Cell Biology Lab: Lab Exam #1

Contamination can arise from:

• Airborne microbes (fungal spores, bacteria).

• Unsterile tools or surfaces.

• Improperly sterilized media.

• Human handling errors (e.g., touching sterile areas).

What are sources of contamination in tissue culture?

• Ocular Lens: Magnifies the image (typically 10x).

• Objective Lenses: Provide different levels of magnification (4x, 10x, 40x, 100x).

• Stage: Holds the specimen slide.

• Condenser Lens: Focuses light onto the specimen.

• Iris Diaphragm: Controls light intensity.

• Coarse/Fine Focus Knobs: Adjust image clarity.

What are the names and functions of the parts of a compound light microscope?

• ocular lens, objective lenses, stage, condenser lens, iris diaphragm, coarse/fine focus knobs

The limit of resolution (R) determines the smallest distance at which two objects can still be distinguished as separate. It is calculated using the equation:

i) R = (0.61 * λ) / NA
Where:

• λ = Wavelength of light (typically 450 nm for blue light).

• NA = Numerical aperture of the lens.

ii) NA = n * sin(θ)
Where:

• n = Refractive index of the medium.

• θ = Half-angle of light entering the lens.

How do you calculate the limit of resolution?

A hemocytometer is used to count cells within a defined grid. The formula used is:

i) Total Cells = (Average Count/Area) * Dilution Factor * Volume Conversion

How do you use a hemocytometer to calculate cell number?

Cells are disrupted using mechanical force (homogenizers, blenders) to release organelles.

How do you homogenize cells?

i) RCF = 1.119 × 10⁻⁵ × (RPM)² × X
Where:

• X = Rotor radius (cm).

How do you calculate RCF from RPM?

DPIP (2,6-dichlorophenol-indophenol) is used as an artificial electron acceptor in place of FAD+ in the electron transport chain. In its oxidized state, DPIP is blue, but when it is reduced by electrons from succinate, it becomes colorless. This color change allows researchers to measure mitochondrial activity by tracking the decrease in absorbance at 600 nm using a spectrophotometer.

What is the function of DPIP in the mitochondrial reaction experiment?

FADH₂ is normally the electron carrier that accepts electrons in the Krebs cycle. However, FADH₂ is not easily detectable spectrophotometrically. DPIP, in contrast, undergoes a visible color change, allowing researchers to quantify succinate oxidation rates using a spectrophotometer.

Why was DPIP used instead of natural electron acceptors like FAD?

By analyzing enzyme kinetics using Michaelis-Menten and Lineweaver-Burk plots, researchers can distinguish between competitive and noncompetitive inhibition:

• Competitive inhibition: The Lineweaver-Burk plot shows an increase in Km but no change in Vmax (lines intersect at the y-axis).

• Noncompetitive inhibition: The plot shows a decrease in Vmax while Km remains constant (lines intersect at the x-axis).

How can you experimentally determine competitive vs. noncompetitive inhibition?

To accurately measure specimen size under a microscope, the ocular micrometer (inside the eyepiece) must be calibrated against a stage micrometer (a slide with a known scale). The calibration steps are:

1. Align the ocular micrometer with the stage micrometer using the lowest objective lens.

2. Determine how many ocular divisions match a known distance on the stage micrometer.

3. Calculate the value of one ocular unit by dividing the stage micrometer length by the corresponding ocular micrometer divisions.

4. Repeat the process for each objective lens, as magnification changes the scale.

5. Use the calibrated values to measure specimens by multiplying the number of ocular units by the conversion factor for that magnification

How do you calibrate the ocular micrometer in a microscope?

Micropipettes must be calibrated to ensure accurate volume measurement. The process involves:

1. Setting the pipette to a known volume (e.g., 100 µL).

2. Pipetting distilled water onto an analytical balance and recording the weight. Since water has a density of 1.0 g/mL at room temperature, the expected weight should match the volume (e.g., 100 µL = 0.100 g).

3. Comparing the measured weight to the expected value. If discrepancies exist, the pipette may need recalibration.

4. Adjusting the pipette using the manufacturer’s calibration tool if the deviation exceeds the acceptable error range.

How do you calibrate a micropipette?

A spectrophotometer must be blanked before measuring sample absorbance. The steps are:

1. Turn on the spectrophotometer and allow it to warm up (if required).

2. Select the correct wavelength based on the experiment (e.g., 600 nm for DPIP reduction).

3. Prepare a blank solution, which contains all reagents except the substance being measured (e.g., a cuvette with buffer but no DPIP).

4. Insert the blank into the spectrophotometer and set absorbance to zero. This removes background absorbance, ensuring accurate sample readings.

5. Measure sample absorbance and ensure cuvettes are clean and properly aligned.

How do you calibrate a spectrophotometer?

Totipotency is the ability of a single cell to develop into a complete organism, meaning it retains all the genetic instructions necessary for forming an entire plant.

What is totipotency?

This principle was demonstrated in the carrot tissue culture experiment by taking small sections of carrot root (explant) and culturing them in a controlled environment. The cells first formed a mass of undifferentiated tissue called a callus before differentiating into roots and shoots, eventually regenerating into a complete plant.

How is totipotency demonstrated in carrot tissue culture?

Two different media were used to facilitate this process: a callus induction medium containing the synthetic plant hormone auxin (2,4-D) to stimulate cell division and de-differentiation, and a shoot development medium that lacked auxin to allow the cells to re-differentiate into specialized plant tissues.

Why were 2 different media used for the culture?

The incubation conditions were carefully controlled with indirect or fluorescent lighting set to a cycle of 16 hours of light and 8 hours of darkness, while the temperature was maintained between 24 and 27°C to optimize growth.

What incubation conditions were used for the carrot culture?

For plant cells to grow successfully in culture, they require a combination of essential components.

• Inorganic salts provide macronutrients such as nitrogen, phosphorus, potassium, magnesium, sulfur, and calcium, along with micronutrients like iron, manganese, zinc, copper, molybdenum, and boron.

• A carbon source, typically sucrose, is necessary for energy production since cultured plant cells do not perform photosynthesis efficiently.

• Growth regulators, including auxins and cytokinins, control cell division and differentiation. Vitamins such as B1 (thiamine), B6 (pyridoxine), and B3 (nicotinic acid) support metabolic processes

• While a gel matrix, usually agar, offers a solid support system for cell growth.

What components are required for growth of plant cells in culture?

In tissue culture, a callus is a mass of undifferentiated plant cells that forms when an explant is cultured in an appropriate medium.

A clone refers to a genetically identical group of cells or organisms derived from a single ancestor, ensuring uniformity in experimental results.

An explant is the small piece of plant tissue used to initiate culture growth, which, under the right conditions, can regenerate into a whole plant.

Differentiation is the process by which unspecialized cells develop into distinct types of plant tissue, such as roots, leaves, or stems.

In contrast, de-differentiation occurs when specialized cells revert to an unspecialized state, allowing them to proliferate and later re-differentiate into new structures.

Definitions from the carrot lab such as: callus, clone, explant, differentiation, de-differentiation. Be able to use these terms properly to describe the experiment.

Maintaining a sterile environment is critical in plant tissue culture to prevent contamination by bacteria, fungi, and other microorganisms. This was achieved using several key techniques, including working in a laminar flow hood to limit airborne contaminants and using only autoclaved media and tools, which were sterilized at 121°C and 15 psi to eliminate all microorganisms. Metal instruments such as forceps and scalpels were flamed in an alcohol burner before use.

What techniques and tools were used to maintain a sterile culture during preparation?

To eliminate all microorganisms. Metal instruments such as forceps and scalpels were flamed in an alcohol burner before use to further ensure sterility.

Why were things autoclaved? Flamed?

Contamination sources include airborne particles carrying fungal spores, unsterilized tools or hands, and improper sealing of culture containers.

What are the sources of different types of contamination?

R = 0.61 pi / NA

Calculate the limit of resolution. Know and understand the equations.

Using a compound light microscope, prokaryotic and eukaryotic cells can be distinguished by their size and structural differences. Prokaryotic cells, such as bacteria, are much smaller, typically ranging from 0.1 to 5 micrometers in diameter, and lack a nucleus or membrane-bound organelles. Their genetic material is found in a nucleoid region, and they often have rigid cell walls and external appendages like flagella. In contrast, eukaryotic cells, which include plant and animal cells, are larger (10–100 micrometers), have a well-defined nucleus, and contain organelles such as mitochondria and the endoplasmic reticulum.

Within eukaryotic cells, plant cells can be differentiated from animal cells by the presence of a rigid cell wall, chloroplasts for photosynthesis, and a large central vacuole. Animal cells lack these structures but have centrioles and lysosomes instead.

How would you distinguish between prokaryotic and eukaryotic cells? Animal and plant cells using a light microscope?

What are the distinguishing features and relative sizes of each?

A hemocytometer is a specialized counting chamber used to estimate cell concentrations in a liquid suspension. To use it, a sample is pipetted into the chamber, and cells are counted in five specific squares (four corner squares and the center square). The total number of cells is then used in the formula: PUT FORMULA HERE.

How do you use a hemocytometer to calculate cell number?

Be able to calculate cell number if given data.

To measure objects under a microscope accurately, the ocular micrometer must first be calibrated using a stage micrometer, which has a known scale. The two scales are aligned, and the number of ocular units corresponding to a specific distance on the stage micrometer is determined. The calibration factor is calculated using the equation:

1 ocular unit = stage micrometer distance (um) / number of ocular spaces

If 100 micrometers align with 50 ocular units, then each ocular unit represents 2 micrometers, allowing measurements of specimen size in micrometers.

How do you calibrate the ocular micrometer (know the calibration steps).

How do you calculate the size of objects using an ocular micrometer during calibration? Be able to do these calculations if provided data.

The relative centrifugal force (RCF) can be calculated using the equation RCF=1.119×10−5(rpm)2(X)

What is the equation for RCF or how to determine RPM?

• X is the radius in cm.

Differential centrifugation is a technique used to separate cellular components based on size and density by spinning samples at increasing centrifugal forces. Initially, low-speed centrifugation at 600 x g pellets the largest organelles, such as nuclei. The supernatant is then spun at higher speeds (e.g., 15,000 x g), which pellets mitochondria. Further centrifugation at even greater forces (e.g., 100,000 x g) isolates microsomes (fragments of the endoplasmic reticulum and ribosomes). The final supernatant contains soluble cytosolic proteins.

What is the basis for separation of organelles during centrifugation?

In spectrophotometry, blanking the instrument is essential to eliminate background absorbance from solvents or reagents. This is done by placing a cuvette containing only the solvent (e.g., water or buffer) into the spectrophotometer and setting its absorbance to zero. The absorbance of experimental samples is then measured at a specific wavelength corresponding to the peak absorbance of the analyte.

When doing the spectrophotometry, \ why do we blank the machine?

Abs = log(1/T%)

For instance, if the T% is 63% then Abs = log(1/0.63)

The absorbance of experimental samples is then measured at a specific wavelength corresponding to the peak absorbance of the analyte

How do you convert %T to Abs? How do you decide what wavelength to use?

Cold temperatures slow down enzymatic activity, reducing the rate at which proteins, nucleic acids, and other biomolecules participate in degradation.

Why were tubes, samples, and reagents frequently kept on ice?

Buffers are also used extensively to maintain a stable pH and ionic environment, preventing unwanted chemical changes that could alter experimental results. By stabilizing pH, buffers help enzymes retain their functional shapes and prevent denaturation.

Why were buffers used during the experiment?

Cells are homogenized to break open membranes and release organelles while minimizing damage to cellular structures. In this experiment, a tissue homogenizer was used to disrupt the cells mechanically, creating a homogenate containing all cellular components in suspension.

Organelles were then separated using differential centrifugation, where successive spins at increasing speeds caused different-sized cellular structures to sediment at different rates. The nuclear fraction was pelleted first at 600 x g, followed by mitochondria at 15,000 x g, and microsomal fragments at even higher speeds.

How do you homogenize cells? How were organelles separated? How were proteins separated from nucleic acids?

Proteins were separated from nucleic acids using perchloric acid (PCA) treatment, where the cold PCA precipitated proteins while leaving nucleic acids in solution. Hot PCA treatments were used to further hydrolyze nucleic acids, allowing for their quantification separately from proteins.

What were the results of the cold and hot PCA treatments?

For example, when preparing protein and DNA standard curve samples, a known concentration of protein or DNA stock solution is diluted to a range of concentrations, allowing for the generation of a calibration curve to quantify unknown samples.

How do you use stock solutions?

This follows the dilution equation:

C1V1=C2V2

where C1​ and V1​ are the concentration and volume of the stock solution, and C2​ and V2 are the desired concentration and final volume of the diluted solution.

If 1 mL of a 10 mg/mL stock solution is diluted to a final volume of 10 mL, the new concentration would be 1 mg/mL, demonstrating a 10-fold dilution.

How do you calculate the volume of stock required to supply a given amount of a compound (example: making a protein and DNA standard curve samples), or the amount of compound supplied if a given volume is added?

A standard curve is a graphical representation of known concentrations of a substance plotted against its absorbance values, typically generated using spectrophotometry. Beer’s Law describes the relationship between absorbance and concentration with the equation.

A standard curve allows for the determination of unknown sample concentrations by measuring their absorbance and applying the equation of the trendline (e.g., y=mx+b) obtained from the standard curve. By solving for xxx, the concentration of an unknown sample can be determined.

How do you draw? How do you use a standard curve? Beer’s Law plays a role here.

A=ecl

• A - absorbance

• e - molar extinction coefficient

• c - concentration (usually 1 cm)

What is Beer’s Law? What do each of the terms represent? Be able to use the line equation from a standard curve to calculate a value.

Dilution factors are used to account for sample dilutions when calculating final concentrations. The dilution factor (DF) is given by:

DF = Final Volume/Initial Volume

• For example, if 1 mL of a sample is added to 9 mL of water, the dilution factor is 10. If the measured concentration in the diluted sample is 0.2 mg/mL, the actual concentration in the original sample is calculated by multiplying by the dilution factor:

  Actual concentration=0.2×10=2.0 mg/mL

How do you calculate dilution factors? Be able to use them correctly for any dilutions neeed.

To determine the amount of protein in an assay sample, absorbance values are measured and compared to a standard curve. Once protein concentration is determined, it can be used to calculate the total protein content in an extract and in the original sample.

Be able to use a standard curve to determine the amount of protein in an assay sample. From this data, calculate the amount of protein in an extract and in the whole sample.

% Recovery = (Final protein amount/Initial protein amount) * 100

How do you calculate percent recovery?

The Bradford protein assay is a colorimetric assay used to quantify protein concentration by measuring the absorbance of Coomassie Brilliant Blue dye, which binds to proteins. The dye shifts from red (470 nm) to blue (595 nm) upon binding to amino acids, particularly arginine, lysine, and histidine. The intensity of the blue color correlates with protein concentration, which is quantified using a standard curve. The Bradford assay is preferred for its simplicity and rapidity but is sensitive to detergent interference.

What are the basic principles of the Bradford protein assay? What did you measure? Just know how and why these work.

Blanks are used in spectrophotometry to correct for background absorbance caused by solvents or reagents. Zeroing the spectrophotometer ensures that only the absorbance of the sample of interest is measured. For redox and photosynthesis assays, blanks typically contain all reagents except the analyte, ensuring accurate readings.

• Our BLANKS lacked DPIP, but included buffer, succinate, and mitochondria.

How do we properly prepare Blanks for the redox and photosynthesis assays and why do we zero the spectrophotometer?

Aerobic respiration occurs in the mitochondria of eukaryotic cells and involves glycolysis, the Krebs cycle, and oxidative phosphorylation.

Photosynthesis takes place in the chloroplasts, where light energy is used to fix carbon into glucose via the Calvin cycle. Succinate oxidation occurs in the mitochondrial matrix as part of the Krebs cycle, where succinate is converted to fumarate, producing FADH₂, which feeds into the electron transport chain.

Outline the overall processes/pathways involved in aerobic respiration and photosynthesis. Where in eukaryotic cells do these occur? Where does succinate oxidation occur?

Mitochondria were isolated from lima beans using homogenization and differential centrifugation. The homogenate was first centrifuged at low speed to remove cell debris and nuclei, followed by high-speed centrifugation to pellet mitochondria. The mitochondrial suspension was then used for metabolic experiments.

How were mitochondria isolated from lima beans for the reactions?

DPIP (2,6-dichlorophenol-indophenol) is a redox dye used to measure succinate oxidation by changing color as it accepts electrons, shifting from blue to colorless. Buffers were used to maintain pH stability, while the mitochondrial suspension provided the enzyme succinate dehydrogenase, which catalyzes succinate oxidation.

What was the function of DPIP in this experiment? Buffer? Mitochondrial suspension?

Positive controls contain the variable being tested and should produce a measurable effect, while negative controls lack the variable and ensure no unexpected results occur. For example, a reaction with mitochondria and DPIP serves as a positive control, while a reaction without mitochondria ensures DPIP does not reduce spontaneously.

Explain the difference between the sample being tested and a negative or positive control.

(Initial Absorbance - Final Absorbance)/time

This quantifies how quickly DPIP is reduced.

How do you calculate succinate oxidation rates from changes in absorbance of DPIP?

Enzyme specificity is determined by the shape and chemical properties of the active site, allowing only specific substrates to bind. The lock-and-key model suggests a perfect fit, while the induced fit model explains how enzymes slightly adjust to accommodate the substrate. Specificity is also influenced by hydrogen bonding, ionic interactions, and hydrophobic forces, as well as environmental factors like pH and temperature.

Describe what determines specificity of an enzyme for its substrate.

Malonate is a competitive inhibitor of succinate dehydrogenase, meaning it mimics succinate and binds to the enzyme’s active site without reacting. This prevents succinate from binding, reducing enzyme activity. Since competitive inhibition is reversible, adding more succinate can outcompete malonate and restore enzyme function.

Describe how inhibitors like malonate interfere with the function of an enzyme like succinate dehydrogenase.

The Michaelis-Menten plot is a hyperbolic curve that shows how reaction velocity (V) changes with substrate concentration ([S]), helping determine Vmax​ and Km​. The Lineweaver-Burk plot is a linear transformation (double reciprocal plot) of this data, making it easier to analyze enzyme inhibition. The x-intercept gives −1/Km​, and the y-intercept gives 1/Vmax⁡​.

What is the difference between Michaelis Menton plot and Lineweaver Burke plot? Be able to identify them based on the data graphed. Interpret the Vmax and Km values from them.

Competitive inhibition occurs when an inhibitor competes with the substrate for the active site, increasing Km​ but leaving Vmax⁡​ unchanged. Noncompetitive inhibition occurs when an inhibitor binds to an allosteric site, reducing Vmax⁡ while leaving Km​ unchanged.

Distinguish between competitive and noncompetitive inhibition.

By measuring enzyme activity at different substrate concentrations:

• If Kmincreases but Vmax⁡​ stays the same → Competitive inhibition.

• If Vmax​ decreases but Km​ stays the same → Noncompetitive inhibition.
  In Lineweaver-Burk plots, competitive inhibitors shift the x-intercept, while noncompetitive inhibitors shift the y-intercept.

How can you experimentally determine between competitive and noncompetitive inhibition by using Km and Vmax for an enzyme? How does an inhibitor change these values?

• BE SURE TO KNOW HOW TO ANALYZE THIS DATA AND EXTRAPOLATE KM AND VMAX OR CALCULATE VALUES FROM A GRAPH.

Since cuvettes are made of glass or quartz, they may have minor variations in thickness, clarity, or surface imperfections, which can affect light transmission and lead to measurement errors.

Steps to Match Cuvettes

1. Use the Same Type of Cuvette – Ensure all cuvettes used in an experiment are made from the same material (glass or quartz) and have the same path length (usually 1 cm).

2. Fill Each Cuvette with the Same Solvent – Add the same blank solution (e.g., water or buffer) to multiple cuvettes and place them in the spectrophotometer.

3. Measure the Absorbance of Each Cuvette – Insert each cuvette into the spectrophotometer and measure its absorbance at a chosen wavelength (e.g., 600 nm for bacterial cultures). Ideally, the absorbance of a blank solution should be close to zero for all cuvettes.

4. Identify Any Variations – If cuvettes give different absorbance readings when filled with the same blank solution, they have inherent optical differences.

5. Select a Matched Pair – Choose two cuvettes that give similar absorbance values (as close to zero as possible) and use these for all measurements to minimize errors.

6. Always Insert Cuvettes in the Same Orientation – Some cuvettes have a clear side and a frosted side. The clear sides must always face the light source to ensure consistent optical transmission.

In spectrophotometry, how do you match cuvettes?