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Pipettes, glassware and temperature checks

15 min

  • Verify temperature at the working sample position
  • Follow the to-contain or to-deliver marking on volumetric equipment
  • Choose a pipette type for the volume and for viscous, volatile, or foaming liquids
  • Calculate delivered volume from water mass and the stated conversion factor

Read the full reference

Try first

Try first

You check a heating block set to 37 °C. A calibrated reference probe sits in a tube of water in a working well. After it settles, it reads 36.4 °C. Its calibration certificate gives a correction of +0.3 °C at this temperature. The block's display reads 37.0 °C. Which temperature do you record for the sample position?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

The next section explains it.

Get the idea

Measure where the specimen sits

A water bath or heating block display reports the controller's own sensor. The temperature at the specimen can differ with position, gradients and drift, so the check is made at the sample position.1

  1. Let the bath and a calibrated reference probe equilibrate.
  2. Read the probe at the working setpoint in representative rack positions.
  3. Apply the probe's calibration correction.
  4. Compare each corrected reading with the procedure's acceptance interval.1

The probe's calibration record names the reference device, the correction factors and their uncertainty. A "NIST traceable" label by itself is a claim.2

Read the marking before you drain

To-contain (TC) ware holds its stated volume. To-deliver (TD) ware releases its stated volume when its drainage procedure is followed.3

  • A continuous etched band or double ring near the top marks a blowout pipette. Its last drop is expelled.
  • A pipette without that mark is self-draining. The drop left in its tip is part of its calibration.1

Using one drainage rule for every pipette adds or leaves behind the same volume on every transfer.

Match the pipette to the liquid

An air-displacement pipette moves liquid through an air cushion. Viscosity, volatility, foaming and temperature differences change what that cushion delivers. A positive-displacement pipette moves its piston in contact with the liquid and handles these liquids better. Reverse pipetting is another option, and either change is verified locally. The pipette's operating range must cover the volume.1

Weigh water to find the volume

Gravimetric calibration weighs replicate deliveries of purified water. The factor Z converts mass to volume and folds in water density and air buoyancy.4

mean delivered volume = mean mass × Z

systematic error = mean delivered volume − selected volume

At 22.0 °C and 101.3 kPa, Z is 1.0033 µL/mg.4 A 100 µL pipette with a mean mass of 99.29 mg delivers 99.29 mg × 1.0033 µL/mg = 99.62 µL, a systematic error of −0.38 µL. Reading the milligrams as microliters overstates the error as −0.71 µL. The laboratory compares the systematic and random errors with its acceptance limits.5

References
  1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
  2. Bruce SS, Possolo A, Watters RL Jr. Metrological Traceability: Frequently Asked Questions and NIST Policy. NIST Technical Note 2156. National Institute of Standards and Technology; 2021. Accessed September 26, 2026.
  3. ASTM International. Standard Specification for Laboratory Glass Volumetric Apparatus. ASTM E694-18(2024). ASTM International; 2024.
  4. International Organization for Standardization. Piston-operated volumetric apparatus — Part 6: Gravimetric reference measurement procedure for the determination of volume. ISO 8655-6:2022. International Organization for Standardization; 2022. Accessed September 26, 2026.
  5. International Organization for Standardization. Piston-operated volumetric apparatus — Part 2: Pipettes. ISO 8655-2:2022. International Organization for Standardization; 2022. Accessed September 26, 2026.

Watch one

You check a 500 µL air-displacement pipette at its nominal setting.

  • Ten deliveries of purified water weigh 4,974.0 mg in total.
  • The water and air are at 22.0 °C and the pressure is 101.3 kPa, so Z is 1.0033 µL/mg.
  • The laboratory's procedure accepts a systematic error within ±4.0 µL at this setting.

Does the pipette pass for systematic error?

  1. Find the mean mass: 4,974.0 mg ÷ 10 = 497.40 mg.

    The check judges the pipette on the average of its replicate deliveries.

  2. Convert to volume: 497.40 mg × 1.0033 µL/mg = 499.04 µL.

    Z corrects for water density and air buoyancy, so each milligram on the balance stands for slightly more than 1 µL.

  3. Subtract the selected volume: 499.04 µL − 500 µL = −0.96 µL, which is −0.19% of the setting.

    The sign shows the direction, and a negative error means the pipette delivers too little.

  4. Compare with the limit: −0.96 µL lies within ±4.0 µL.

    The procedure's limit decides pass or fail for this setting.

The pipette delivers 499.04 µL, a systematic error of −0.96 µL, and it passes the systematic error check.

Your turn

Problem 1 of 3

A 37 °C water bath's display reads 37.0 °C. Which evidence shows whether the specimens in its rack are at the required temperature?

Incorrect. The display reports the bath's own sensor at its mounting point. A steady reading shows a stable controller. Measure the temperature where the specimens sit with a calibrated reference probe.

Correct. The reference probe equilibrates in the bath, its calibration correction is applied, and readings at representative sample positions are compared with the procedure's acceptance interval.

Incorrect. A probe read before it equilibrates reports a temperature between the room and the bath. One central spot can also miss a cooler position near the edge when uniformity matters.

Hint
  1. Where is the display's sensor, and where are the specimens?
  2. A probe needs time in the bath before its reading describes the bath.

Review Temperature-controlled equipment

Problem 2 of 3

A Class A volumetric pipette marked TD has no etched band or double ring near its top. After its stated drainage time, a small volume remains in the tip. What happens to it?

Correct. Without a band or double ring, the pipette is self-draining. Its TD calibration delivers the stated volume by gravity and already allows for the liquid left in the tip.

Incorrect. Blowout belongs to a pipette marked with a continuous etched band or double ring. Blowing out a self-draining pipette delivers more than its stated volume.

Incorrect. The TD calibration already allows for the liquid left in the tip. Rinsing it out adds that liquid, so the vessel receives more than the stated volume.

Hint
  1. Look for the mark near the top of a pipette that calls for blowout.
  2. A TD calibration is made with the pipette drained the way its marking describes.

Review Function and class

Problem 3 of 3

You check a 50 µL pipette at its nominal setting. Ten deliveries of purified water give a mean mass of 49.62 mg. For the recorded water temperature and air pressure, Z is 1.0033 µL/mg. What mean volume did the pipette deliver, to two decimal places?

Show the answer

49.78 µL

Mean delivered volume = 49.62 mg × 1.0033 µL/mg = 49.78 µL. The systematic error is 49.78 µL − 50 µL = −0.22 µL.

Review Piston pipettes

Use it

  • You set up a manual enzyme immunoassay on the special chemistry bench.
  • Step 1 adds 20 µL of a viscous, glycerol-based conjugate to each tube.
  • Step 2 incubates the tubes for 30 min in a heating block set to 37 °C. The procedure accepts 36.0 to 38.0 °C in every well used.
  • Step 3 adds 1.0 mL of stop solution with a 1 mL serologic pipette marked TD, with a double ring near the mouthpiece.
  • This morning's check with a calibrated reference probe, correction applied, read 37.1 °C in a center well and 35.6 °C in a corner well. The display reads 37.0 °C.
Decision 1 of 3

Which device do you use for the 20 µL of conjugate?

Viscosity changes what an air cushion delivers. The dial reads 20 µL, and a different volume reaches each tube.

Used an air-displacement pipette regardless of the liquid

Viscosity, volatility, foaming, and temperature differences change what an air cushion delivers, so a correct-looking setting can deliver the wrong volume. For these liquids, use a positive-displacement pipette or reverse pipetting, verified locally.

Twenty microliters lies outside this pipette's operating range, so its accuracy at that setting is unknown. It also keeps the air cushion that a viscous liquid upsets.

The piston moves the liquid directly, which improves delivery of viscous liquids. Its range covers 20 µL, and the local verification shows it delivers this conjugate correctly.

Review Piston pipettes

Decision 2 of 3

What do you do with the heating block?

The corner well reads 35.6 °C after correction, below the 36.0 °C limit. Tubes there would incubate too cool, and their reactions would run slower than the calibration assumed.

The block fails at a well that the run uses. A block that passed its check at every position gives each tube the same incubation, and the failed check goes into the equipment record for repair.

The whole block warms with the setpoint. The center well, at 37.1 °C now, would rise past 38.0 °C, and the unevenness between wells would remain.

Review Temperature-controlled equipment

Decision 3 of 3

The stop solution drains from the serologic pipette, and a small drop stays in the tip. What do you do with it?

The double ring near the mouthpiece marks a blowout pipette. Its TD calibration counts the last drop as part of the 1.0 mL, so it is expelled into the tube.

Leaving the drop is the rule for a self-draining pipette. This one carries a double ring, so leaving the drop delivers less than 1.0 mL to every tube.

Applied one drainage rule to every pipette

Blowing out a self-draining pipette adds the liquid its calibration leaves in the tip, and leaving the residue in a blowout pipette delivers too little. Either error repeats with every transfer as a systematic volume error. The marking sets the drainage rule, whether TC or TD, with or without an etched band or double ring.

A touch against the wall ends delivery from a self-draining pipette. The double ring calls for blowout, so the drop that remains belongs in the tube.

Review Function and class

Each item in this setup showed what it needed through something you could check:

  • The conjugate's viscosity chose the positive-displacement pipette.
  • The corner well's corrected reading of 35.6 °C, below the 36.0 °C limit, settled the heating block. The display and the center well could not show it.
  • The double ring near the mouthpiece made the serologic pipette a blowout pipette.

Keep

Sources checked