BIOL 111: Lab 4 - Separation of Cell Components

BIOL 111: General Biology Lab Notes

Lab 4: Separation of Cell Components

Course Information

• Professor: Dr. Nicole Vega-Cotto

• Email: nicole.vegacotto@ucc.edu

• Office Hours: By appointment

• Course & Section: BIOL – 111 – 300

• Classroom: Online (asynchronous)

Learning Objectives

• Understand why cell fractionation is used by cell biologists.

• Understand the difference between differential centrifugation and density gradient centrifugation.

• Understand the procedure used for cell fractionation.

• Be able to explain which organelles are found in each fraction and the basis for this separation.

• Be able to predict how particles that differ in density will separate in a hypothetical fractionation process.

• Know the stains used to identify leucoplasts and nuclei.

• Know the method used to identify the presence of mitochondria.

• Know the conditions required to preserve enzymatic activity in the tissue sample.

• Know all terms in this handout that appear in boldface print.

Instructions

• Review the introduction slides.

• Read the information in the lab handout.

• Complete the lab worksheet.

• Complete any other practice problems, Exit ticket, Quiz, and Discussion Post.

Separation of Cell Components: An Overview

• The process of isolating and separating different parts of a cell, such as organelles.

• Achieved through a technique called cell fractionation, which involves disrupting the cell and then separating components based on size and density.

• Methods include homogenization and centrifugation.

• This technique is crucial for scientists to study individual organelles and their functions via microscopy or biochemistry.

1. Homogenization: Breaking Cells

• The initial step where cells are disrupted to release their components into a solution called a homogenate.

• Methods: Can be achieved using a blender or a mortar and pestle.

• Conditions for Preservation: Homogenization is performed in an ice-cold aqueous medium (e.g., salt or sucrose solution) under specific conditions to preserve cellular components and enzymatic activity:

  • Cold temperature: Inhibits the activity of hydrolytic enzymes that could degrade cellular components.

  • Buffer: Prevents changes in pH that could denature proteins or damage organelles.

  • Sugar solution: Prevents the rupturing of cell organelles due to osmotic pressure, as it acts as an isotonic medium.

2. Cell Fractionation: Isolating Cell Parts

• This step involves isolating cellular components from the homogenate.

• Components are separated based on their density and size by spinning them at high speeds.

• Centrifuge: Lab equipment used to separate cell parts by size and density.

Differential Centrifugation Method

• A centrifugation technique where fractions are separated sequentially at increasing speeds.

• This process allows heavier organelles (e.g., nuclei) to pellet first, followed by lighter ones (e.g., mitochondria).

• The homogenate undergoes a series of centrifugations with increasing centrifugal force and time.

Procedure Steps:

1. First Step: Low speed for a short duration.

   • A pellet #1 forms at the bottom, containing larger, denser particles (e.g., nuclei).

   • Supernatant #1 is the solution floating above the pellet, containing smaller, lighter components.

2. Second Step: Higher speed for a longer duration.

   • Supernatant #1 is transferred to a new tube and spun again.

   • A pellet #2 forms (e.g., mitochondria, lysosomes, peroxisomes), and supernatant #2 remains.

3. Subsequent Steps: This process can be repeated for a $3^ ext{rd}$, $4^ ext{th}$ time, etc., with progressively higher speeds and longer durations to separate increasingly smaller components.

Principles of Separation:

• The smaller the cellular component, the greater the centrifugal speed and the longer the centrifugation time required to sediment it.

• Larger particles sediment out first, then medium-sized ones, and finally, the smaller-sized components are deposited at the bottom of the centrifuge tube.

• The smaller the subcellular component, the greater the centrifugal force required to form a pellet.

Ultracentrifuge

• A specialized centrifuge used to separate cell components based on their size and density by spinning samples at extremely high speeds.

• Speed: Up to $80,000 ext{ rpm}$ (revolutions per minute).

• Force: Produces forces up to $500,000$ times gravity.

• Operation: Operates in a vacuum to reduce friction and at $4^ ext{C}$ to prevent cell damage and preserve enzymatic activity.

Density Gradient Method (Equilibrium Density Gradient Centrifugation / Rate-Zonal Centrifugation)

• A type of cell fractionation that utilizes a density gradient within an ultracentrifuge.

• Primarily used to separate macromolecules (e.g., DNA, RNA, proteins).

Procedure:

1. A centrifuge tube is filled with a solution, typically sucrose, where the concentration varies from the bottom to the top.

2. The solution is most concentrated at the bottom, creating a gradient where density decreases towards the top.

3. The cell homogenate is carefully layered on top of the gradient.

Principles of Separation:

• During centrifugation, denser particles spin down to lower levels in the gradient.

• Particles come to rest in an area of the density gradient that is equivalent to their own buoyant density.

• The rate at which each component sediments depends on its size and shape, and it is related to the molecular weight of the component.

• This rate is expressed as the sedimentation coefficient (S value).

• Large S values indicate larger particles, and they sediment faster and reach their equilibrium density quicker.

Procedure for the Isolation of Cellular Components

• Cell components such as nuclei, leucoplasts, chloroplasts, and mitochondria differ in size, shape, density, and chemical composition.

• Under controlled conditions, these components can perform some of their functions outside the cell for a limited period.

• Identification can be achieved through microscope observation, specific staining, and chemical reactions.

Staining and Microscope Observation

• Iodine: Combines with starch to form a black precipitate, useful for identifying starch-containing organelles like leucoplasts.

• Hematoxylin: Stains nuclei, DNA, and RNA an orange/light brown color, allowing for their visualization.

Chemical Reaction to Detect Mitochondria

• The presence of mitochondria can be detected using a chemical reaction involving Tetrazolium.

• Enzymes within the mitochondria cause the color of Tetrazolium to change from clear to red.

Results for Tetrazolium Test

• Pellet 2: Often tests positive, indicating the presence of mitochondria, as they are typically found in this fraction during differential centrifugation.

• Supernatant 2: Tests negative, indicating the absence or very low concentration of mitochondria, as they would have pelleted out.

• Negative Control: Tests negative, confirming the specificity of the reaction.

Two commonly used methods for separating cell components are:

1. Differential Centrifugation Method: This technique separates cellular components sequentially at increasing speeds. Heavier and denser organelles, such as nuclei, pellet first at lower centrifugal forces, while lighter components, like mitochondria, lysosomes, and peroxisomes, require higher speeds and longer durations to sediment.

2. Density Gradient Method (Equilibrium Density Gradient Centrifugation / Rate-Zonal Centrifugation): This method utilizes an ultracentrifuge with a density gradient (e.g., a sucrose solution with varying concentrations). Cellular components separate based on their buoyant density, settling at a level in the gradient that matches their own density. This technique is often used for separating macromolecules like DNA, RNA, and proteins.

1. The homogenate needs to be submitted to a series of centrifugations with increasing centrifugal force because this method, known as differential centrifugation, separates cellular components sequentially based on their size and density. Heavier and denser organelles, such as nuclei, pellet first at lower centrifugal forces, while progressively lighter components (like mitochondria, lysosomes, peroxisomes, and then even smaller components) require higher speeds and longer durations to sediment. This allows for the isolation of different cell parts in distinct fractions.

2. In centrifugation:

• A pellet is the solid material that accumulates at the bottom of the centrifuge tube after spinning, containing larger, denser particles that have sedimented.

• The supernatant is the liquid solution floating above the pellet, which contains the smaller, lighter components that have not yet sedimented.

1. To isolate a very tiny cell component using the differential centrifugation method, you would need to use:

• Speed: High speed

• Duration: Long duration

• Temperature: Cold (e.g., $4^\text{C}$)

Explanation of Settings:

• High speed and long duration: According to the principles of separation in differential centrifugation, the smaller the cellular component, the greater the centrifugal speed and the longer the centrifugation time required to sediment it. Tiny components have very low mass and density, so a significantly higher centrifugal force is needed over an extended period to overcome their inertia and form a pellet.

• Cold temperature (e.g., $4^\text{C}$): This condition is crucial for preserving the integrity of cellular components and enzymatic activity. Cold temperatures inhibit the activity of hydrolytic enzymes that could degrade cellular components and prevent damage to cells and proteins during the high-speed spinning process, as outlined in the homogenization and ultracentrifuge sections.