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📝 Differential Centrifugation and Cell Fractionation (8 MCQs)

📖 From Principles of Biochemistry • 1. The Foundations of Biochemistry • 8 questions available

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What is Differential Centrifugation and Cell Fractionation?

Definition:
Differential centrifugation is a specific type of cell fractionation technique that separates cellular components by subjecting a homogenate to a series of increasing centrifugal forces, where organelles are sequentially pelleted based on their size and density, allowing for the isolation of crude fractions containing nuclei, mitochondria, lysosomes, microsomes, and ribosomes for further analysis.

Working:
This technique works by centrifuging the homogenate at low speed (e.g., 1000g) to pellet heavy components like nuclei and unbroken cells, then the supernatant is centrifuged at medium speed (e.g., 10,000g) to pellet mitochondria and lysosomes, followed by high speed (e.g., 100,000g) to pellet microsomes, and finally ultracentrifugation to pellet ribosomes, with the sedimentation rate being proportional to the square of the radius and the density difference, as described by the equation $v = \frac{2r^2(\rho_p - \rho_m)g}{9\eta}$ , where $\rho_p$ is particle density, $\rho_m$ is medium density, and $\eta$ is viscosity.

Example:
A simple example is isolating the mitochondrial fraction from rat liver, where after homogenization, the sample is centrifuged at 600g for 10 minutes to remove nuclei, the resulting supernatant is centrifuged at 10,000g for 15 minutes to pellet mitochondria, and the pellet is washed and resuspended for studies on oxidative phosphorylation, demonstrating the stepwise enrichment of specific organelles.

Reason:
Differential centrifugation is essential for studying organelle function, as it provides a simple and cost-effective method to obtain enriched fractions for biochemical assays, enzyme analysis, and proteomics, and its understanding is fundamental for students to appreciate how cellular components are isolated and studied in research and diagnostic laboratories.

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📝 All Differential Centrifugation and Cell Fractionation MCQs

Q1. A cell homogenate contains nuclei, mitochondria, lysosomes, and soluble proteins. After a low-speed spin, the pellet is removed and the supernatant is centrifuged at a higher speed. Which interpretation best explains why this sequential strategy works?

A.Larger and denser structures generally sediment before smaller structures under progressively stronger centrifugal conditions ✅

B.All organelles have identical sedimentation rates, but centrifugation separates them randomly

C.Soluble proteins always sediment before membrane-bound organelles because they are chemically smaller

D.The first centrifugation chemically destroys large organelles, allowing smaller organelles to pellet later

💡 Difficulty: medium | ✅ Correct: A

📖 Explanation: Differential centrifugation separates cellular components primarily according to differences in sedimentation behavior. Large structures such as nuclei sediment at relatively low centrifugal forces, whereas smaller organelles require greater force and longer centrifugation. The process therefore uses sequential spins to enrich different fractions.

Q2. A researcher wants to isolate mitochondria from a homogenized tissue sample. The first centrifugation produces a pellet containing nuclei and unbroken cells. The researcher discards the pellet and performs a faster second spin. Why is this second pellet more suitable for mitochondrial analysis than the original pellet?

A.The second pellet is enriched in smaller organelles that remained suspended after the initial low-speed centrifugation ✅

B.The second spin converts nuclear material into mitochondria

C.The higher speed dissolves membrane fragments while preserving only soluble proteins

D.The first pellet contains only soluble proteins, so mitochondria cannot have been present there

💡 Difficulty: medium | ✅ Correct: A

📖 Explanation: The initial low-speed centrifugation removes much of the nuclear and cellular debris fraction. Mitochondria are smaller and therefore remain largely in the supernatant. Increasing the centrifugal force then causes many mitochondria to sediment, producing a fraction enriched in them.

Q3. A student claims, 'If a mitochondrial pellet is obtained after the second centrifugation, every mitochondrion in the original homogenate must be present in that pellet.' Which criticism is most scientifically appropriate?

A.Some mitochondria may remain in the supernatant because sedimentation is incomplete and conditions are not perfectly selective ✅

B.All mitochondria must remain in the first pellet because mitochondria are larger than nuclei

C.Mitochondria cannot sediment because they are surrounded by membranes

D.The pellet necessarily contains only mitochondria, so recovery is always 100%

💡 Difficulty: hard | ✅ Correct: A

📖 Explanation: A differential centrifugation pellet is an enriched fraction rather than a perfectly pure or complete collection. Sedimentation depends on particle size, density, shape, viscosity, centrifugal force, and duration. Consequently, some target organelles can remain suspended while other contaminants pellet.

Q4. A laboratory compares two centrifugation steps. In Step 1, most nuclei are removed. In Step 2, the mitochondrial fraction increases while the amount of soluble protein decreases. Which conclusion is best supported by these observations?

A.Step 2 is enriching particulate organelles that sediment more readily than soluble proteins under those conditions ✅

B.Step 2 is converting soluble proteins into mitochondria

C.Step 1 must have removed all mitochondria before Step 2 began

D.The decrease in soluble protein proves that the mitochondrial fraction is completely pure

💡 Difficulty: medium | ✅ Correct: A

📖 Explanation: The observations are consistent with enrichment of particulate components during the higher-speed centrifugation. Soluble proteins generally remain in the supernatant under conditions that sediment many organelles. However, enrichment does not imply absolute purity because other particles may sediment together.

Q5. A graph of pellet composition versus centrifugation speed shows nuclear markers dropping sharply after the first spin, mitochondrial markers rising during an intermediate-speed spin, and soluble-protein markers remaining high in the final supernatant. What does the graph most strongly indicate?

A.Different cellular components have different sedimentation behaviors, allowing sequential enrichment ✅

B.All components sediment at exactly the same speed but are measured differently

C.The intermediate-speed spin destroys soluble proteins and creates mitochondria

D.Nuclear material has the greatest solubility because its marker decreases first

💡 Difficulty: hard | ✅ Correct: A

📖 Explanation: The changing marker distributions indicate that cellular components respond differently to increasing centrifugal conditions. Large structures are removed earlier, intermediate-sized organelles become enriched at higher speeds, and many soluble molecules remain suspended. This pattern is characteristic of differential separation rather than chemical transformation.

Q6. Two protocols are tested on identical homogenates. Protocol X uses one very high-speed centrifugation, while Protocol Y uses several progressively faster centrifugations, removing each pellet before the next step. If the goal is to obtain separate enriched fractions, why is Protocol Y generally preferable?

A.Sequential removal reduces overlap between components that sediment at different rates and improves fractionation ✅

B.A single high-speed spin guarantees that each organelle forms a completely separate pellet

C.Progressive centrifugation prevents every organelle from sedimenting

D.Sequential centrifugation changes the density of organelles so they become chemically distinct

💡 Difficulty: hard | ✅ Correct: A

📖 Explanation: A single very high-speed spin can sediment many different cellular components together, producing a mixed pellet. Sequential centrifugation removes rapidly sedimenting material first and then subjects the remaining supernatant to stronger conditions, improving enrichment of successively smaller components.

Q7. A mitochondrial preparation shows strong mitochondrial enzyme activity but also contains a substantial amount of a lysosomal marker. A student concludes that the centrifugation procedure failed completely. Which evaluation is most accurate?

A.The procedure produced enrichment but not complete purity, because organelles with overlapping sedimentation properties can co-sediment ✅

B.The lysosomal marker proves that mitochondria cannot be present

C.Any detectable contaminant means differential centrifugation has no separation value

D.The lysosomal marker must have been created chemically during centrifugation

💡 Difficulty: easy | ✅ Correct: A

📖 Explanation: Differential centrifugation is primarily an enrichment method, not an absolute purification method. Mitochondria and lysosomes can have overlapping sedimentation characteristics under some conditions, allowing both to appear in the same pellet. Marker assays are therefore useful for assessing enrichment and contamination.

Q8. A hypothetical organelle has a sedimentation behavior very similar to mitochondria. A researcher increases centrifugation speed but observes that both organelles continue appearing together in the pellet. Which modification would most directly address the separation problem?

A.Use a separation method based on a property other than sedimentation rate, such as density ✅

B.Increase the speed indefinitely until only one organelle remains

C.Discard the supernatant after every spin without analyzing it

D.Reduce all centrifugation speeds to zero

💡 Difficulty: easy | ✅ Correct: A

📖 Explanation: When two organelles have similar sedimentation behavior, simply increasing centrifugal force may cause both to sediment together. A method exploiting a different physical property, particularly buoyant density, can provide better resolution. This illustrates why differential centrifugation is often followed by more selective purification techniques.

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