bisc-220 lab final

[Safety] What PPE does this course require during lab experiments?

Long pants, a buttoned lab coat, gloves, and shoes covering the entire foot with closed backs; goggles when required. Contain long hair and loose clothing.

[Safety] What should you do before leaving the lab or touching personal electronics?

Remove gloves before handling electronics or writing utensils. Remove both gloves and lab coat before leaving the lab.

[Safety] What should you do if glass breaks or a spill occurs?

Notify the instructor immediately. Never pick up broken glass with bare hands; follow the instructor's cleanup directions.

[Safety] Where do blood-contaminated slides and coverslips go in this course?

The red biohazard sharps container.

[Safety] Where do blood-contaminated gloves or paper towels go?

The biohazard non-sharps container with a red bag.

[Safety] Where does clean, uncontaminated broken glass go?

The designated puncture-resistant clean-glass container, not regular trash.

[Safety] Where do hazardous-chemical-contaminated non-glass materials go?

The designated solid chemical waste container; contaminated glass or blades go in the chemical sharps container.

[Safety] What takes priority when waste has both biological and chemical hazards?

Chemical waste disposal takes priority under this course's guidelines.

[Safety] Where do disposable plastic pipette tips go?

Choose by contamination: regular trash for nonhazardous materials, biohazard non-sharps for biological hazards, or solid chemical waste for hazardous chemicals. Plastic tips do not go in sharps bins in this course.

[Safety] Where do Benedict's, Biuret, iodine, and chromatography solvent liquid waste go?

Their appropriately labeled chemical waste containers; follow the current lab-specific instructions and keep incompatible waste streams separate.

[Pipetting] How many microliters are in 1 mL?

1,000 microliters (µL). For example, 750 µL = 0.750 mL.

[Pipetting] What does P1000 mean, and what range is specified for the course's P1000?

Its maximum volume is 1,000 µL; the manual specifies a recommended range of 100-1,000 µL for this instrument.

[Pipetting] Why must you identify the pipette model before reading its display?

Digit place values depend on the model. A three-digit display of 100 corresponds to 1,000 µL on the illustrated P1000 but 100 µL on a P200.

[Pipetting] Which plunger stop is used before drawing up a sample?

The first stop. Immerse the tip, then slowly release the plunger to aspirate the set volume.

[Pipetting] Which plunger stop is used to finish dispensing a sample?

The second stop, which pushes out the remaining liquid. Keep the plunger depressed until the tip is clear of the receiving liquid.

[Pipetting] What happens if you release the plunger while the tip is still submerged after dispensing?

Liquid is drawn back into the tip, removing part of the sample you just dispensed.

[Pipetting] Why should a micropipette remain upright with its tip pointed down?

Liquid can flow into and damage the internal mechanism if the pipette is tilted or inverted.

[Pipetting] Why must you use a tip and stay within the pipette's volume range?

A tip protects the internal mechanism from liquid; operating outside the specified range can damage the pipette and produce inaccurate volumes.

[Pipetting] Why should you change tips between different samples?

To prevent carryover and cross-contamination.

[Pipetting] What is an aliquot, and why use one?

A measured portion removed from a stock solution. Working from aliquots reduces repeated access to the stock and the risk of contamination or spills.

[Carbohydrates] What are the four major classes of biological macromolecules?

Carbohydrates, proteins, lipids, and nucleic acids. Lipids are grouped with macromolecules but generally are not true polymers of repeating monomers.

[Carbohydrates] What are the building blocks of carbohydrates, proteins, and nucleic acids?

Monosaccharides build carbohydrates; amino acids build proteins; nucleotides build nucleic acids.

[Carbohydrates] What happens during dehydration synthesis?

Smaller molecules join through a covalent bond while a water molecule is released.

[Carbohydrates] What happens during hydrolysis?

Water is used to break a covalent bond, splitting a larger molecule into smaller molecules.

[Carbohydrates] Which monosaccharides make up maltose, sucrose, and lactose?

Maltose = glucose + glucose; sucrose = glucose + fructose; lactose = glucose + galactose.

[Carbohydrates] What bond links monosaccharides in carbohydrates?

A glycosidic linkage.

[Carbohydrates] How do starch, glycogen, and cellulose differ in function?

Starch stores glucose in plants; glycogen stores glucose in animals and many microbes; cellulose provides structural support in plant cell walls.

[Carbohydrates] How do starch and glycogen differ in branching?

Both contain glucose with alpha linkages, but glycogen is more highly branched. Starch includes unbranched amylose and branched amylopectin.

[Carbohydrates] Why can humans digest starch but not cellulose?

Human digestive enzymes can hydrolyze starch's alpha linkages but lack cellulase to hydrolyze cellulose's beta-1,4 linkages.

[Carbohydrates] What does Benedict's reagent detect?

Reducing sugars, including glucose and some disaccharides; it does not detect every carbohydrate.

[Carbohydrates] What is a reducing sugar?

A sugar with an available reducing group that can reduce copper ions in Benedict's reagent under the test conditions.

[Carbohydrates] What are negative and positive Benedict's test results?

Negative remains blue. Positive can be green, yellow, orange, or brick-red precipitate after heating, generally increasing with reducing-sugar concentration.

[Carbohydrates] What chemical change produces Benedict's red precipitate?

Reducing sugars reduce Cu2+ to Cu+, producing insoluble copper(I) oxide, Cu2O.

[Carbohydrates] Why is Benedict's test only semiquantitative in this lab?

Color gives an approximate concentration category rather than a precise concentration.

[Carbohydrates] How is the Benedict's test performed in this manual?

Mix 1 mL sample with 2 mL reagent and heat in a boiling-water bath for about 5 minutes, removing fully red samples early.

[Carbohydrates] What does Lugol's iodine detect, and what is a positive result?

Starch; the yellow-amber reagent becomes dark blue or blue-black through interaction with the starch structure.

[Carbohydrates] Does Lugol's test require boiling?

No. The starch color change should occur promptly after adding iodine.

[Carbohydrates] What results are expected for glucose with Benedict's and Lugol's tests?

Benedict's positive after heating; Lugol's negative because glucose is not starch.

[Carbohydrates] What results are expected for unhydrolyzed sucrose with Benedict's and Lugol's tests?

Both negative: sucrose is nonreducing and is not starch.

[Carbohydrates] What results are expected for unhydrolyzed starch with Benedict's and Lugol's tests?

Typically Benedict's negative under this lab's conditions and Lugol's positive.

[Carbohydrates] What is an appropriate negative control for the carbohydrate tests?

DI water tested with the same reagent volumes and procedure as the samples.

[Carbohydrates] What are appropriate positive controls for Benedict's and Lugol's tests?

Known glucose for Benedict's and known starch for Lugol's.

[Carbohydrates] Why does heating sucrose with HCl make a later Benedict's test positive?

Acid hydrolysis breaks sucrose into glucose and fructose, which are reducing sugars.

[Carbohydrates] What do the 0-minute and 5-minute sucrose hydrolysis samples compare?

The effect of prior acid-heating time on production of reducing sugars; both aliquots then receive the Benedict's heating step.

[Carbohydrates] An unknown is Benedict's positive both before and after hydrolysis. Does this prove sucrose is present?

No. Glucose alone could produce both positives; glucose mixed with sucrose could also do so. These results cannot distinguish those possibilities.

[Carbohydrates] An unknown is Benedict's negative before hydrolysis, positive after hydrolysis, and Lugol's negative. What is consistent with this result?

A hydrolyzable nonreducing sugar such as sucrose, within the candidate substances used in this lab.

[Proteins] What covalent bond links amino acids in proteins?

A peptide bond.

[Proteins] What does the Biuret assay detect?

Peptide bonds in proteins and sufficiently long peptides, rather than free amino acids.

[Proteins] What color changes occur in the Biuret assay?

Reagent alone is blue; proteins produce violet or lavender, while smaller peptides can produce pinkish color.

[Proteins] Why does a more concentrated protein sample generally produce a darker Biuret color?

It contains more peptide bonds per unit volume available to interact with copper ions in the alkaline reagent.

[Proteins] How is Biuret both qualitative and quantitative?

A color change indicates protein presence; absorbance compared with known standards estimates concentration.

[Proteins] What standard protein and starting concentration are used in this lab?

Bovine serum albumin (BSA), starting at 2 mg/mL.

[Proteins] What is a serial dilution?

A series in which each diluted solution becomes the starting solution for the next dilution.

[Proteins] What happens when 4 mL protein solution is mixed with 4 mL water?

The total volume doubles while protein amount stays constant, so concentration is halved: a twofold dilution.

[Proteins] What are the BSA concentrations in stock tubes S1-S5 before adding Biuret?

S1 = 2; S2 = 1; S3 = 0.5; S4 = 0.25; S5 = 0.125 mg/mL.

[Proteins] Why must each serial-dilution tube be mixed before transferring to the next?

To make the solution uniform so the transferred portion has the intended concentration.

[Proteins] What equation describes dilution?

C1V1 = C2V2, where V2 is the final total volume. Dilution factor = V2/V1 = C1/C2.

[Proteins] What are the final standard concentrations after mixing 1 mL of S1-S5 with 1 mL Biuret?

1, 0.5, 0.25, 0.125, and 0.0625 mg/mL, respectively. Adding reagent causes another twofold dilution.

[Proteins] What incubation and wavelength are used for the Biuret assay?

Incubate for 20 minutes at room temperature, then measure absorbance at 540 nm.

[Proteins] What is in the Biuret blank?

1 mL DI water plus 1 mL Biuret reagent, with no protein.

[Proteins] Why blank the spectrophotometer?

To set a baseline accounting for absorbance from the reagent, solvent, and cuvette so measured absorbance reflects the sample.

[Proteins] What does a spectrophotometer measure in this experiment?

How much light at 540 nm is absorbed by the Biuret-treated sample.

[Proteins] What belongs on each axis of a protein standard curve?

x-axis: known protein concentration, mg/mL; y-axis: measured absorbance at 540 nm, which is unitless.

[Proteins] How do you calculate concentration from a standard curve y = mx + b?

Substitute absorbance for y and solve x = (y - b)/m. Use the same concentration definition used for the standards.

[Proteins] A standard curve is A = 0.40C + 0.02. What concentration gives A = 0.22?

C = (0.22 - 0.02)/0.40 = 0.50 mg/mL on that curve's concentration basis.

[Proteins] If the curve uses final assay concentrations and an unknown measures 0.50 mg/mL, what was its concentration before equal-volume Biuret addition?

1.0 mg/mL. Multiply the measured final concentration by the twofold dilution factor.

[Proteins] Should you apply another factor of two if the standard curve already uses concentrations before Biuret addition?

No. That curve directly estimates the unknown's concentration before Biuret addition. Match the calculation to the curve's x-axis definition.

[Proteins] What should you do if an unknown's absorbance exceeds the range of the standards?

Dilute the unknown, assay it again within the standard range, and account for the additional dilution factor.

[Proteins] How should cuvettes be handled and oriented?

Hold them near the top, wipe optical faces with a Kimwipe, orient the clear optical faces with the beam, and avoid fingerprints or bubbles in the light path.

[Proteins] Why can fingerprints, bubbles, or an incorrect cuvette orientation distort absorbance?

They interfere with light transmission independently of the sample's protein concentration.

[Proteins] What special tip-reuse order is allowed for the Biuret standards in this manual?

Blank, then lowest to highest concentration: blank -> tube 5 -> 4 -> 3 -> 2 -> 1. Use a separate tip for the unknown.

[Scientific method] What is a hypothesis?

A testable, falsifiable proposed explanation for an observation or relationship.

[Scientific method] What is a prediction?

A specific expected experimental result if a hypothesis is correct, often phrased as an if-then statement.

[Scientific method] What makes a hypothesis testable and falsifiable?

Its variables can be measured, and a possible observation could contradict the proposed explanation.

[Scientific method] Does matching a prediction prove a hypothesis?

No. The results support it under the tested conditions; alternative explanations and future tests remain possible.

[Scientific method] What is an independent variable?

The factor the investigator deliberately changes, such as milk type, substrate type, or light exposure.

[Scientific method] What is a dependent variable?

The measured response, such as glucose concentration, CO2 volume, or number of floating leaf discs.

[Scientific method] What are controlled variables?

Other factors kept consistent so they do not explain differences between treatment groups.

[Scientific method] What are levels of treatment?

The specific values or categories of the independent variable being tested.

[Scientific method] What is the purpose of a positive control?

To show that the experimental system can produce the expected measurable response.

[Scientific method] What is the purpose of a negative control?

To establish background or absence of the expected response when the relevant treatment or component is missing.

[Scientific method] How is a control treatment different from a controlled variable?

A control treatment is a comparison group; a controlled variable is a condition kept consistent across groups.

[Scientific method] Why include independent replicates?

To assess reproducibility and variation among independent experimental units rather than relying on one result.

[Scientific method] What do mean and standard deviation describe?

Mean describes the average; standard deviation describes the spread of replicate values around their mean.

[Scientific method] Which observations should be used for SD of replicate fermentation results at 12 minutes?

The independent replicate CO2 measurements for the same substrate at 12 minutes, not the different time points from one flask.

[Scientific method] Where do the independent and dependent variables usually appear on a graph?

Independent variable on x; dependent variable on y. Label both with names and units where applicable.

[Scientific method] When should you use a line graph versus a bar graph?

Use a line graph for a response over a continuous variable such as time; use a bar graph for separate categories such as substrate types.

[Scientific method] What does a histogram display?

The frequency distribution of numerical observations grouped into intervals, or bins.

[Scientific method] Where should table titles and figure legends go in this course?

A table title goes above its table; a figure legend goes below its figure. Number them consecutively and include enough context to understand them.

[Scientific writing] What distinguishes a primary research source from a review article?

A primary article reports original work performed by its authors; a review synthesizes and interprets findings from other studies.

[Scientific writing] What are peer review and critical reading meant to evaluate?

Whether methods, evidence, and reasoning adequately support the conclusions; publication alone does not remove the need to evaluate evidence.

[Scientific writing] What belongs in an introduction and a methods section?

Introduction: background, question, hypothesis, and purpose. Methods: enough detail about design and procedures to allow replication.

[Scientific writing] How do results and discussion sections differ?

Results describe observations and trends with data; discussion interprets them, relates them to the hypothesis and literature, and addresses limitations and next steps.

[Scientific writing] What is an abstract, and what belongs in references?

An abstract briefly summarizes the study. References list the works actually cited; this course specifies APA style for lab reports.

[Enzymes] How do enzymes speed reactions?

They lower activation energy, allowing reactions to proceed faster without being consumed in the overall reaction.

[Enzymes] What is an active site?

The region of an enzyme where its substrate binds and catalysis occurs.

[Enzymes] What does the induced-fit model describe?

Substrate binding causes a small change in enzyme shape that helps catalysis.

[Enzymes] Why does lactase break down lactose but not sucrose?

Its active site recognizes lactose's structure and bond arrangement; sucrose does not fit productively.

[Enzymes] What reaction does lactase catalyze?

Lactose + water -> glucose + galactose, by hydrolyzing lactose's beta-glycosidic bond.

[Enzymes] What reaction does sucrase catalyze?

Sucrose + water -> glucose + fructose.