Q.29 Determine the correctness or otherwise of the following Assertion (a) and the Reason (r)
Assertion (a): MTT assay is used to determine cell viability based on the principle of colour formation by DNA fragmentation.
Reason (r): MTT assay is used to determine cell viability based on the colour development by converting tetrazolium soluble salt to insoluble salt.
- (A) Both (a) and (r) are true and (r) is the correct reason for (a)
- (B) Both (a) and (r) are true and (r) is not the correct reason for (a)
- (C) (a) is true but (r) is false
- (D) (a) is false but (r) is true
MTT Assay Assertion-Reason Analysis: DNA Fragmentation vs Tetrazolium Reduction
The MTT assay measures cell viability through mitochondrial activity, not DNA fragmentation. Understanding its true mechanism clarifies why the assertion-reason pairing is incorrect.
Correct Answer
Answer: (D) (a) is false but (r) is true
Assertion (a) incorrectly states MTT assay uses DNA fragmentation for color formation. Reason (r) correctly describes the actual mechanism: mitochondrial dehydrogenases reduce yellow soluble MTT to purple insoluble formazan, measured colorimetrically at 570 nm.
Option Analysis Table
| Option | Assertion (a) | Reason (r) | Relationship | Correct? |
|---|---|---|---|---|
| (A) | True | True | (r) explains (a) | ❌ Wrong: (a) false |
| (B) | True | True | (r) unrelated to (a) | ❌ Wrong: (a) false |
| (C) | True | False | – | ❌ Wrong: (r) true |
| (D) | False | True | – | ✅ Correct |
Assertion Analysis
Assertion (a): FALSE
MTT assay does NOT rely on DNA fragmentation. DNA fragmentation indicates apoptosis (programmed cell death), measured by other assays like TUNEL, Comet assay, or DNA laddering on gels. MTT measures metabolic activity of live cells.
Reason (r): TRUE
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow soluble tetrazolium salt. Live cells’ mitochondrial NAD(P)H-dependent oxidoreductases cleave the tetrazolium ring, forming purple water-insoluble formazan crystals. These are solubilized (DMSO/acid) for absorbance reading.
Reaction Mechanism
MTT (yellow, soluble) →[NAD(P)H-dehydrogenases]→ Formazan (purple, insoluble)
Absorbance at 570 nm ∝ viable cell number
Complete MTT Assay Principle (6 Steps)
- Live cells have active mitochondria with dehydrogenases
- MTT added (0.5 mg/mL typically)
- Enzymatic reduction produces formazan crystals
- Solubilization with DMSO or isopropanol
- Colorimetric reading (OD570 – OD630)
- Viability % = (Sample OD / Control OD) × 100
Common Confusions
- DNA fragmentation = apoptosis marker (TUNEL assay)
- MTT/formazan = mitochondrial metabolism marker
- LDH release = membrane integrity (another viability assay)
- ATP content = luminescence-based viability (CellTiter-Glo)
Clinical/Research Applications
- Cytotoxicity screening (drugs, nanoparticles)
- Cell proliferation studies
- IC50 determination
- High-throughput 96-well format
The assay’s sensitivity to mitochondrial inhibitors and its inability to distinguish live/apoptotic/necrotic states are known limitations, but its principle remains tetrazolium reduction, not DNA fragmentation.
