Lab 2 Notes: Micropipette Use and Dilutions

Part 1: Unit Conversions

• This lab uses the metric system for measurements.

• Metric units are multiples of ten (or powers of ten) of each other, which simplifies conversions by moving the decimal point.

• Basic metric base units:

  • length: meters (m)

  • mass/weight: grams (g)

  • volume: liters (L)

• Common prefixes used in this course:

  • kilo- (k): 1,000 × base unit (e.g., 1 kg = 1000 g)

  • centi- (c): 1/100 of base unit (e.g., 1 cm = 0.01 m; 1 m = 100 cm)

  • milli- (m): 1/1000 of base unit (e.g., 1 mL = 0.001 L; 1 L = 1000 mL)

  • micro- (µ): 1/1000 of milli- (e.g., 1 µg = 0.001 mg; 1 mg = 1000 µg)

• General rules for converting units:

  • Larger to smaller units: multiply by the conversion factor.

  • Smaller to larger units: divide by the conversion factor.

• Quick checks:

  • When converting to smaller units, you should end up with a larger numeric value.

  • When converting to larger units, you should end up with a smaller numeric value.

• Practice conversions:
  1) 450 m = km
  2) 2500 mL = L
  3) 200 µg = mg
  4) 0.68 kg = mg
  5) 3.5 L = µL
  6) 783 cm = mm

• Quick reference conversions:

  • 1 cm = 10 mm; 1 mm = 0.1 cm

  • 1 mL = 0.001 L; 1 L = 1000 mL

  • 1 g = 1000 mg; 1 mg = 1000 µg

Part 2: Micropipette Introduction and Pipetting Practice

• Compare two sizes of micropipettes: P1000 and P200.

  • Reading the amount: the top and bottom digits indicate the selected volume.

  • Example (shown in the text): Top: 0 2 2 0; Bottom: 0 0, which corresponds to 200 μL for both P1000 and P200 in this example.

  • Volume limits (as given):

  • P1000: 200 mL to 1000 mL (or 1 mL)

  • P200: 20 mL to 200 mL

  • Note: While the pipets can be set outside these ranges, accuracy decreases and damage may occur.

• How to set a micropipette:

  • Rotate the wheel at the top until the desired numbers are read.

• Pipette tips:

  • Uses disposable plastic tips; tips are sterile and should touch only the liquid and the container.

  • Never touch the internal tip with hands; touch only liquid and destination.

• Pipetting steps:

  • Hold the pipette upright in your dominant hand.

  • Attach a disposable tip to the pipette.

  • Depress plunger to the first stop to draw up liquid.

  • Slowly release to the first stop to pick up liquid.

  • Depress the plunger to the second stop to dispense the liquid.

  • Keep the tip upright after drawing liquid; do not allow the tip to be level with or above the handle.

• Practice tasks:

  1. Pipette 200 μL, 500 μL, and 1 mL of water into a dish placed on the balance. Record weights in the table.

  2. Pipette 200 μL, 500 μL, and 1 mL of ethanol into a dish placed on the balance. Record weights in the table.

Part 3: Density Measurements and Table

• Density concept: mass per unit volume, defined as
  ho = rac{m}{V}

• Experimental setup:

  • Measure weights of 200 μL, 500 μL, and 1000 μL samples for water and ethanol.

  • Convert those masses to densities and compare to reported densities.

• Table 1: Calculating Density

  • Columns: Water; Ethanol

  • Weights to record (in grams):

  • Weight of 200 μL (g)

  • Weight of 500 μL (g)

  • Weight of 1000 μL (g)

• Step: Convert measured mass (from balance) to density using the corresponding volume per row.

• Notes:

  • The density of water at room temperature is ~1.00 g/mL; ethanol is ~0.789 g/mL (values vary with temperature).

  • Convert final density to g/mL and compare to online values.

  • The density calculation highlights the relation between mass and volume for small liquid samples.

Part 4: Solutions, Concentrations, and Dilutions

• Solutions and concentrations:

  • Solutions are homogeneous mixtures of two or more substances.

  • Concentration describes the relative amount of each substance in a solution.

• Common concentration descriptors used in this course: 1) Percent – volume/volume (v/v): % of solute volume relative to total solution volume.

  • Example: 50% ethanol means half the total volume is ethanol.

  • If 2 solutions have different end volumes but differ in ethanol content, the one with higher ethanol volume fraction has higher concentration.
    2) Percent – weight/volume (w/v): mass of solute per 100 mL of solution (grams per 100 mL).

  • Example: To make a 2% NaCl solution in water, you can mix 2 g NaCl with enough water to reach 100 mL total (or 4 g NaCl in 200 mL total).
    3) Mass per volume: mass of solute per total volume (e.g., 20 g/L, 0.02 g/mL, 20 mg/mL for a 2% NaCl solution).
    4) Cells per volume: number of cells per unit volume (e.g., 100 cells/µL = 100,000 cells/mL).
    5) Molarity (M): moles of solute per liter of solution, defined as M = rac{n}{V} where n = ext{moles} and V = ext{volume in L}.

  • Example: To make 1 M NaCl, use 58.44 g NaCl (molar mass of NaCl) per 1 L of solution: 58.44 rac{g}{mol} imes 1 ext{ mol} ext{ in } 1 ext{ L}

  • Alternative: 29.22 g NaCl in 0.5 L of water also gives 1 M (since 29.22 g / 0.5 L = 58.44 g/mol).

• Dilutions:

  • Goal: change concentration by adding solvent; dilutions can only decrease concentration.

  • Key equation: C1 V1 = C2 V2

  • C1: starting concentration; V1: volume of starting solution taken

  • C2: ending concentration; V2: final total volume after dilution

  • Required data: three of the four variables to solve for the fourth.

  • If you know C1, C2, and V2, solve for V1: V1 = rac{C2 V2}{C1}

  • Units for concentration and volume can be in any consistent units (e.g., M, mM, g/mL, etc.), but C1, C2 must use the same unit and V1, V2 must use the same unit.

• Practical steps for performing a dilution:

  • Determine C1, C2, and V2 (or any 3 of the 4).

  • Compute V1 using the formula above.

  • Mix V1 of the starting solution with enough solvent to reach V2 total volume.

Part 5: Dilution Example (NaCl)

• Example: Make 100 mL of 0.5 M NaCl from a 5 M stock.

• Setup:

  • C1 V1 = C2 V2

  • 5 ext{ M} imes V_1 = 0.5 ext{ M} imes 100 ext{ mL}

  • Solve: V_1 = rac{0.5 ext{ M} imes 100 ext{ mL}}{5 ext{ M}} = 10 ext{ mL}

• Dilution procedure: add 10 mL of 5 M NaCl to a container and bring the total volume to 100 mL with water (i.e., add 90 mL water).

Practice Problems

1) HeLa cells:

• Given: cell count = 85,000 cells/mL. Target = 40,000 cells/mL. Final volume V2 = 10 mL.

• Use C1 V1 = C2 V2 with: C1 = 85{,}000 ext{ cells/mL},\n C2 = 40{,}000 ext{ cells/mL},
  V_2 = 10 ext{ mL}

• Solve: V1 = rac{C2 V2}{C1} = rac{40{,}000 imes 10}{85{,}000} ext{ mL} \
  ightarrow V_1 \ ext{≈ } 4.71 ext{ mL}

• Volume of cell culture media to add: V{ ext{media}} = V2 - V_1 \
  ightarrow ≈ 10 - 4.71 = 5.29 ext{ mL}

2) Yeast cells:

• Given: 115,000 cells/mL. Target = 30,000 cells/mL. Final volume V2 = 20 mL.

• V1 = rac{30{,}000 imes 20}{115{,}000} ext{ mL} \ ightarrow V1 \ ext{≈ } 5.22 ext{ mL}

• Media volume: V_{ ext{media}} ≈ 20 - 5.22 = 14.78 ext{ mL}

3) MCF7 cells:

• Given: 92,000 cells/mL. Target = 20,000 cells/mL. Final volume V2 = 5 mL.

• V1 = rac{20{,}000 imes 5}{92{,}000} ext{ mL} \ ightarrow V1 \ ext{≈ } 1.09 ext{ mL}

• Media volume: V_{ ext{media}} ≈ 5 - 1.09 = 3.91 ext{ mL}

4) HeLa cells (85,000 cells per 100 μL): target 40,000 cells/mL; final volume V2 = 10 mL.

• Stock concentration: 85,000 cells per 100 μL = 850,000 cells/mL.

• V1 = rac{40{,}000 imes 10}{850{,}000} ext{ mL} \ ightarrow V1 \ ext{≈ } 0.471 ext{ mL}

• Media volume: V_{ ext{media}} ≈ 10 - 0.471 = 9.529 ext{ mL}

5) HeLa cells with varying NaCl concentration to 1 mL total:

• Stock HeLa: 85,000 cells per 100 μL ⇒ 850,000 cells/mL.

• Target: 40,000 cells in 1 mL final volume.

• Volume of cell suspension: V_{ ext{cells}} = rac{40{,}000 imes 1}{850{,}000} ext{ mL} ≈ 0.5 ext{ mL}

• For each NaCl final concentration, use C1 = 1 M NaCl stock:

  • 0.5 M NaCl: V_{ ext{NaCl}} = rac{0.5 imes 1}{1} = 0.5 ext{ mL}

  • 0.25 M NaCl: V_{ ext{NaCl}} = 0.25 ext{ mL}

  • 0.10 M NaCl: V_{ ext{NaCl}} = 0.10 ext{ mL}

  • 0.05 M NaCl: V_{ ext{NaCl}} = 0.05 ext{ mL}

• PBS volumes to reach 1 mL total:

  • 0.5 M: PBS = 0 mL

  • 0.25 M: PBS = 0.25 mL

  • 0.10 M: PBS = 0.40 mL

  • 0.05 M: PBS = 0.45 mL

6) Yeast cells and ethanol dilution series:

• Yeast stock: 4,000 cells per 100 μL = 40,000 cells per mL.

• Final volume per tube: 1 mL.

• For EtOH concentrations from stock 50%:

  • 25% EtOH: use V_{ ext{EtOH}} = rac{0.25 imes 1}{0.50} = 0.50 ext{ mL}

  • 12.5% EtOH: V_{ ext{EtOH}} = rac{0.125 imes 1}{0.50} = 0.25 ext{ mL}

  • 10% EtOH: V_{ ext{EtOH}} = rac{0.10 imes 1}{0.50} = 0.20 ext{ mL}

  • 5% EtOH: V_{ ext{EtOH}} = rac{0.05 imes 1}{0.50} = 0.10 ext{ mL}

• For each tube, remaining volume to 1 mL is PBS: adjust accordingly.

Part 6: Serial Dilution and Dye Absorbance (Dye A and Dye B)

• Serial dilution concept:

  • A 1 into 10 serial dilution yields each step at 1/10 of the previous concentration.

  • The text describes a 1 into 10 serial dilution chain starting from a stock of 100% dye.

• Setup for this activity:

  • Set up a 1 into 5 dilution series using the provided 100% dye as stock.

  • You should make 5 dilutions, the first being a 1 into 5 from the stock.

  • Final volume per tube: 800 μL.

• Dilution scheme (example, volumes chosen to achieve 1:5 steps with final 800 μL per tube):

  • Tube 1 (1:5): 160 μL stock + 640 μL diluent = 800 μL; concentration = 0.20 × C_stock

  • Tube 2 (1:25): take 160 μL from Tube 1 into 640 μL diluent = 800 μL; concentration = 0.20 × 0.20 = 0.04 × C_stock

  • Tube 3 (1:125): 160 μL from Tube 2 into 640 μL diluent = 800 μL; concentration = 0.20 × 0.20 × 0.20 = 0.008 × C_stock

  • Tube 4 (1:625): 160 μL from Tube 3 into 640 μL diluent = 800 μL; concentration = 0.0016 × C_stock

  • Tube 5 (1:3125): 160 μL from Tube 4 into 640 μL diluent = 800 μL; concentration = 0.00032 × C_stock

• Estimation of unknown dye concentration:

  • Use the dilution series as a guide to estimate the unknown dye concentration by color comparison or absorbance.

• Graphing:

  • Plot absorbance at 595 nm (A595) vs dilution concentration (relative to stock).

  • Data provided (absorbance minus blank):

  • Dilution #1: 0.504

  • Dilution #2: 0.101

  • Dilution #3: 0.020

  • Dilution #4: 0.004

  • Dilution #5: 0.001

  • Use a scatter plot and draw a best-fit line.

  • Subtract blank absorbance prior to graphing (done already here).

• How the graph is used:

  • The line relates absorbance to concentration for this dye.

  • Unknown dye concentration can be estimated by finding the concentration corresponding to the measured absorbance on the calibration curve.

Summary of Key Formulas and Concepts

• Density: \rho = \frac{m}{V}

• Concentration descriptors:

  • % (v/v): \%20\text{(v/v)} = \frac{V{ ext{solvent component}}}{V{ ext{solution}}} \times 100\% (for liquid components)

  • % (w/v): \%20\text{(w/v)} = \frac{m{ ext{solute}}}{V{ ext{solution}}} \times 100\%

  • Mass per volume: \text{mass per volume} = \frac{m}{V} (e.g., g/mL, mg/mL)

  • Cells per volume: e.g., 100 cells/µL ⇔ 100{,}000 cells/mL

  • Molarity: M = \frac{n}{V} with n = \frac{m}{Mr} and Mr = \text{molar mass}

• Dilution equation: C1 V1 = C2 V2

• Unit consistency rule: use the same concentration unit for C1 and C2, and the same volume unit for V1 and V2.

• Practical notes:

  • Dilutions lower concentration, not raise it.

  • When performing serial dilutions, the concentration decreases by a constant factor each step (e.g., 1:5 reduces by a factor of 5).

  • For tiny volumes, convert to µL if needed to avoid pipetting errors.

References and Real-World Relevance

• Understanding unit conversions is fundamental in any experimental workflow when preparing solutions, buffers, and reagents.

• Accurate micropipetting and dilution calculations are critical for reliable quantitative experiments (e.g., cell culture, enzyme assays, dye assays).

• Density measurements validate pipetting accuracy and help correlate mass measurements to volume, important in gravimetric analyses.

• Serial dilutions and absorbance measurements are common in spectroscopy, ELISA, dye quantification, and standard-curves development.