Cell Viability Testing A Lecture for 7th semester.pptx

1. Cell Culture Techniques:
Cell Viability Testing
Dr. Showkat Ahmad Wani

2. Introduction
• Cell culture means growing cells outside the body in artificial
conditions.
• We must check if our cultured cells are healthy, alive, or dead.
• Cell Viability Testing tells us how many cells are alive and
healthy.

3. What is Cell Viability?
• Cell viability = Ability of cells to survive and grow.
• Viable (alive) cells: intact membrane, active metabolism.
• Non-viable (dead) cells: damaged membrane, no activity.

4. Why Test Cell Viability?
1. To check cell health before experiments.
2. To know the effects of drugs or toxins.
3. To decide when to subculture.
4. To ensure consistency of results.

5. Methods of Cell Viability Testing
There are two main types:
1. Dye Exclusion Methods
2. Metabolic Activity-Based Methods

6. Dye Exclusion Methods
• Principle: Live cells exclude dyes; dead cells absorb dyes.
• Examples:
• Trypan Blue Exclusion Test
• Erythrosin B Test
• Nigrosin Staining

7. Trypan Blue Exclusion Test
• 1. Mix equal volumes of cell suspension and 0.4% Trypan Blue.
• 2. Wait 2–3 minutes.
• 3. Load on hemocytometer and observe.
• 4. Count:
• - Clear cells = Live
• - Blue cells = Dead
• Cell Viability (%) = (Live cells / Total cells) × 100

8. b) Erythrosin B or Nigrosin Staining
• These dyes work similarly to trypan blue.
• Dead cells take up the stain, live ones do not.
• Often used when trypan blue is not available.

9. Metabolic Activity-Based Tests
• Measure metabolic activity (only live cells show it).
• Examples:
• MTT Assay
• Resazurin (Alamar Blue) Test
• ATP Assay

10. MTT Assay
• • MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
• • Live cells convert yellow MTT to purple formazan.
• • Color intensity number of live cells.
∝
• • Read absorbance using spectrophotometer.

11. b) Resazurin (Alamar Blue) Test
• A blue dye that turns pink when reduced by live cells.
• Simple and non-toxic (cells can be used again after the test).

12. ATP Assay (Principle)
• ATP = energy molecule produced only by live cells.
• Reaction: Luciferin + Luciferase + ATP → Light (Luminescence).
• Light intensity amount of ATP number of living cells.
∝ ∝

13. Factors Affecting Cell Viability
• Contamination (bacteria, fungi)
• Wrong temperature or pH
• Lack of nutrients
• Toxic substances or high drug dose
• Mechanical damage during handling

14. Morphological Observation
• Under the microscope:
• Live cells: clear, round/spread, intact.
• Dead cells: shrunk, detached, dark.
• Gives rough idea, confirm with tests.

15. Safety Precautions
• Handle all dyes carefully (MTT toxic).
• Wear gloves, mask, and lab coat.
• Dispose waste in biohazard bins.

16. Thank You