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

Lab Techniques & Instrumentation

Set up a calibration log with these columns: instrument ID, date and time, standards with lot and expiry, temperature, slope (percent), offset (mV), limits, pass or fail, action, initials.

Calibration 1 at 25 °C: pH 7.00 reads +4 mV and pH 4.01 reads +175 mV. Calculate the slope in mV per pH unit and as a percentage of 59.16. Record it in the log.

Calibration 2 at 25 °C: pH 7.00 reads -6 mV and pH 4.01 reads +150 mV. Calculate the slope percentage and record it.

Using limits of 95 to 105 percent slope and ±30 mV offset, mark each calibration pass or fail.

For the failing calibration, write the action you would take: what label goes on the meter, who you tell, and what you check next (cleaning, reconditioning, buffer quality).

Write one sentence on which sample results might need review if the failing meter had been used the previous afternoon.

Find the calibration or service sticker on any instrument you have access to (a thermostat, a scale or, in your teaching lab, a balance or pH meter) and note what information it shows.

If you are in a lab course, ask your instructor to demonstrate a two-point pH calibration and compare their log entry with yours.

> **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

An instrument is only as trustworthy as its last calibration. A pH meter that drifted 0.2 units overnight, a balance that was knocked out of level, or a thermometer that reads 1 °C low can all produce results that look perfectly normal and are wrong. Calibration, verification and maintenance, and the records that prove they happened, are what let a lab stand behind its numbers. Auditors check these logs early in any inspection.

## Three words that are often mixed up

- **Calibration:** comparing an instrument's readings with known reference standards, and usually adjusting it so it reads correctly. Example: calibrating a pH meter with pH 4, 7 and 10 buffers.
- **Verification (or check):** confirming that the instrument still reads within limits, without adjusting it. Example: weighing a certified check weight on a balance each morning.
- **Maintenance:** keeping the instrument working: cleaning, replacing parts, servicing. Some is daily (wiping a balance pan), some yearly by an engineer (preventive maintenance).

**Traceability** means each reference standard is linked, through an unbroken chain of calibrations, to national or international standards (in the US, often through NIST). Certificates for buffers, weights and thermometers state this traceability.

## Calibrating a pH meter

A pH electrode produces a voltage that changes with pH. At 25 °C an ideal electrode changes by **59.16 mV per pH unit**. Real electrodes age, so the meter is calibrated against buffers of known pH.

Typical steps (your SOP and the meter's manual come first):

1. Check the buffers are in date and at room temperature, and note their lot numbers.
2. Rinse the electrode with deionised water and blot (do not wipe) it dry.
3. Place it in **pH 7.00** buffer first (this sets the offset, or zero point). Wait for a stable reading and accept.
4. Rinse, blot, and place it in **pH 4.01** (or 10.01) buffer. This sets the **slope**. For samples across a wide range, use a three-point calibration with 4, 7 and 10.
5. Read the slope and offset the meter reports and compare them with your acceptance limits. A common rule of thumb is a slope of **95 to 105 percent** and an offset within about ±30 mV, but follow your lab's limits.
6. Verify with a buffer not used in the calibration, if your SOP requires it.
7. Store the electrode in its **storage solution** (often KCl), never in deionised water, which damages it over time.
8. **Log it.**

### Worked example: is this electrode acceptable?

At 25 °C the meter reads -2 mV in pH 7.00 buffer and +168 mV in pH 4.01 buffer.

```text
slope = (168 - (-2)) / (7.00 - 4.01) = 170 / 2.99 = 56.9 mV per pH unit
percent of ideal = 56.9 / 59.16 x 100 = 96.1 percent
offset = -2 mV
```

The slope is within 95 to 105 percent and the offset is small, so the calibration passes. A slope of 88 percent would mean the electrode needs cleaning, reconditioning or replacement.

## What goes in the log

A calibration record should let anyone reconstruct what happened. Typical fields:

| Field | Example |
|---|---|
| Instrument ID | PH-03 |
| Date and time | 2026-10-23 08:12 |
| Standards used, lot and expiry | pH 7.00 lot 51A, exp 2027-03; pH 4.01 lot 49C, exp 2027-01 |
| Temperature | 22.4 °C |
| Results | slope 96.1 percent, offset -2 mV |
| Pass or fail against limits | Pass (limits 95 to 105 percent, ±30 mV) |
| Action taken | None |
| Initials | AB |

## When an instrument fails

- Do not use it. Label it **out of service** (a tag or sticker) so others do not either.
- Record the failure and what you did.
- Tell your supervisor.
- Ask the important question: **what was measured since the last good calibration?** Those results may need review or repeat testing. This is often handled as a deviation, which you will meet in the Lab Data & Quality course.

## Preventive maintenance and service

Larger instruments (HPLC, centrifuges, biosafety cabinets, spectrophotometers) have scheduled **preventive maintenance**, often by the manufacturer's engineer, with a sticker showing the next due date. Biosafety cabinets and fume hoods are certified on a schedule. Check the sticker before you use any instrument; an overdue one may not be used for reportable work.

## Common mistakes

- Calibrating with expired or contaminated buffers.
- Wiping a pH electrode (static charge and scratches) instead of blotting it.
- Storing the electrode in deionised water.
- Writing the log at the end of the day from memory.
- Using an instrument with an expired calibration sticker "just this once".

## Quick recap

- Calibration adjusts against traceable standards; verification checks without adjusting; maintenance keeps it working.
- pH: offset at pH 7, slope with 4 or 10; accept a slope around 95 to 105 percent (per your SOP).
- Log every calibration completely and at the time you do it.
- A failed instrument is tagged out, and results since the last good calibration are reviewed.

For traceability and calibration services, see NIST: https://www.nist.gov/calibrations (Checked on 2026-10-23.)

## Try it

Open the **Do** tab to judge calibration data and design a calibration log on paper.


Calibrate a pH meter, judge its slope and offset, and keep a calibration and maintenance log.

Calibration and maintenance

Using a pH Meter

Instrument's Calibration in the Clinical Laboratory: Impact on patient care

## Why it matters

Ordinary light microscopes show cells and tissues, but many lab questions need more. Where in the cell is this protein? Is this bacterium alive? What does a virus or a crack in a metal grain boundary look like at 10 nanometres? Fluorescence and electron microscopy answer those questions, and they are central to biochemistry, cell biology, clinical microbiology and materials science. You may operate these instruments as a technician or depend on core facilities that do.

## The resolution limit of light

**Magnification** makes an image bigger. **Resolution** is the smallest distance at which two points still look separate. Magnifying beyond the resolution limit only makes a blur bigger.

For a light microscope, the Abbe limit is approximately:

```text
d = wavelength / (2 x NA)
```

where NA is the numerical aperture of the objective. With green light (about 500 nm) and a good oil-immersion objective (NA 1.4), d is about 500 / 2.8 = **about 180 nm**. Most viruses and all individual proteins are smaller than that.

## Fluorescence microscopy

A **fluorophore** absorbs light of one wavelength and emits light of a longer wavelength. The difference is the **Stokes shift**. A fluorescence microscope uses this:

1. A bright light source (LED, mercury or xenon lamp, or a laser) passes through an **excitation filter** that selects the wavelength the fluorophore absorbs.
2. A **dichroic mirror** reflects that light onto the sample.
3. The fluorophore emits longer-wavelength light, which passes through the dichroic mirror and an **emission filter** to the camera or eyepiece.
4. Only the labelled structures glow against a dark background.

The excitation filter, dichroic and emission filter are often mounted together as a **filter cube** matched to a fluorophore.

Common labels:

- **DAPI** and **Hoechst** bind DNA and stain nuclei blue.
- **Green fluorescent protein (GFP)** can be genetically fused to a protein so living cells show where it goes.
- **Antibodies tagged with fluorophores** (immunofluorescence) label specific proteins in fixed cells or tissue.

### Confocal and beyond

A widefield fluorescence image includes out-of-focus glow from above and below the focal plane. A **confocal microscope** scans a laser point and uses a **pinhole** to block out-of-focus light, giving sharp **optical sections** that can be stacked into 3D images. **Super-resolution** methods (such as STED, PALM and STORM) use clever tricks with fluorophores to get below the Abbe limit, to tens of nanometres.

### Limits

- **Photobleaching:** fluorophores fade with exposure. Keep light exposure short and image consistently.
- **Phototoxicity:** strong light damages living cells.
- **Autofluorescence:** some tissues glow on their own; controls without the label reveal it.
- You only see what you labelled.

## Electron microscopy

Electrons have a far shorter wavelength than visible light, so electron microscopes resolve down to the nanometre scale or below. The beam must travel in a **vacuum**, so samples must be dry or frozen and stable in vacuum.

| | Transmission EM (TEM) | Scanning EM (SEM) |
|---|---|---|
| How it works | Electrons pass **through** a very thin section | A focused beam scans the **surface**; detectors collect emitted electrons |
| Image | 2D internal structure (organelles, viruses, crystal defects) | 3D-looking surface topography |
| Sample preparation | Fix, embed, cut ultrathin sections (tens of nm), stain with heavy metals | Fix, dry, coat non-conductive samples with a thin metal layer (sputter coating) |
| Typical resolution | Down to below 1 nm | About 1 to 10 nm |

SEM instruments often add **energy-dispersive X-ray spectroscopy (EDS)**, which identifies elements in a spot on the sample. That makes SEM a workhorse in materials science for fracture surfaces, coatings and particles.

**Cryo-electron microscopy (cryo-EM)** freezes samples so fast that water forms glass-like ice instead of crystals, preserving molecules close to their natural state. It is now a major method for determining protein structures.

## Choosing a method

| Question | Better choice |
|---|---|
| Where is protein X in living cells, over time? | Fluorescence (GFP), confocal for 3D |
| What is the internal fine structure of a mitochondrion or virus? | TEM |
| What does the fracture surface of a steel part look like, and what elements are present? | SEM with EDS |
| What is the 3D structure of a purified protein complex? | Cryo-EM |

## Common mistakes

- Confusing magnification with resolution.
- Interpreting a fluorescence image without no-label or no-primary-antibody controls.
- Comparing images taken with different exposure settings.
- Forgetting that EM sample preparation (drying, staining, coating) can create artefacts.

## Quick recap

- Light microscopes are limited to about 200 nm resolution.
- Fluorescence uses excitation and emission filters with labels such as DAPI, GFP and antibodies; confocal adds sharp optical sections.
- TEM looks through thin sections; SEM scans surfaces; both need vacuum and careful preparation.
- Pick the method by the question: living or fixed, inside or surface, molecules or materials.

These instruments are operated only after training by the facility that runs them.

## Try it

This lesson is quiz-only. Open the **Do** tab to check your understanding.


Compare fluorescence, confocal and electron microscopy, and choose the right method for a question.

Advanced microscopy

Widefield and Confocal Fluorescence Microscopy

Electron Microscopy (TEM and SEM)

Clear a table and wipe it with an ordinary household cleaner. Mark out a "work zone" with tape, with a clean area on one side and a "waste" area on the other.

Set out two clean bottles or jars with lids (the "medium bottle" and the "flask"), a measuring spoon or syringe without a needle, and a label for the flask with today's date and your initials.

Fill the medium bottle with tap water and close it. Write the planned steps on a card before you start.

Wash your hands. Open the medium bottle and hold its lid in your other hand, inner side facing down, without touching the inside.

Transfer a measured amount of water to the flask without touching the rims, then close both containers. Notice whenever your hand passes over an open container.

Put the used spoon or syringe straight into the waste area.

Repeat the whole transfer three times, aiming for no hand-over-open-container moves and the shortest open time.

Write down every moment that would have been a contamination risk in a real lab, and how you would change your layout to avoid it.

If you are taking a lab course, ask your instructor to watch your technique during your next supervised aseptic transfer.

> **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

Aseptic technique is the set of habits that keeps unwanted microbes out of your work, and keeps your work's microbes where they belong. A single contaminating bacterium or fungal spore can ruin a cell culture that took weeks to grow, give a false positive in a clinical culture, or contaminate a batch in a biomanufacturing facility. Good technique also protects you and others. Every biological technician, medical laboratory technologist and biochemist is expected to perform it automatically.

## Sterile versus aseptic

- **Sterile** means free of all living microorganisms. Items are made sterile by autoclaving (steam under pressure), filtration, irradiation or similar.
- **Aseptic** technique means working in a way that keeps sterile items sterile and prevents contamination during handling.

You cannot make your bench sterile, but you can avoid contaminating sterile media, tips and cultures while you work.

## Where you work

- **Biosafety cabinet (BSC).** Cell culture and work with many microorganisms is done in a Class II BSC, which draws air through HEPA filters to protect the work, you and the room. Do not block the front or rear grilles, work at least several centimetres inside the cabinet, and move slowly so you do not disrupt the air curtain. Bunsen burners are generally **not** used in a BSC: the heat disrupts airflow and can damage the filter.
- **Open bench with a flame.** Many microbiology teaching labs work on the bench beside a Bunsen burner, which creates an updraft, and flame loops and tube mouths. Follow what your lab's SOP specifies.

The biosafety level of the agent (BSL-1 to BSL-4) decides which containment and practices are required. See the CDC/NIH guide *Biosafety in Microbiological and Biomedical Laboratories (BMBL)*: https://www.cdc.gov/labs/bmbl/ (Checked on 2026-10-23.)

## The core habits

1. **Prepare.** Plan the steps, gather everything you need, and label tubes and plates before you start, so the cabinet is not crowded and open containers wait as little as possible.
2. **Hands and PPE.** Wash hands, put on a clean lab coat and gloves as your SOP requires. Spray gloves with 70 percent ethanol and let them dry.
3. **Disinfect the work surface** before and after, usually with 70 percent ethanol (which works best with some water present and needs contact time) or another approved disinfectant.
4. **Disinfect items going into the cabinet** by wiping or spraying bottles and racks.
5. **Open containers only when needed and close them promptly.** Hold caps in your hand or place them face down on a disinfected surface, as your SOP specifies. Never touch the inside of a cap, the neck of a bottle or a sterile tip.
6. **Never pass hands or arms over open containers.** Particles fall from you onto whatever is below.
7. **Use each sterile pipette or tip once.** If it touches anything non-sterile, discard it and take a new one.
8. **Work in a logical flow**, from clean to dirty, and keep waste at the side.
9. **Clean up.** Discard waste into the correct biohazard containers, disinfect the surface again, remove gloves and wash hands.

## Worked example: an aseptic transfer of medium

In a BSC, a technician moves 10 mL of sterile medium into a labelled sterile flask:

1. Wipes the medium bottle and flask with 70 percent ethanol and places them in the cabinet.
2. Attaches a sterile 10 mL serological pipette to the pipette controller without touching the tip.
3. Opens the bottle, keeps the cap in the other hand, draws 10 mL, recaps the bottle.
4. Opens the flask, dispenses down the inside wall without touching the neck, recaps the flask.
5. Discards the pipette into the waste container without touching anything else on the way.

Every step keeps sterile surfaces from touching non-sterile ones and keeps containers open as briefly as possible.

## Checking your technique

- **Negative controls:** incubate a tube or plate of medium you opened but did not inoculate. Growth means your technique or materials introduced contamination.
- **Settle plates and environmental monitoring** are used in cleanrooms and biomanufacturing.
- In cell culture, watch for cloudy medium, sudden colour changes and floating particles.

## Common mistakes

- Talking, coughing or reaching over open containers.
- Overcrowding the cabinet and blocking its grilles.
- Touching the tip of a pipette to the outside of a bottle neck and carrying on.
- Leaving bottles open while you fetch something.
- Skipping the negative control.

## Quick recap

- Sterile is a state; aseptic is a set of working habits.
- Work in a BSC (no flame) or beside a flame, as your SOP says.
- Disinfect, open briefly, never touch sterile surfaces, never reach over open containers, single-use tips.
- Prove your technique with negative controls.

Aseptic work with real cultures is learned only in your teaching lab or workplace, after biosafety training and under supervision.

## Try it

Open the **Do** tab to rehearse the motions of an aseptic transfer using only clean, empty containers and tap water. No microbes, media or cultures are involved.


Understand the habits of aseptic transfer that keep cultures and reagents free of contamination.

Aseptic technique

Aseptic Technique

Sterile Technique

Calibrate and log instruments, compare fluorescence and electron microscopy, and practise the habits of aseptic technique.