MLS 407 | Pipetting Concepts

Accurate test results are dependent upon ___ and ____ measurement of specific volumes of patient specimens and reagents

accurate; precise

T/F: All measuring devices used in the testing process have a potential of error (inaccuracy)

True; error potential is additive

What are considerations when selecting laboratory supplies?

• Formulation

• Heat tolerance

• Chemical tolerance

• Inert-ness

• Proper Use

• Maintenance and Care

• Cost

Tolerance of Accuracy

• Class A

  • preferred in clinical labs

  • guaranteed by manufacturer to deliver stated volume within error tolerance of ASTM standards when used properly

  • accurate and precise

  • Example: Error ~0.1% of stated capacity = 0.1 mL error for 100 mL

• Class B

  • Twice the tolerance limits

  • Used when durability is needed

  • Example: Error ~0.5-1% of stated capacity = 0.5-1.0 mL for 100 mL

What class would these belong to?

• Glass graduated cylinder

• Polypropylene (PP) graduated cylinder

• Polymethylpentene (PMP) graduated cylinder

• Glass volumetric flask with stopper

• A

• B

• A

• A

T/F: Erlenmeyer flasks can be used for volumteric measurements

False; they can only be used for mixing and transferring

T/F: Beakers can only be used for mixing and transporting, not volumetric measurements

True

What do TC and TD mean? and where would you find these labeled?

TD = to deliver

TC = to contain

On volumetric pipettes

What does TC 20C mean?

To contain at 20C; at 20C, the pipette will have the specified volume contained in it

If you allowed liquid in a TC pipette to drain out of it and into a receiving vessel, you would need to what?

get every last drop of liquid out of the pipette to have that volume transferred to the receiving vessel. the original vessel contains the specified volume, but may not deliver that exact volume accurately

What does TC 20D mean?

To deliver at 20C

At 20oC, the specified volume listed on the pipette will be transferred to the receiving vessel when the liquid is allowed to drain freely from the pipette

After allowing the liquid to freely drain out of a TD pipette into a receiving vesse, is it necessary to get every last drop out of the pipette?

No, there will be residual liquid left, but that is expected

What is a serological pipette?

Used for measuring and delivering volumes (1-25mL) of liquid

These are TD pipettes

Have size color codes on top

What should you use for volumes:

• Smaller than 1 mL

• Larger than 25 mL

• micropipette

• serological/volumetric pipette

What do frosted rings or double stripes indicate on a serological pipette?

Indicates the pipette requires a “blow out” step to deliver the entire contents of the pipette (including the residual volume)

How are increments labeled on a serological pipette?

1/10 = 0.1 mL increments

1/100 = 0.01 mL increments

What is a safety bulb?

Device used to fill a serological pipette with liquid and deliver the residual volume of liquid left in the tip after the liquid is allowed to drain freely out of the pipette into the receiving vessel

Use a safety bulb to force the residual liquid out of the pipette tip into the receiving vessel with ___ puff(s) of air

One

How do you choose which serological pipette to use?

Choose the pipette size that best approximates the intended delivery volume

How do you use a serological pipette?

With a safety bulb, fill to 0 mark with proper meniscus alignment. Then, allow contents to freely drain. Then, stop the flow and bring meniscus to the targeted volume mark. Blow out

How to use a safety bulb?

1. Expel air out of bulb

2. Place bulb on top of pipette, keeping the bulb expelled of air

3. Slowly release to aspirate liquid into pipette, NOT into bulb; ~1-2 inches above calibration mark

4. Remove safety bulb from top of pipette and place index finger on top of pipette to stop flow

5. Wipe outside of pipette

6. Gently release finger pressure, controlling liquid flow out of pipette

7. STOP liquid flow when meniscus is properly aligned to calibration mark

8. After meniscus is in proper alignment, release finger pressure from top of pipette, allowing liquid to freely drain into receiving vessel

9. When liquid stops draining, place bulb on top of pipette and introduce one puff of air, then remove bulb

10. Touch pipette tip to inside of receiving vessel to capture residual fluid by capillary action. Any liquid remaining will stay in pipette

11. Removing pipette without touching or dragging tip along inside wall of vessel

If your eye is positioned above the meniscus, the amount of liquid measured will be falsely ___

LOW

If your eye is positioned below the meniscus, the amount of liquid measured will be falsely ___

HIGH

If a pipette tip has graduations what do you do?

Blow out

What are volumetric pipettes?

Pipettes designed with an open-ended bulb in the center of a glass/plastic tube, which holds the bulk of the fluid.

There are no volume graduation markings to the tip.

Delivers single, specified volume of liquid

Are serological or volumetric pipettes more accurate?

Volumetric. These are used when greatest accuracy is required (ex: preparation of calibrators, standards)

What are the most common sizes for volumetric pipettes?

5 mL, 10 mL

T/F: The color codes are the same for both volumetric and serological pipettes

False

Do volumetric pipette tips have frosted rings on top?

No, which means no blow out step required

How can you eliminate sources of error when pipetting?

• Choose correct pipette size

• Use correct technique

• Wipe excess fluid off outside pipette properly

• Meniscus properly aligned

• All samples/equipment at room temperature

• Hold pipette vertical

• No bubbles

What is the range for micropipettes?

1 uL to 5000 uL (most often up to 1000 uL)

What does “automatic” mean for micropipettes?

Implies the parts of the pipette used to aspirate and dispense volumes is an integral part of the pipette

What happens if you use a 1.0 mL micropipette 5 times to deliver 5.0 mL of liquid?

Each time you pipette, there is a potential of delivering an inherent amount of error. By doing it 5 times, you introduce 5X the amount of error. Use a different pipette.

Single Stop Micropipette

there is only ONE plunger stop to fill pipette tip and dispense liquid into receiving vessel

Double Stop Micropipette

there are TWO plunger stops to fill pipette tip and dispense liquid into receiving vessel

Fixed Volume Micropipette

• This pipette delivers one specific volume

• Used in laboratories with limited variety of testing procedures

• Limitation: fixed volume

Variable Volume Micropipette

• This pipette is able to deliver various volumes by changing the indicator dial to the desired volume setting

• Used in larger laboratories having a larger testing menu

• Limitation: cost; must check calibration at each volume used for testing

Forward Pipetting Technique

Routinely used when pipetting aqueous solutions

Clinical Laboratory: serum, plasma, urine, body fluids

Reverse Pipetting Technique

Not routinely used when pipetting aqueous solutions.

Error: Increases delivery volume

Clinical Laboratory: used when pipetting viscous, dense, volatile solutions such as whole blood, solvents, alcohols

How deep should the micropipette dip be in a liquid?

2-3 mm (small)

4-6 mm (large)

T/F: When delivering the same sample and same sample volume in multiple tubes, use the same tip for improved precision

True

How long should you wait for the sample to fill a micropipette tip?

1 second

What are barrier pipettes and tips?

• These types of pipettes and micropipette tips contain a filter acting as a liquid barrier

• Prevents contamination between samples

• Prevents contamination of equipment

• Used when protection from aerosol and liquid contamination is required

• Clinical Laboratory: Polymerase Chain Reaction (PCR) techniques

What are air-displacement pipettes?

• recommended for aqueous samples and general lab work

• always have a cushion of air (dead volume) between pipette piston and liquid sample

• piston is permanent part of pipette

• when the push-button is pressed, the piston inside moves down to let air out (displaced)

• the volume of air displaced = volume liquid aspirated

What are positive-displacement pipettes?

• recommended for problem samples (viscous, dense, volatile, radioactive, corrosive)

• direct contact of piston with sample (no air cushion)

• disposable piston

• works like syringes

• with no elastic air cushion, aspiration force is constant, unaffected by physical properties of sample

What would be the appropriate immersion depth for these volumes?

1. 0.1-1 uL

2. 1 - 100 uL

3. 101 - 1000 uL

4. 1001 uL - 10 mL

1. 1 mm

2. 2-3 mm

3. 2-4 mm

4. 3-6 mm

What is the difference in technique between forward and reverse mode on air-displacement micropipettes?

1. Forward: go to first top before aspiration

2. Reverse: go to second stop before aspiration

What are the most used laboratory instruments?

variable volume air displacement single channel pipettes because of its versatility and wide volume range

What are multichannel pipettes commonly usd for?

microplate applications like ELISA, PCR, or cell culture

What is the most commonly used function of electronic pipettes?

aliquoting a reagent into multiple doses (stepper mode)

What are serological pipettes used for in a lab?

cell and tissue culture applications; general laboratory liquid dosage

What is a bulk reagent dispenser useful for?

it is reliable and easy for dispensing reagents directly from reagent bottle

What causes a micropipette to become contaminated in its interior? And what should you when this happens?

Causes

• laying a filled micropipette on its side

• holding a filled micropipette upside down

• snap back plunger

Action

• take it out of service and notify instructor

What causes this error: Distorts flow of liquid, resulting in an inaccurate volume of liquid delivered to RV (receiving vessel)

Allowing pipette tip to come in contact with the liquid already present in the RV