Core bench skills and instruments: balances, glassware, pipetting, buffers, centrifuges, chromatography, spectrophotometry, calibration, microscopy and aseptic technique.

Lab Techniques & Instrumentation

Gather a kitchen scale that reads to 1 g (or 0.1 g), a clear measuring jug or cup with millilitre markings, a small bowl, a notebook and a pen.

Put the bowl on the scale and press tare (zero). Write down what tare does in your own words.

Fill the jug to the 100 mL mark with tap water. Crouch so your eye is level with the mark and read the bottom of the curved surface. Then look from above and note how the reading seems to change (parallax).

Pour the 100 mL into the tared bowl and record the mass in grams. Water is close to 1.00 g per mL at room temperature, so 100 mL should weigh about 100 g.

Empty and dry the bowl, then repeat the measurement four more times. Record all five masses in a table, writing down every digit the scale shows.

Calculate the mean of your five masses and the difference between the mean and 100 g. That difference is your accuracy (bias).

Calculate the range (largest minus smallest). That is a simple measure of your precision.

Write one sentence on which source of error you think was biggest: the jug's markings, your reading of the meniscus, or the scale's readability.

Optional, only in your teaching lab and only if your instructor sets it up: repeat the exercise with a Class A volumetric pipette and an analytical balance, and compare your precision.

> **Awareness level only.** This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.


## Why it matters

Almost every lab result starts with a mass or a volume. If the 250.0 mg you weighed was really 247 mg, or the 100 mL flask was filled past the line, every number downstream carries that error: the concentration of your standard, the calibration curve built from it, and the patient or product result read from that curve. Balances and volumetric glassware are the first instruments you will be trained on, and the habits you build with them follow you through your whole career.

## Accuracy, precision and readability

- **Accuracy** is how close a measurement is to the true value.
- **Precision** is how close repeated measurements are to each other.
- **Readability** is the smallest step the display shows.

A balance can be precise but inaccurate (it always reads 0.3 mg high) or have fine readability but poor precision (the last digit jumps around in a draught). Calibration and good technique address different parts of this.

## Balances

| Type | Typical readability | Typical use |
|---|---|---|
| Top-loading (precision) balance | 0.01 g or 0.001 g | Buffer salts, media, larger masses |
| Analytical balance | 0.0001 g (0.1 mg) | Standards, reference materials |
| Microbalance | 0.001 mg or finer | Very small samples, filters |

Good weighing practice on an analytical balance:

1. **Check it is level.** Most balances have a bubble level. An unlevel balance reads wrong.
2. **Check the daily verification.** Many labs weigh a certified check weight each day and log it. If the log shows a failure, the balance is out of service.
3. **Close the draught shield doors** before reading. Air currents move the last digit.
4. **Let warm or cold items reach room temperature.** A warm beaker creates convection currents and reads light.
5. **Use a weighing boat or paper, and tare it.** Taring sets the display to zero with the container on the pan.
6. **Weigh by difference** for hygroscopic or valuable material: weigh the closed vial, remove some material, weigh the vial again. The mass transferred is the difference, and nothing left on a weighing boat is lost from the calculation.
7. **Keep the pan clean.** Spilled material changes later readings and can damage the mechanism.

### Worked example: weighing by difference

| Reading | Mass (g) |
|---|---|
| Vial with sample, before | 12.4831 |
| Vial with sample, after transfer | 12.2337 |
| **Transferred** | **0.2494** |

You report 0.2494 g (249.4 mg), with all four decimal places the balance gave you.

## Volumetric glassware

Glassware falls into two groups:

- **Volumetric (precise):** volumetric flasks, volumetric (transfer) pipettes and burettes. They are calibrated to a single volume or a fine scale.
- **Approximate:** beakers, Erlenmeyer flasks and, to a lesser extent, graduated cylinders. Beaker markings are only rough guides, often within about 5 percent. Never use a beaker to measure a volume that matters.

Volumetric glassware is made to a **class**. Class A has the tightest tolerances, and Class B tolerances are roughly twice as wide. The tolerance is printed on the glass. For example, a 100 mL Class A volumetric flask is typically marked ±0.08 mL.

Two markings tell you how the glass was calibrated:

- **TC (to contain):** the flask holds the stated volume when filled to the line. Volumetric flasks are TC.
- **TD (to deliver):** the stated volume comes out when the pipette drains, leaving a small film behind. Most transfer pipettes are TD, and you do not blow out the last drop unless the pipette is marked for it.

### Reading a meniscus

Water forms a curved surface (a meniscus) that dips in the middle. Read the **bottom of the meniscus** with your eye level with the line. Looking from above or below causes **parallax error**. A white card with a dark stripe held behind the glass makes the bottom of the curve easier to see.

### Significant figures

Record every certain digit plus one estimated digit. A 50 mL burette has 0.1 mL graduations, so you read it to 0.01 mL, for example 23.46 mL. Writing "23.5 mL" throws away information you actually measured.

## Common mistakes

- Measuring an important volume with a beaker or graduated cylinder instead of a volumetric pipette or flask.
- Filling a volumetric flask to the line before the solid has fully dissolved, then topping up later.
- Reading the meniscus from above.
- Weighing a sample straight from the oven.
- Rounding a balance reading in the notebook.
- Using a balance that failed its daily check.

## Beyond this course

This course teaches the knowledge behind bench work. Real bench skills come from supervised practice, and the careers it serves have formal requirements:

- **Medical laboratory technologist / scientist:** a NAACLS-accredited program with clinical rotations (https://www.naacls.org), ASCP Board of Certification MLS certification (https://www.ascp.org/content/board-of-certification), and a state licence in states that require one.
- **Biological technician:** an associate or bachelor's degree in biology or biotechnology with supervised lab hours. In regulated manufacturing, GMP training happens on the job (FDA CGMP: https://www.fda.gov/drugs/pharmaceutical-quality-resources/current-good-manufacturing-practice-cgmp-regulations).
- **Chemist:** a bachelor's in chemistry with hands-on lab courses; ACS-approved programs are preferred by many employers (https://www.acs.org/education/policies/acs-approval-program.html).
- **Biochemist or materials scientist:** a bachelor's with real lab or instrument coursework for technician roles; an MS or PhD for independent research.

Requirements vary by state and employer, so check the official sources. (Checked on 2026-10-23.)

## Quick recap

- Accuracy, precision and readability are different properties.
- Level, verify, close the doors, tare, and weigh by difference when it matters.
- Use Class A volumetric glassware for exact volumes, and read the bottom of the meniscus at eye level.
- Record every digit the instrument gives you, plus one estimated digit for scales you read by eye.

Real lab training covers your institution's balances and glassware in person. For lab safety rules, see OSHA's laboratory page, https://www.osha.gov/laboratories (Checked on 2026-10-23.)

## Try it

Open the **Do** tab to practise accuracy, precision and meniscus reading with a kitchen scale, a measuring jug and plain tap water.


Weigh and measure to the stated precision: balances, weighing by difference, volumetric glassware and reading a meniscus.

Balances and volumetric glassware

How to Use an Analytical Balance

The Volumetric Pipet and Pipetting Technique

Copy these ten weighings for a P200 set to 50 µL into a table or spreadsheet: 49.2, 50.4, 49.8, 50.1, 48.9, 50.3, 49.6, 50.0, 49.4, 50.2 (mg).

Calculate the mean mass. Convert it to volume using 1.003 µL per mg.

Calculate the systematic error in µL and as a percentage of 50 µL.

Calculate the sample standard deviation and the CV (standard deviation divided by mean, as a percentage).

Suppose your lab's limits for this pipette at 50 µL are ±1.0 percent systematic error and 0.6 percent CV. Decide whether the pipette passes, and write one sentence explaining why.

List three technique errors from the lesson that could explain a high CV.

In your teaching lab, with your instructor's permission and supervision, practise forward pipetting plain water: set a volume, attach a tip, find the first and second stops, aspirate vertically and dispense against a tube wall ten times.

> **Awareness level only.** This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.


## Why it matters

Biology and clinical labs move small volumes all day: 2 µL of DNA into a reaction, 50 µL of serum into a cuvette, 200 µL of standard into each well of a plate. A micropipette makes this possible, but it is easy to use badly. Holding it at an angle, plunging too fast or using the wrong stop can each cause errors of several percent, and those errors are invisible until a control fails.

## How an air-displacement micropipette works

Most lab micropipettes are **air-displacement** pipettes. The plunger moves a piston inside the barrel. When you release the plunger, the piston rises and the air cushion beneath it pulls liquid into a disposable tip. The liquid never touches the pipette itself, only the tip.

Pipettes come in sizes. Common names follow the maximum volume:

| Pipette | Typical range |
|---|---|
| P2 | 0.2 to 2 µL |
| P10 | 1 to 10 µL |
| P20 | 2 to 20 µL |
| P200 | 20 to 200 µL |
| P1000 | 100 to 1000 µL |

Choose the **smallest pipette that covers your volume**. Accuracy and precision are best in the upper part of a pipette's range. Pipetting 15 µL with a P20 is better than with a P200. Never turn the volume dial past the pipette's limits: it can damage the mechanism.

## The two stops

Press the plunger slowly and you will feel two points of resistance:

- **First stop:** the calibrated volume. You press to here before aspirating.
- **Second stop (blow-out):** pushes out the last drop when dispensing.

### Forward pipetting (the standard method)

1. Set the volume and attach a fresh tip firmly.
2. Hold the pipette vertically. Press the plunger to the **first stop**.
3. Immerse the tip just below the surface, a few millimetres (about 1 mm for very small volumes, a few mm for large ones). Too deep coats the outside of the tip with extra liquid.
4. Release the plunger **slowly and smoothly**. Wait a second for the liquid to finish entering the tip.
5. Withdraw the tip, touching it against the wall of the vessel if your SOP allows, to remove drops on the outside.
6. Dispense by pressing to the first stop with the tip against the wall of the receiving vessel, then to the **second stop** to blow out the rest.
7. Keep the plunger pressed while you remove the tip from the liquid, then release it. Eject the tip.

### Reverse pipetting

For viscous liquids (glycerol, serum) or liquids that foam (detergent solutions), labs often use **reverse pipetting**: press to the second stop before aspirating, then dispense only to the first stop, leaving the excess in the tip. It reduces bubbles and film errors. Your SOP will say which method to use.

### Habits that improve accuracy

- **Pre-wet the tip** by aspirating and dispensing the liquid two or three times before the measured transfer.
- Let the pipette, tips and liquid reach the same temperature.
- Keep a consistent rhythm and plunger speed.
- Change tips between samples to avoid carry-over.
- Never lay a pipette on its side or turn it upside down with liquid in the tip: liquid can run into the barrel and contaminate it.

## Checking a pipette: the gravimetric method

Labs check pipettes by weighing water, because 1 µL of water at about 20 °C weighs very close to 1 mg. The full procedure (ISO 8655) corrects for temperature and air pressure, but the idea is simple.

### Worked example

Ten 100 µL dispenses of water weigh (in mg): 99.6, 100.1, 99.8, 99.9, 100.2, 99.7, 99.9, 100.0, 99.8, 99.6.

- **Mean:** 99.86 mg. Converting with a factor of about 1.003 µL per mg gives about 100.16 µL.
- **Systematic error (accuracy):** 100.16 minus 100 = +0.16 µL, or +0.16 percent.
- **Random error (precision):** the standard deviation is about 0.20 mg, so the coefficient of variation (CV) is 0.20 / 99.86 = about 0.20 percent.

Your lab compares these numbers with acceptance limits for that pipette size. If a pipette fails, it is labelled out of service and sent for adjustment.

## Common mistakes

- Aspirating at an angle instead of vertically.
- Pressing past the first stop before aspirating (this draws up too much in forward pipetting).
- Letting the plunger snap back, which splashes liquid into the barrel and forms bubbles.
- Using a P1000 at 50 µL.
- Reusing a tip between different samples.
- Skipping the scheduled pipette check because the pipette "seems fine".

## Quick recap

- Pick the smallest pipette that covers the volume, and stay within its range.
- First stop to aspirate, second stop to blow out (forward pipetting).
- Vertical, shallow, slow and smooth; pre-wet the tip; change tips.
- Pipettes are checked by weighing water: mean gives accuracy, CV gives precision.

## Try it

Open the **Do** tab to analyse a pipette check on paper, then practise the plunger motions with plain water if you have access to a teaching lab.


Use a micropipette accurately: choosing the right size, the two stops, forward and reverse pipetting, and gravimetric checks.

Pipetting

How to Use a Micropipette

Proper Pipetting Technique

> **Awareness level only.** This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.


## Why it matters

Buffers and reagent solutions are the background of nearly every assay. An enzyme assay at the wrong pH, a cell wash at the wrong salt concentration or a standard made at the wrong molarity can make a whole run invalid. The good news is that the maths is short and the same few formulas cover most lab work.

## Molarity and the core formula

**Molarity (M)** is moles of solute per litre of solution. To find the mass you need:

```text
mass (g) = molarity (mol/L) x volume (L) x molar mass (g/mol)
```

### Worked example: 500 mL of 0.150 M NaCl

NaCl has a molar mass of 58.44 g/mol.

```text
mass = 0.150 x 0.500 x 58.44 = 4.383 g
```

Weigh 4.383 g, dissolve it in less than 500 mL of water in a beaker, transfer quantitatively to a 500 mL volumetric flask (rinsing the beaker into the flask), and make up to the line.

**Check the label for the exact form.** Many salts come as hydrates. Magnesium chloride hexahydrate (MgCl2·6H2O, 203.30 g/mol) needs more than twice the mass of anhydrous MgCl2 (95.21 g/mol) for the same molarity. Using the wrong molar mass is one of the most common prep errors.

## Dilutions

When you dilute a stock, the amount of solute stays the same:

```text
C1 x V1 = C2 x V2
```

To make 250 mL of 0.10 M from a 2.0 M stock: V1 = 0.10 x 250 / 2.0 = 12.5 mL of stock, made up to 250 mL. A **serial dilution** repeats a fixed step (for example 1:10 each time) to reach very low concentrations accurately.

Other units you will meet: **percent w/v** (grams per 100 mL, so 0.9 percent NaCl is 0.9 g per 100 mL) and **mg/mL** or **µg/mL** for standards.

## Buffers

A **buffer** resists changes in pH. It is a mixture of a weak acid (HA) and its conjugate base (A-). The **Henderson-Hasselbalch equation** links pH to their ratio:

```text
pH = pKa + log10( [A-] / [HA] )
```

Rearranged, the base-to-acid ratio you need is:

```text
[A-] / [HA] = 10 ** (pH - pKa)
```

A buffer works best within about **one pH unit of its pKa**. Outside that range it has little capacity to absorb added acid or base.

| Buffer system | pKa (about 25 °C) | Useful range |
|---|---|---|
| Acetate | 4.76 | 3.8 to 5.8 |
| Phosphate (H2PO4- / HPO4 2-) | 7.21 | 6.2 to 8.2 |
| Tris | 8.06 | 7.1 to 9.1 |

### Worked example: 1 L of 0.10 M phosphate buffer, pH 7.40

1. Ratio: 10 ** (7.40 - 7.21) = 10 ** 0.19 = 1.549.
2. Fraction that is base: 1.549 / (1 + 1.549) = 0.608. Base = 0.0608 M, acid = 0.0392 M.
3. Masses for 1 L, using anhydrous salts:
   - Na2HPO4 (base, 141.96 g/mol): 0.0608 x 1 x 141.96 = 8.63 g
   - NaH2PO4 (acid, 119.98 g/mol): 0.0392 x 1 x 119.98 = about 4.71 g (4.707 g before rounding the molarity)

The calculation gets you close. Real solutions differ from the ideal equation because of ionic strength and temperature, so the standard practice is:

- Dissolve the components in about 80 percent of the final volume.
- Measure with a **calibrated pH meter** at the temperature you will use the buffer, and adjust with small additions of acid or base as your SOP specifies.
- Make up to the final volume **after** adjusting the pH.
- Label it: name, concentration, pH, date, your initials, expiry and lot numbers of the components.

Tris is a classic trap: its pKa falls by about 0.03 units per °C, so a Tris buffer set to pH 8.0 at room temperature is noticeably more alkaline in a cold room.

## Common mistakes

- Using the molar mass of the anhydrous salt when the bottle is a hydrate (or the reverse).
- Making up to volume before adjusting the pH.
- Choosing a buffer whose pKa is more than one unit away from the target pH.
- Setting the pH at room temperature for a buffer used at 4 °C or 37 °C.
- Leaving bottles unlabelled "just for today".

## Quick recap

- mass = M x V x molar mass; dilute with C1V1 = C2V2.
- Henderson-Hasselbalch gives the base-to-acid ratio: 10 ** (pH - pKa).
- Pick a buffer with a pKa within one unit of your target, then confirm with a calibrated pH meter.
- Label every solution completely.

## Try it

Open the **Do** tab and finish a small Python buffer calculator. The code already runs: you write one line, the Henderson-Hasselbalch ratio.


Calculate masses, dilutions and Henderson-Hasselbalch ratios to prepare solutions and buffers.

Preparing solutions and buffers

Solution Preparation

Preparing Tris Buffer

Weigh and measure to the stated precision, use a micropipette accurately, and calculate and prepare solutions and buffers.