Calculations for seeding density, multiwell plates, and complete media preparation.
2.1 Terminologies and principles
Key terminologies:
- Seeding Density: Number of cells placed per unit area or volume at culture initiation.
- Confluence: Percentage of culture surface covered by adherent cells.
- Complete Medium: Basal medium supplemented with serum, growth factors, and antibiotics.
- Basal Medium: Chemically defined medium containing essential nutrients without supplements.
- Fetal Bovine Serum (FBS): Complex biological supplement providing growth factors, hormones, and attachment factors.
Principles:
- Optimal Seeding Density: Critical for proper cell growth, attachment, and experimental reproducibility.
- Media Component Proportions: Components calculated as percentage of final total volume (v/v).
- Pipetting Accuracy Margin: Adding excess (typically 10%) accounts for mechanical error in volume transfers.
2.2 Calculating Seeding Density and Volumes
Cells needed per well = desired density × final volume.
Always prepare 10–20% excess volume to account for pipetting losses.
Worked example:
A researcher plans to seed a mammalian cell line into a 24-well culture plate. Each well will contain a final volume of 1.0 mL of complete growth medium. The required seeding density is 2.5 × 10⁴ cells per well. The available cell suspension has a concentration of 1.2 × 10⁶ cells/mL.
To account for pipetting losses, an additional 15% excess volume of cell suspension must be prepared.
a) Calculate the number of cells required to seed one well.
b) Calculate the number of cells required to seed all 24 wells (without excess).
c) Calculate the total number of cells required after including the 15% excess.
d) Calculate the total volume (in mL) of the cell suspension needed to seed the plate, including the excess volume.
e) Determine the volume of cell suspension (in µL) that should be added to each well.
Step-by-step calculations
Given
- Number of wells = 24
- Final volume per well = 1.0 mL
- Desired seeding density = 2.5 × 10⁴ cells/well
- Cell suspension concentration = 1.2 × 10⁶ cells/mL
- Excess volume for pipetting losses = 15%
a) Cells required per well
Formula:
Cells per well = Desired seeding density
Cells per well = 2.5 × 10⁴ cells
b) Cells required for 24 wells (without excess)
Cells for all wells = Cells per well × Number of wells
Cells = (2.5 × 10⁴ cells/well) × 24 wells
Cells = 6.0 × 10⁵ cells
c) Total cells required including 15% excess
Excess factor = 1 + 0.15 = 1.15
Total cells required:
Total cells = 6.0 × 10⁵ cells × 1.15
Total cells = 6.9 × 10⁵ cells
d) Total volume of cell suspension required (mL)
Formula:
Volume required (mL) = Total cells required / Cell concentration
Substitute values:
Volume = (6.9 × 10⁵ cells) / (1.2 × 10⁶ cells/mL)
Volume = 0.575 mL
Rounded appropriately:
Total volume required ≈ 0.58 mL
e) Volume of cell suspension per well
Total volume prepared = 0.575 mL
Volume per well:
Volume per well = Total volume / Number of wells
Volume per well = 0.575 mL / 24
Volume per well = 0.02396 mL
Convert to microlitres:
0.02396 mL × 1000 µL/mL ≈ 24 µL
Practical preparation:
- Prepare diluted cell suspension at correct concentration in larger volume (e.g., 2-5 mL)
- Use multi-channel pipette for uniform distribution
- Add medium after cells to ensure even suspension
2.2 Media preparation
Media components are calculated as percentages of final volume.
Example: 89% basal medium, 10% FBS, 1% antibiotics.
Worked example:
A laboratory technician is required to prepare 500 mL of complete cell culture medium. The medium formulation consists of the following components:
· Basal medium: 89% (v/v)
· Fetal bovine serum (FBS): 10% (v/v)
· Antibiotic–antimycotic solution: 1% (v/v)
a) Calculate the volume (in mL) of each component required to prepare 500 mL of the complete medium.
b) Verify that the calculated component volumes add up to the final required volume.
c) If the same medium is required in a volume of 1.0 L, calculate the volume of each component needed.
Step-by-step calculations
Given
- Final volume of medium (Vf) = 500 mL
- Composition:
- Basal medium = 89%
- Fetal bovine serum (FBS) = 10%
- Antibiotics = 1%
a) Volume of each component for 500 mL medium
Formula:
Volume of component = Percentage × Final volume / 100
- Basal medium:
Volume = 89 × 500 / 100
Volume = 445 mL
- FBS:
Volume = 10 × 500 / 100
Volume = 50 mL
- Antibiotic–antimycotic solution:
Volume = 1 × 500 / 100
Volume = 5 mL
b) Verify total volume
Total volume = 445 + 50 + 5 = 500 mL ✅
(Matches the final required volume)
c) Volume of each component for 1.0 L (1000 mL) medium
- Basal medium:
Volume = 89 × 1000 / 100 = 890 mL
- FBS:
Volume = 10 × 1000 / 100 = 100 mL
- Antibiotic–antimycotic solution:
Volume = 1 × 1000 / 100 = 10 mL
Total volume = 890 + 100 + 10 = 1000 mL ✅
Summary
| Component | Volume (500 mL) | Volume (1.0 L) |
| Basal medium | 445 mL | 890 mL |
| FBS | 50 mL | 100 mL |
| Antibiotic–antimycotic | 5 mL | 10 mL |
| Total | 500 mL | 1000 mL |
Mixing protocol:
- Measure basal medium first
- Add serum under sterile conditions
- Add antibiotics last
- Mix gently without creating bubbles
Practice Questions
- Why add 10% excess volume?
2. Calculate FBS volume for 250 mL at 10%.
Answers
- To compensate pipetting losses.
25 mL.