigenada al 3%
• Pinces
• Bisturí
• Tub d'assaig de coll ample
• Gradeta
• Aigua destil·lada

Procediment: 

1. Tallem un tros de patata, un de pastanaga, un de tomàquet, un de fetge i un de cor, TOTS de la mateixa mida i per que capiguen dins el tub d'assaig
2. Pesem tots els trossos dels diferents teixits i apuntem el seu pes (a nosaltres ens va donar: la patata 0,5 g, la pastanaga 0,4 g, el tomàquet 0'4g, el cor 0,7g i el cor també 0.7g)
3. Posarem els 5 teixits cadascún en un tub d'assaig diferent i tots amb 5ml d'aigua destil·lada i marquem amb un permanent el que hi ha en cada tub.
4. Posem 2ml d'aiguan oxigenada en cada tub i marquem l'alçada que assoleixen les bombolles en cadascun. Mesurarem l'alçada en mm.

Resultats:
Els teixits que presenten més activitat són els animals, i d'entre els dos el que més el fetge, després el cor. Els teixits vegetals presenten molta menys activitat que els animals, la patata la que més, després la pastanaga i el tomàquet casi be no presenta activitat, per no dir que no en presenta cap.

Preguntes: 
-Variable dependent i independent? 
La dependent és l'alçada de les bombolles i la independent són els diferents teixits.
-Problema que es vol investigar?
Quin teixit presenta més activitat de la catalasa?

PART 2- Com afecten diferents factors en l'activitat de la catalasa?

Materials:

• Fetge
• Aigua oxigenada al 3%
• Pinces
• Bisturí
• Tub d'assaig de coll ample
• Gradeta
• Pinzes de fusta
• Vec de bunsen i encenedor
• NaCl
• HCl al 10%
• Aigua destil·lada

Procediment: 

1. Tallem 4 trossos de fetge a temperatura ambient, TOTS de la mateixa mida i per que capiguen dins el tub d'assaig
2. Pesem tots els trossos i apuntem el seu pes (a nosaltres ens va donar: fetge 1 1,6g, el fetge 2 1,6g, el fetge 2 1'3g, el fetge 4 1,2g)
3. El primer tros de fetge el posem en un tub d'assaig amb 5ml d'aigua destil·lada. El segon tros de fetge el posem en una dissolució de NaCl durant 10min i el tercer tros també 10 minuts submergit pero en HCl. Per últim el cuart tros el bullim 10 min.
4. Finalment posem tots els trossos en els seus respectius tubs i amb 5ml d'aigua destil·lada.
5. Tallem l'últim tros de fetge que necessitarem en aquesta pràctica que està congelat, intentant que sigui de la mateixa mida que els altres i l'aboquem dins el seu tub d'assaig amb 5ml d'aigua destil·lada també
6. Posem 2ml d'aiguan oxigenada en cada tub i marquem l'alçada que assoleixen les bombolles en cadascun. Mesurarem l'alçada en mm.

Resultats:
Els teixits que presenten més activitat són els animals, i d'entre els dos el que més el fetge, després el cor. Els teixits vegetals presenten molta menys activitat que els animals, la patata la que més, després la pastanaga i el tomàquet casi be no presenta activitat, per no dir que no en presenta cap.

Preguntes: 
-Variable dependent i independent? 
La dependent és l'alçada de les bombolles i la independent les condicions en les que està el fetge.
-Problema que es vol investigar?
Com afecten diferents factors en l'activitat de la catalasa?
-Quina és la funció

Photo Lab Time

L16. CELLS ORGANELLS

                                       
                                          Chloroplasts of Vallisneria

                                          
                                           Carrot Chromoplasts : 100X

                                                     Potato amyloplasts, stained with lugol: 10 x 40 = 400X

                                                    Chromoplasts red cabbage: 15 x 10 = 150X

                                       
                                         Tomato Chromoplasts: 4 x 10= 40X

                                       
                                         Stoma of a red cabbage; 15 x 40= 600X

                                          
                                         Chromoplasts red cabbage; 15 x 40= 600X

               Potato amyloplasts, stained with lugol: 10 x 100 = 1000X

All the photos are taken from Maria Luna's blog because I was ill that day of class so I couldn't do this observations.  

L15. LIFE IN A DROP OF WATER

In this picture we can see a flagel, moving quickly through the water.

You can see the video: 

http://www.youtube.com/feature=player_embedded#t=30

L14. GRAM STAINING

1. Objectives

- Differenciate the yogurt bacteria and relate the staining procedure with the structure of the cells.

2. Material

- 1 Slide
- 1 Cover slip
- Tongs
- Needle
- Gram stain: crystal violet, iodine and safranin.
- Descolorize reagent: ethanol 96%
- Microscope
- Yogurt

3. Procedure

- PROKARIOTIC CELL OBSERVATIONS: Gram Stainning
1. Prepare a heat-fixed sample of the bacteria to be stained.
2. Cover the smear with crystal violet for an exposure of 1 min.
3. Rinse with distilled water.
4. Apply Iodine solution for 1 min.
5. Rinse the sample with distilled water.
6. Decolorize using ethanol. Drop by drop until the purple stops flowing. It is very important that you: wash immediately with distilled water.
7. Cover the sample with the safrain stain for an exposure time of 45 seconds.
8. Rinse the sample with distilled water.
9. Gently dry the slide with paper.

Gram-negative: stain pink of reddish color.
Gram-positive: stain purple color.

L.13 EPIDERMIS CELLS

1.  Objectives:

• Identify the shape of epidermis cells.
• Identify and explore the parts of a stoma.
• Measure dimensions of the entire cell and the stoma.

2. Materials:

•  1 Slide
•  1 Cover slip
•  Distilled water
•  10% Salt water
•  Scissors
•  Needle

3. Procedure:

Plant cells observation:

1. Cut the stalk of the leek.
2. In the place of the cut, pull out the transparent part of the epidermis and using forceps.
3. Using the brush, place the peel onto the slide containing a drop of tap water.
4. Take a cover slip and place it gently on the peel with the aid of a needle.
5. View it in the microscope.
6. Describe the change in the shape of the cells.
7. Draw a diagram with the parts of a stome: stoma,cell guards,epidermis cells.

Salt treatment:

1. Prepare a 10% of salt solution.
2. Put the salt with a dropper on the left part of the slide.
3. Place a piece of cellulose paper in the opposite part of the cover slip, and let the dissolution to go though your sample.

Conclusions and coment

4. Questions

1.        What is the major function of a cell membrane?

• Keep separate internal environment of the phospholipid layer and transport functions played by proteins. The combination of active transport and the passive transport endoplasmic make a selective barrier membrane that allows the cell differentiation medium.

2.      What is the major function of the cell wall?

• The cell wall protects the cell contents, and gives rigidity, works as a mediator in all relations of the cell with the environment and acts as a cellular compartment. In plants, defines the structure and provides support to the tissue and many more parts of the cell.

3.      How does salt affect the cells shape? And the stomes?

• Salt wrinkle cells and stomes (turgency)

L12. ANIMAL CELLS vs PLANT CELLS

Last Monday we compared animal and plant cells by looking trough 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.

1. Objectives

- Identify the major components of cells.
- Differenciate between animal and plant cells,
- Measure dimensions of the entire cell and the nucleous.

2. Materials

- Toothpick
- 2 Slides
- 2 Covers slips
- Distilled water
- Methylene blue
- Iodine
- Onion
- Glycerine
- Two whatch glasses
- Dropper
- Needle

3. Procedure

3.1 Plant cells observation:

1. Pour some distilled water into watch glass.
2. Peel off the leaf from half a piece of onion and using forceps, pull out a piece of transparent onion peel (epidermis) from the leaf.
3. Put the epidermis in the watch glass containing distilled water.
4. Take a few drops of iodine solution ( or safranin) in a dropper and transfer into another watch glass.
5. Using a brush ( or a needle), transfer the peel into the watch glass containing the dye, Let this remain in the safranin solution (or iodine) for 30 seconds, so that the peel is stained.
6. Take the peel from the iodine solution and place it in the watch glass containing distilled water.
7. Take a few drops of glycerine in a dropper and pour 2-3 drops at the center of a dry glass slide.
8. Using the brush, place the peel onto the slide containing glycerine.
9. Take a cover slip and place it gently on the peel with the aid of a needle.
10. Remove the extra glycerine using cellulose paper.
11. View it in microscope.

3.2 Plant cells observation:
1. Gently scrape the inner side of the cheek using a toothpick, wich will collect some cheek cells.
2. Place the cells on a glass slide that has water on it.
3. Mix the water and the cheek cells using a needle and spread them.
4. Dry the sample under the light to fix the sample on the slide,
5. Take a few drops of methylene blue solution using a dropper and add this to the mixture on the slide,
6. After 2-3 minutes remove any excess water and satin from the slide using cellulose paper.
7. Take a clean cover slip and lower it carefully on the mixture with the aid of a needle.
8. Using the top of the needle, press the cover slip gently to spread the epithetial cells,
9. Remove any extra liquid around the cover slip using cellulose paper.

4. Results

PLANT CELLS:

MRcell: 6,9/400= 0,017cm
0,17x10000= 172,5microns

MRnucleous; 400=0,7x10000/X
X=7000/400=17,5 microns

CHEEK CELLS:
400= 1,5x10000microns/X
X=1,5x10000microns/400=37,5

400=0,3x10000microns/X
X= 0,3x10000microns/400=7,5microns

L11. DNA EXTRACTION

1- Introduction 

DNA is a nuclic acid that encodes the genetic instructions used in the development and fuctioning of all living organsims and many viruses.
Nucleic acids are biopolymers formed by simple units called nucleotids. Each nucleotide is composed by a nitrogen-containing nucleobase (G, T, C, A) as well as a monosaccharide (deoxyribose) and a phosphate group.
Most DNA molecules consist of two strands coiled around each other to form a double helix. The two strands run in opposite directions to each other and are therefore anti-parallel. Moreover the bases of the two opposite strands unit according to base pairing rules : A-T and G-C.

2- Material

- 1L Erlenmeyer flask.

- 100mL beaker.

- 10mL graduated cylinder.

- Small funnel.

- Glass stirring rod.

- 10mL pipet.

- Knife.

- Safety goggles.

- Cheesecloth.

- Kiwi.

- Pineapple juice (1mL/5mL).

- Distilled water.

- 90% Ethanol ice-cold.

- 7mL DNA buffer.

- 50mL dish soap.

- 15g NaCl.

- 900mL tap water.

3- Procedure 

Put the ethanol in freezer you will need it really cold later. 

Prepare the buffer in a 0,5L beaker: add 450 mL of a tap water, 25 mL of dish soap and 7g NaCl. 

1- Pell the kiwi and chop it to small pieces. Place the pieces of the kiwi in one 600mL beaker and smash with a fork until it becomes a juice puree.

2- Add 8mL of buffer to the mortar.

3- Mash the kiwi puree carefully for 1 minutewithout creating many bubbles.

4- Filter the mixture: put the funnel on top of the graduated cylinder. Place the cheesecloth on top of the funnel. 

5- Add beaker contain carefully on top of the cheesecloth to fill the graduated cylinder. The juice will drain through the cheesecloth but the chucks of kiwi will not pass through into the graduated cylinder.

6- Add the pineaple juice to the green juice (you will need about 1mL of pineaple juice to 5mL of the green mixture DNA solution). This step will help us to obtain a purer solution DNA. Pineaple juice contains an enzyme that breaks down proteins.

7- Tilt the graduated cylinder and pour in an equal amount of ethanol with an automatic pipet. Put the ethanol through the sides of the graduated cylinder very carefully.You will need about equal volumes of DNA solution to ethanol.

8- Place the graduated cylinder so that it is eye level. Using the stirring rod, collect DNA at the boundary of ethanol and kiwi juice. Do not stir the kiwi juice; only stir in the above ethanol layer!!

9-  The DNA Precipitate looks like long, white and thin fibers.

10- Gently remove the stirring rod and examine what DNA looks like.

4- Questions

1- What did the DNA looks like? 

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

2- Why do you mash the kiwi? Where it is located inside the cells?
Because we  wanted to liberate the DNA that is located inside the nucleus.
 

3- Explain what is the function of every compund of the buffer (soap and salt).
 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 have to do with our method of extraction?  

This means that we can only see the DNA in the part of the ethanol beucause if it touches the water it will dissolve.

L10. PROTEINS AND EVOLUTION

1- Introduction

Genes are made of DNA and are inherited from parent to offspring. 
Soma DNA sequences code form RNA which turns codes for the amino acid sequence of proteins. Cytochrome C is a protein involved in using energy in the cell. Cytochrome C is found in most, if not all, known eukaryotes. 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. 

Cytochrome C is associated with the inter membrane of the mitochondrion. It is a small protein from eucaryote cell.  

2-Procedure 

We compare the protein "Cytochrome C": 

3-Conclusions

-Mamales: horse, whale and donkey

-Birds: chicken and penguin

-Reptils: snake

-Insect: moth

-Fungi: yeast

-Plant: wheat

	Horse	Donkey	Whale	Chiken	Penguin	Snake	Moth	Yeast	Wheat
Horse	0
Donkey	0	0
Whale	5	4	0
Chiken	11	10	9	0
Penguin	13	12	10	3	0
Snake	21	29	18	18	19	0
Moth	24	23	22	23	23	26	0
Yeast	40	39	39	40	39	40	46	0
Wheat	38	37	36	39	39	37	40	43	0

(taken from laura's blog)

(taken from myriam's blog)