Absorbance Measurement and Fermentation Basics
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Questions and Answers

What should you do at the 2-minute mark during the absorbance measurement?

Stop the timer and turn off the lamp.

How often should the absorbance of Cuvette #3 be measured after the initial reading?

Every 2 minutes for a total of 14 minutes.

What is the purpose of using DCMU in Experiment II?

To investigate its effect as an inhibitor on the Hill reaction of photosynthesis.

How do you prepare a 1:10 dilution of DCMU from the stock solution?

<p>Add 0.2 mL of DCMU stock to 1.8 mL of RO water and vortex to mix.</p> Signup and view all the answers

What should be added to Tube #2 in the preparation phase?

<p>2 mL buffer, 1 mL DCPIP, and 1 mL Stock DCMU.</p> Signup and view all the answers

Why is it important to vortex each test tube after adding the components?

<p>To ensure thorough mixing of the solutions for accurate results.</p> Signup and view all the answers

What is the final step in handling the cuvettes after the experiment is completed?

<p>Rinse the tubes and cuvettes and leave them upside down to dry.</p> Signup and view all the answers

Why were alcoholic beverages popular during the Middle Ages in Europe?

<p>They provided nutritious, carbohydrate-rich, and safe options for consumption when food was scarce or unsafe.</p> Signup and view all the answers

What is the role of Saccharomyces in the production of both alcoholic beverages and bread?

<p>Saccharomyces ferments carbohydrates to produce ethanol and carbon dioxide, which is essential for alcohol production and causes bread to rise.</p> Signup and view all the answers

What happens to the ethanol produced during the fermentation of bread?

<p>The ethanol evaporates during the baking process.</p> Signup and view all the answers

What is the significance of carbohydrates in the ethanol production process?

<p>Carbohydrates serve as the energy and carbon source for yeast during fermentation.</p> Signup and view all the answers

What is required for carbohydrates other than glucose to be used in fermentation?

<p>They must first be broken down into simple sugars like glucose.</p> Signup and view all the answers

Explain the chemical reaction for the fermentation process detailed in the content.

<p>The reaction represents glucose being converted into ethanol and carbon dioxide in the presence of water.</p> Signup and view all the answers

How has the process of fermentation evolved in modern times?

<p>It is now used to produce ethanol fuel, maximizing efficiency and resource utilization.</p> Signup and view all the answers

Which plant materials are mentioned as substrates for fermentation?

<p>Sugarcane, corn, and barley are mentioned as substrates for fermentation.</p> Signup and view all the answers

What is the primary purpose of adding sodium carbonate at the end of the reaction?

<p>Sodium carbonate is added to stop the reaction by inactivating the enzyme.</p> Signup and view all the answers

How should absorbance values greater than 1.000 be reported?

<p>They should be reported as 1.00.</p> Signup and view all the answers

What is the first step to prepare the reaction tubes before adding the enzyme?

<p>Add 3.5 mL phosphate buffer and 0.5 mL of the appropriate ONPG solution to each tube.</p> Signup and view all the answers

Why is it important to stagger the addition of enzyme to the reaction tubes?

<p>Staggering the addition helps prevent all tubes from completing the reaction at the same time, allowing for accurate measurement of each sample.</p> Signup and view all the answers

What role does the blank tube play in this experiment?

<p>The blank tube is used to zero the spectrophotometer, ensuring accurate measurement of absorbance for the reaction tubes.</p> Signup and view all the answers

What should be done if absorbance measurements fall below 0.000?

<p>They should be reported as 0.000.</p> Signup and view all the answers

Explain the importance of rinsing the cuvette between samples in this experiment.

<p>Rinsing the cuvette prevents cross-contamination between samples, which can skew absorbance readings.</p> Signup and view all the answers

What is the purpose of securely attaching the micropipette tip to the barrel?

<p>To create an airtight seal that prevents air leaks and ensures accurate measurement.</p> Signup and view all the answers

Describe the function of the three positions of the control button on the micropipette.

<p>The three positions are 'At Rest' (idle), 'Stop 1' (measures the selected volume), and 'Stop 2' (blows out the last drops).</p> Signup and view all the answers

What is the correct method to immerse the micropipette tip in a liquid during measurement?

<p>Immerse the tip approximately 3 mm into the liquid while holding the micropipette vertically.</p> Signup and view all the answers

Explain why the control button should be allowed to glide back slowly after immersing in the liquid.

<p>Gliding back slowly helps create a consistent and accurate volume measurement without allowing air bubbles.</p> Signup and view all the answers

What is the significance of 'blow-out' at Stop 2 when dispensing liquid?

<p>The blow-out ensures any remaining liquid in the tip is expelled completely.</p> Signup and view all the answers

What steps should be taken to safely eject the micropipette tip after use?

<p>Hold the micropipette over the disposal bag and depress the eject button carefully to shoot the tip off.</p> Signup and view all the answers

Why is it important to avoid exceeding the maximum or minimum volumes with a micropipette?

<p>Exceeding volume limits can damage the micropipette and lead to inaccurate measurements.</p> Signup and view all the answers

What potential problem can affect experiments using a spectrophotometer, according to the content?

<p>The presence of other absorbing molecules in the solution can interfere with the measurements.</p> Signup and view all the answers

What is the purpose of preparing serial dilutions of the enzyme stock solution?

<p>The purpose is to determine a proper concentration of b-galactosidase that allows for measurable enzyme activity without being too diluted or too concentrated.</p> Signup and view all the answers

How should the enzyme dilutions be labeled for the experiment?

<p>They should be labeled according to their dilution factors, such as 1:10, 1:100, 1:1000, etc.</p> Signup and view all the answers

Why is it important to stagger the time enzyme addition to each tube?

<p>Staggering the time ensures that each reaction has a distinct incubation time, allowing for accurate measurement of absorbance at the same time interval.</p> Signup and view all the answers

What role does sodium carbonate play in the enzyme activity measurement?

<p>Sodium carbonate is used to stop the reaction after a set incubation period, allowing for consistent measurement of absorbance.</p> Signup and view all the answers

What should be done with the cuvette before measuring the absorbance in the spectrophotometer?

<p>The cuvette must be wiped with a Kimwipe to ensure that there are no smudges or contaminants affecting the absorbance reading.</p> Signup and view all the answers

How is the concentration of o-nitrophenolate produced determined from absorbance readings?

<p>The concentration is determined using a standard curve that correlates absorbance to o-nitrophenolate concentration.</p> Signup and view all the answers

What formula is used to calculate the reaction rate of o-nitrophenolate formation?

<p>The reaction rate is calculated by dividing the concentration of o-nitrophenolate produced by the time interval, typically in minutes.</p> Signup and view all the answers

What is the significance of using a blank solution when zeroing the spectrophotometer?

<p>Using a blank solution ensures that the spectrophotometer readings account for the background absorbance of the reagents, providing accurate measurements of the samples.</p> Signup and view all the answers

What is the dilution factor for a solution made by combining 1.0 mL of stock solution with 9.0 mL of diluent?

<p>10</p> Signup and view all the answers

Using the dilution formula, how would you express the concentration of a diluted sample (Cd) if the concentration of the undiluted sample (Cu) is 100 mg mL−1 and the dilution is 0.1?

<p>10 mg mL−1</p> Signup and view all the answers

In the formula V1C1 = V2C2, what does V1 represent?

<p>Volume of the original or stock solution.</p> Signup and view all the answers

If you want to prepare 200 µL of a 25 mg mL−1 solution from a 100 mg mL−1 stock solution, what volume of the stock solution (V1) do you need?

<p>50 µL</p> Signup and view all the answers

What are the components of a microcosm that may be affected by fertilizer based on the objectives of the eutrophication experiment?

<p>Terrestrial and aquatic plants, water chemistry, and aquatic microorganisms.</p> Signup and view all the answers

What is the formula used to calculate dilution when determining the concentration of a diluted sample?

<p>Cd = Cu × D</p> Signup and view all the answers

What is the significance of conducting the eutrophication experiment in both terrestrial and aquatic environments?

<p>To compare and analyze how fertilizer impacts different ecosystems.</p> Signup and view all the answers

How is the dilution factor calculated and what does it represent in a preparation?

<p>It is the total volume of the solution; it represents the degree of dilution applied.</p> Signup and view all the answers

Flashcards

Prepare 1:10 DCMU solution

Dilute DCMU stock solution 1:10 using RO water.

Prepare 1:100 DCMU solution

Dilute a 1:10 DCMU solution 1:10 using RO water.

Control Tube 1 (no DCMU)

Contains buffer, water, and DCPIP but no DCMU.

Cuvette Preparation (Steps 3-5)

Transfer chloroplast solution to labeled cuvettes, seal with parafilm, and invert to thoroughly mix.

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Hill Reaction Measurement

Measure change in absorbance of DCPIP over time in the presence and absence of DCMU.

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DCMU

A chemical herbicide that inhibits photosynthesis.

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Absorbance Measurement

Use a spectrophotometer to determine the absorbance of light by a substance.

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Cuvette #3 measurement

Measure absorbance of Cuvette #3 solutions every 2 minutes for 14 minutes.

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Ethanol fermentation

A process where yeast converts carbohydrates into ethanol and carbon dioxide.

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Simple carbohydrates

Sugars like glucose that yeast can directly use for fermentation.

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Complex carbohydrates

Larger molecules like starch that need to be broken down into simple sugars first for fermentation.

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Structural carbohydrates

Components like cellulose, that plants use to build their structures, harder to ferment.

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Yeast

Microscopic organisms that are used for fermentation to produce desirable outputs.

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Fermentation byproducts

The ethanol and CO2 created during the fermentation of carbohydrates by yeast

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Glucose

A simple sugar that is a crucial part of fermentation.

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Ethanol Fuel

Ethanol with a purity of 200 proof (100%) and an additive so alcohol tax is avoided.

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ONPG Solution

A solution containing o-nitrophenyl-β-D-galactopyranoside, used in enzymatic reactions.

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Phosphate Buffer

A solution maintaining a stable pH in enzymatic reactions.

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Enzyme Solution

A solution containing the enzyme to be tested in Part 1.

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Blank Tube

A control tube containing all components except substrate or enzyme, used as a reference for measuring absorbance.

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Reaction Rate

The speed at which the substrate is broken down by the enzyme.

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Spectrophotometer

A device measuring the absorbance of light by a solution.

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Inhibitor A

An unknown chemical substance used to determine its effect on enzyme activity (as inhibitor).

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Sodium Carbonate

A solution used to stop the enzymatic reaction.

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Micropipette Tip Attachment

The tip must be placed securely on the micropipette barrel to ensure a tight seal and prevent leakage of air or tip detachment.

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Micropipette Control Button Positions

The control button has three positions: "At Rest" for inactivity, "Stop 1" to measure pre-selected volume, and "Stop 2" for expelling last drops when transferring liquid.

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Micropipette Measurement Steps

Attach the tip, press to the first stop, immerse vertically, release slowly, slide out of solution, and discard.

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Micropipette Transfer Steps

Position tip carefully in the receiving vessel, press to first stop, press to second stop for complete expulsion, remove the tip slowly.

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Micropipette Tip Disposal

Discard used tips into designated disposal bags.

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Micropipette Volume Units

0.1 milliliter (mL) is equal to 100 microliters (μL), and 1.0 mL is equal to 1000 μL.

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Micropipette Handling

Handle micropipettes carefully to avoid damage or dropping. Store in the provided stands when not in use.

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Spectrophotometer Precautions

Ensure your solution doesn't contain unwanted molecules absorbing light, since this will cause inaccuracies in your experiments.

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Serial Dilution

A method of progressively diluting a solution by a constant factor, creating a series of solutions with decreasing concentrations.

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Enzyme Activity

The rate at which an enzyme catalyzes a specific reaction, measured by the amount of product formed per unit time.

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Why measure enzyme activity?

To determine the optimal concentration of the enzyme for an experiment, ensuring it's neither too diluted nor too concentrated for accurate measurement.

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Standard Curve

A graph that relates the absorbance of a solution to the concentration of the substance being measured.

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o-Nitrophenolate

A yellow-colored product produced by the enzyme b-galactosidase, used to measure enzyme activity.

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Aliquot

A small portion of a larger sample taken for analysis or testing.

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Dilution

The ratio of the volume of the original solution to the total volume of the solution after dilution. It represents the fraction of the original solution present in the diluted solution.

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Dilution Factor

The total volume of the diluted solution, which is the sum of the original solution volume and the diluent volume.

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Concentration of Diluted Sample (Cd)

The concentration of the substance in the diluted solution, calculated by multiplying the undiluted concentration by the dilution.

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Concentration of Undiluted Sample (Cu)

The concentration of the substance before dilution, also known as the stock solution concentration.

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V1C1 = V2C2

A formula used to calculate dilutions, where V1 is the volume of the stock solution, C1 is the concentration of the stock solution, V2 is the volume of the new solution, and C2 is the concentration of the new solution.

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What is the formula for calculating the concentration of a diluted sample (Cd)?

Cd = Cu × D, where Cd is the concentration of the diluted sample, Cu is the concentration of the undiluted sample, and D is the dilution.

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Calculating Dilution

The process of determining the ratio of the original solution volume to the total volume of the solution after dilution.

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Preparing a Diluted Solution

The process of making a solution with a lower concentration by adding a diluent to a stock solution.

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Study Notes

Photosynthesis

  • Photosynthesis is the process by which green plants, algae, and certain bacteria convert light energy and CO2 into organic molecules.
  • The overall process of photosynthesis can be summarized by the equation: 6 CO2 + 12 H2O → C6H12O6 + 6 O2 + 6 H2O.
  • Photosynthesis involves two categories: light-dependent reactions and light-independent reactions (Calvin Cycle).
  • In the light-dependent reactions, sunlight energy is trapped and converted to ATP and NADPH by an electron transport chain (ETC) and chemiosmosis.
  • The products of light-dependent reactions are used to drive the Calvin Cycle, in which CO2 is reduced to carbohydrate.
  • While the light-independent reactions don't directly require light, they are dependent on the products of light-dependent reactions.
  • Light absorbed by photosynthetic pigments funnels energy to the reaction center chlorophyll a in Photosystems I (PSI) and II (PSII).
  • Excited electrons are passed to the primary electron acceptor associated with the photosystem.
  • PSII releases energy to transfer protons from the stroma to the thylakoid space.
  • This generates an electrochemical gradient that powers ATP synthesis.
  • PSII chlorophyll a in becomes a strong oxidizing agent that strips electrons from water to replace the ones lost.
  • Light splitting (photolysis) splits H2O into protons and oxygen.
  • Excited electrons in PSI are transferred to a separate ETC.
  • The electrons are used to reduce NADP+ to NADPH.
  • Electrons lost by PSI are replaced by electrons donated by plastocyanin.

The Hill Reaction

  • In 1937, Robert Hill discovered that isolated chloroplasts produce oxygen when exposed to light and given an electron acceptor.
  • The Hill reaction is formally defined as the reduction of any electron acceptor by electrons and protons from water, with the production of oxygen, when chloroplasts are exposed to light: H2O + A → AH2 + O2.
  • Plant leaves are grinded then centrifuged to separate the chloroplasts from other cellular components.
  • The isolated chloroplasts were suspended in cold sodium-potassium phosphate buffer (pH 7.3).
  • A pigment DCPIP (oxidized blue) will act as a stronger oxidizing agent than NADP+, thus replacing NADP+ in the light-dependent reactions.
  • DCPIP becomes colorless when reduced.
  • The change in light absorbance at 620 nm can be measured, with the aid of a standard curve, to determine the DCPIP oxidized concentration over time.

Isolating Chloroplasts

  • Collect a tube of chloroplast solution from the back bench. Wrap tube in aluminum foil and keep it on ice.
  • This prevents the chloroplasts from overheating.
  • Label 3 test tubes “1” through “3”.
  • Wrap Tube #2 in aluminum foil.
  • Place all 3 tubes in the test tube rack on your bench.
  • Label 3 cuvettes “1” through “3”.
  • Wrap Cuvette #2 in aluminum foil.
  • Place all 3 cuvettes in a cuvette rack.
  • Set up the light source and beaker of water.
  • Ensure the spectrophotometer is on and set to 620 nm.
  • Add 3 mL buffer and 1 mL RO water to Tube #1; do not add DCPIP (control).

Experiment II: Effect of Inhibitor on the Hill Reaction

  • DCMU is a chemical used in many herbicides and an inhibitor of photosynthesis.
  • A stock solution of DCMU is provided.
  • Prepare a 1:10 and a 1:100 dilution of the inhibitor stock solution using RO water.
  • Label 4 test tubes “1” through “4”.
  • Add 2 mL buffer and 2 mL RO water to Tube #1 (control).
  • Add 2 mL buffer, 1 mL DCPIP, and 1 mL of Stock DCMU to Tube #2.
  • Add 2 mL buffer, 1 mL DCPIP, and 1 mL 1:10 DCMU to Tube #3
  • Add 2 mL buffer, 1 mL DCPIP, and 1 mL 1:100 DCMU to Tube #4.

Additional Notes

  • Specific steps and instructions to carry out the experiment are included.
  • Different steps and experimental setups are described for different experiments and controls.
  • Important aspects of proper setup, like the use of foil, are explained
  • Data tables for recording results are provided.

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Description

This quiz explores the procedures and principles behind absorbance measurement along with key fermentation concepts. It covers important steps in experiments involving DCMU, as well as the role of yeast in alcohol production and baking. Test your knowledge of these scientific processes and their historical contexts.

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