Proper Pipetting Technique Reviewer

Comprehensive Exam Reviewer: Proper Pipetting Technique

MLS 420 | Molecular Biology Lab

1. MICROPIPETTES — Overview

Micropipettes are instruments used to measure and transfer liquids ranging from 0.2 to 10,000 µL. They are essential in molecular biology, diagnostics, and virtually all scientific laboratories.

Key Conversions to Remember:

100 µL = 0.1 mL
1,000 µL = 1 mL
10,000 µL = 10 mL

⚠ If bubbles appear during aspiration, the volume is no longer accurate — air has displaced the liquid.

2. TYPES OF MICROPIPETTE

A. By Number of Channels

Type	Description
Single-Channel	1 channel; handles one sample at a time
Multi-Channel	Handles 8, 12, or 16 samples simultaneously; ideal for high-throughput labs; dispenses only one/same quantity at a time

B. By Volume Adjustment

Type	Description
Fixed Volume	No adjustment; aspirates only one set volume; like a volumetric flask — no graduations in between; more accurate but more expensive
Variable Volume	Manually adjustable within a range (e.g., 100–1,000 µL); includes a volume lock feature

C. By Operating Mechanism

Type	Description
Mechanical	Standard piston-driven; operated by thumb pressure (1st and 2nd stop); used in most lab sessions
Electronic	Digital controls; programmable; reduces human error; ideal for labs requiring reproducibility

📝 Reproducibility = the ability to produce the same results even when different individuals perform the pipetting.

3. COMPONENTS OF A MICROPIPETTE

External Parts

Part	Function
Plunger	Controls liquid aspiration and dispensing
Tip Ejector	Removes the disposable pipette tip
Volume Adjustment Dial	Sets the desired volume
Volume Display	Shows the set volume
Shaft	Holds the pipette tip in place
Disposable Pipette Tip	Autoclavable attachment for liquid uptake and dispensing

Internal Parts

Part	Function
Piston	Drives liquid aspiration and dispensing
Friction Ring	Regulates smooth piston motion
Springs	Returns piston to original position after dispensing
O-ring	Ensures a tight seal; prevents air/liquid leakage

⚠ Dropping a pipette can damage internal parts, affecting accuracy. Never drop the pipette.

4. COMMON MICROPIPETTE SIZES (MEMORIZE!)

Pipette	Volume Range
P2	0.2 – 2 µL
P10	1 – 10 µL
P20	2 – 20 µL
P100	20 – 100 µL
P200	20 – 200 µL
P1000	100 – 1,000 µL

5. HOW A MICROPIPETTE WORKS

Micropipettes operate on the principle of air displacement.

Position	Name	Action
Position 1	Rest	Pipette is at rest; no pressure applied
Position 2	1st Stop (Filling)	Plunger depressed to first stop; air is displaced; creates a vacuum that draws liquid up when slowly released while tip is submerged
Position 3	2nd Stop (Dispensing)	Plunger pushed further; forces a burst of air to fully expel all liquid from the tip

The 1st stop = aspiration/filling. The 2nd stop = dispensing only — ensures complete expulsion of liquid residue.

6. STEPS OF MICROPIPETTING

Set the Volume — Adjust the volume setting on the micropipette
Attach the Pipette Tip — Securely attach a disposable tip to the pipette
Aspirate the Liquid — Create a vacuum to draw liquid into the tip
Dispense the Liquid — Release liquid from the tip into the container
Eject the Tip — Remove the used tip from the pipette

📝 Always set the volume BEFORE attaching the pipette tip to avoid contaminating the tip through contact with the lab gown or table.

7. PROPER WAY TO USE A PIPETTE (Step-by-Step)

Choose the right pipette for the volume needed
Check for a lock — unlock before use to avoid damage/noise
Set the volume
Lock the pipette
Attach an appropriate pipette tip
Press to the 1st stop, submerge tip, slowly release to aspirate
Press to the 2nd stop to fully dispense

Ergonomic tip: Place the index finger under the ejector to keep the pipette secure. The index finger stabilizes the ejector so it doesn't interfere when pressed.

8. ERRORS TO AVOID

Error	Correct Practice
Misaligned or improperly secured tips	Ensure tips are properly attached to avoid leaks
Inconsistent plunger pressure	Apply consistent pressure throughout
Submerging tip too deep or too shallow	Submerge only 2–3 mm below the liquid surface
Holding pipette at incorrect angle	Keep the pipette vertical at all times

9. BEST PRACTICES

Practice	Why It Matters
Pre-wet the pipette tip before use	Equilibrates the tip with the sample; improves accuracy
Pipette slowly	Prevents bubbles and ensures consistent aspiration
Use the correct pipette and tip size	Ensures precision; avoids overloading

10. DISPOSABLE PIPETTE TIPS

Autoclavable attachments for liquid uptake and dispensing
Purchased separately from the pipette
Must match the pipette type for accurate results
Although autoclavable, they are commonly disposed of — especially after contact with strong chemicals or blood
May be classified as sharps — dispose per stockroom/lab guidelines

11. PROPER HANDLING AND CARE

Never force the volume dial past its intended range
Return dial to highest setting before storing — releases tension on the internal spring
Store vertically in a stand — horizontal storage can allow liquid to enter the pipette body
Clean exterior with a lint-free cloth using mild detergent or 70% ethanol
Avoid dropping — may require early recalibration
Never aspirate liquid into the body — harsh chemicals cause buildup and inaccurate results
Calibrate regularly — every 3–6 months

QUICK REVIEW: KEY FACTS TO REMEMBER

Micropipettes work on air displacement
Bubbles = inaccurate volume
1st stop = aspiration | 2nd stop = dispensing
Always pre-wet tips before use
Tip immersion depth = 2–3 mm
Hold pipette vertically during use
Store pipette vertically when not in use
Calibration interval = every 3–6 months
Multi-channel pipettes can handle 8, 12, or 16 samples simultaneously but only one volume at a time
Fixed volume pipettes are more accurate but more expensive

Reviewer based on MLS 420 Lab Lecture by Channey Tinapay, RMT | Notes by Libanan, Neri, Sullano | BSMLS 3E