Midterm paper

LAB 1: CELL BIOLOGY Protocol Slides PCB 3702L FIU

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LAB 1 PROTOCOL OBJECTIVES

1. Describe which cell structures are present on a cheek sample.

2. Explain cell membrane transport, focusing on osmosis.

3. Explain the factors that might affect the enzymes normal functions.

MICROSCOPE BASICS 1. Turn ON the Microscope’s light switch

2. Adjust the illumination

3. Place objective lens on the LOWEST magnification (x4)

4. Pull on the stage clip to load the specimen slide onto the stage

5. Adjust the stage using the stage control knobs in order to find the specimen on the slide

6. First, use the COARSE adjustment knob to bring the stage up until the specimen is in sight (should look like a clump)

7. Then, use the FINE adjustment knob to slowly bring the stage further up until the specimen becomes clearer (more features will become noticeable)

8. IF you need to see a more magnified version of the specimen, carefully move up to the next magnification lens and REPEAT steps 6 & 7 until you have a clear view of the specimen

Always grip the microscope by the arm and put your hand beneath its base. Always hold the scope upright. Do not bump it against anything.

ACTIVITY 1: CHEEK CELL WET MOUNT

CHEEK CELL WET MOUNT PROCEDURES

1 Place a drop of 0.9% NaCl

saline solution onto a microscope slide.

2

Choose one member of your group and with a

toothpick, carefully scrape the lining of your cheek to

obtain a sample of epithelial cells.

3

Transfer the sample to the saline solution on your

microscope slide and mix using the toothpick.

4

Add a drop of methylene blue stain to your

microscope slide. Again, use the toothpick to stir.

5

Apply a coverslip to your sample and gently blot excessive fluid with a

Kimwipe (tissue).

Examine the stained epithelial cells using a

microscope.

CHEEK CELL WET MOUNT QUESTIONS What kind of cell are we staining?

• Diploid, Somatic Cell

• Stratified Squamous Epithelial Cell

What parts of the cell were visible under the microscope?

­ Nucleus, cytoplasm, cell membrane

List 2 organelles that were not visible but should have been in the cheek cell.

Methylene blue is a cationic, basic stain. For this reason, methylene blue binds

strongly to negatively charged cell components. How might this explain its role in

staining the nucleus?

• Binds to the negatively charged molecules in the cell such as DNA/RNA

DNA Structure

nucleus

APPLICATION QUESTIONS One adaptive trait of viruses is the ability to envelope themselves in

host-derived membrane after viral progeny budding from cells. How

might this help viruses proliferate?

• The host membrane might help viruses avoid detection by the host’s immune system

A patient is deficient in the HMG-CoA reductase enzyme responsible

for cholesterol synthesis. A cell biologist notices an odd morphology to

this patient’s cell. What is the connection?

• Cholesterol regulates membrane fluidity, depending on environmental conditions

In some neurological disorders such as amyotrophic lateral sclerosis

(ALS) where motor neurons in the brain and spinal cord die, the nuclear

pores of neurons become clogged. In relation to the central dogma of

molecular biology, what is one product that would be unable to exit

the nucleus, and which step of gene expression would be inhibited?

• mRNA will be unable to exit the nucleus

• Translation would be inhibited

mRNA

mRNA

ACTIVITY 2: CELL MEMBRANE TRANSPORT

What is the difference between passive and active transport?

TONICITY OF SOLUTIONS Describes the ability of a solution to change the volume of cells by altering their water content via osmosis. It depends not only on relative solute concentrations, but also on solute permeability

•Isotonic solutions cause no net movement of water

What is the effect of a hypertonic solution? A hypotonic solution?

SHEEP’S BLOOD ACTIVITY PROCEDURES

1. Obtain three test tubes and label them 1, 2 and 3.

2. Add 1 mL of 0.9% NaCl to the test tube labeled 1, 1 mL of 10% NaCl to test tube labeled 2, and 1 mL of deionized (DI) water to the test tube labeled 3.

3. Using a pipet, place 3 drops of sheep’s blood into each test tube.

4. After carefully stirring the solutions and waiting five minutes, add 1 drop from each of the test tubes onto microscope slides.

5. Examine the slides at 40x and record your observations.

SHEEP’S BLOOD ACTIVITY - RESULTS Which solution is hypotonic?

Which solution is hypertonic?

Which concentration of NaCl lysed the cells?

Which of the three solutions most closely approximated the solute concentration in a red blood cell? How do you know?

0.9% NaCl DI Water10% NaCl

0.9 % NaCl 10 % NaClDI Water

OSMOSIS IN ERYTHROCYTES

IsotonicHypotonic Hypertonic

Full video can be found here: https://www.youtube.com/watch?v=A8cI6FkcG4c

APPLICATION QUESTIONS

A high concentration of ions from dietary salt in blood plasma leads to increased water retention in the body, which increases blood volume and consequently, blood pressure.

A mutation in the CFTR gene causes a dysfunction of the salt and water balance. This results in dehydration of mucous secretions and excessive loss of salt in sweat.

BLOOD PRESSURE How might these results relate to the effect of a salty diet on blood pressure?

CYSTIC FIBROSIS Why might the absence of chloride ion channels in cystic fibrosis patients result in the common symptom of excessively thick mucous secretions?

ACTIVITY 3: METABOLISM & ENZYMES

ENZYMES Serving as biological catalysts, enzymes catalyze chemical reactions by

accelerating the conversion of reactants into products. This is done by

lowering the amount of activation energy required for a reaction to occur

Enzymes are not used up or changed at the end of a given reaction

Enzymes are pH and temperature sensitive, with each enzyme having

optimal activity within a specific pH and temperature range

Enzymes do not alter the equilibrium constant and do not affect the overall

∆G of a reaction

How are they produced?

• Enzymes are usually proteins but can be RNA (Ribozymes)

• Proteins are synthesized by ribosomes on the rough ER; they are then

modified by the Golgi apparatus

Which factors can affect the performance of an enzyme?

• pH, temperature, salinity, inhibitors, activators, enzyme concentration,

substrate concentration, cofactors, etc. Central Dogma of Biology

m

ACTIVITY 3: METABOLISM & ENZYMES

How might temperature affect the rate of reaction of certain enzymes? What if it is too hot or too cold?

What can pH do to the rate of reaction of certain enzymes? What if this enzyme lives in the stomach? The small intestine? Blood?

In cyanide poisoning, cyanide binds to and inhibits the cytochrome c oxidase enzyme of the electron transport chain. Which organelle would be affected and how would its function be impaired?

What enzyme is responsible for converting hydrogen peroxide into water and oxygen?

ENZYMES: LOCK-AND-KEY

VS. INDUCED FIT MODELS • Generally accepted model

• Binding of substrate induces a conformational change in the active site of the enzyme

• Active site is the “lock” and the substrate fits like a “key”

• Both the substrate and enzyme active site should fit like lock and key to initiate a reaction