OD600 Measurement by Spectrophotometer
Quantifying bacterial culture density by measuring optical density at 600 nm — the standard proxy for cell concentration in liquid culture. Used to gate induction timing in recombinant protein expression, log-phase harvests, and any workflow where cell density matters. Written for a novice audience; assumes basic pipetting technique.
Version History
Version 0.1.2 Latest locked
Effective: 2026-05-21F12.v2 declared outputs: OD600 (MCO:0000077) in absorbance units AU (UO:0000269). Bridge change; protocol body, parameters, equipment, materials, references identical to v0.1.1. Second F12.v2-shape bump on garage-genomics side — declaration-driven matrix will auto-populate this column on any new Process against this version. Property CURIE matches measurement_type (single-output procedure).
Version 0.1.1 Viewing
Effective: 2026-04-20All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0
Effective: 2026-04-20Initial release. Atomic technique supporting procedure 59 (OpenTaq Cellular Reagent Production) and any workflow gated on culture density. Content written from standard spectrophotometry practice.
Procedure Details
- Live culture handling. All steps with live culture must be in a biosafety cabinet (PC1/CL1) if the strain is a GMO. Autoclave any waste cuvettes and tips.
- Spectrophotometer. Ensure no leaks from the cuvette — spilled culture in the instrument is a decontamination headache.
- UV source (if any). Most OD600 measurements use visible light and are safe; some dual-beam instruments include a UV source — consult the instrument manual and wear eye protection per vendor guidance.
What you need:
- The spectrophotometer (any instrument that reads 600 nm will work — ranging from a £200 benchtop unit to a £20,000 research spec). Turn it on and allow 5 minutes of warm-up. Select the OD or absorbance mode at 600 nm wavelength.
- Cuvettes. Plastic 1 mL disposable cuvettes are standard (acrylic, optical-grade). Polystyrene cuvettes also work but some are not UV-compatible — not an issue at 600 nm. Verify the cuvette's optical path length (usually 1.0 cm — the instrument assumes this).
- Blank solution. The same growth media your culture is in (for example, LB or Superior Broth). Keep it at room temperature; refrigerated media gives lower readings until it warms.
- Live culture. Should be mixed thoroughly just before sampling — settled cells give a lower reading.
- Pipette + sterile tips. For taking a 1 mL aliquot of culture and 1 mL of blank.
Mental model: OD600 measures how much light at 600 nm is blocked (absorbed + scattered) by particles in suspension. Bacteria are those particles. The relationship between OD and cell count is approximately linear in the 0.1–1.0 range and becomes non-linear above 1.0 (where dilution becomes necessary).
Rule of thumb conversions (strain-dependent, use as rough guide only):
- OD600 = 0.1 ≈ 8 × 10⁷ E. coli cells/mL
- OD600 = 1.0 ≈ 8 × 10⁸ E. coli cells/mL
- OD600 = 0.4–0.7 = mid-log phase (typical induction window)
- Setup (5 min): turn on instrument, warm-up, prepare blank.
- Per-measurement (1 min): sample, blank, measure, record.
- For induction monitoring: measure every 30 min from OD600 0.1 upward until target OD600 reached.
-
Micropipette
Liquid handling
Specs: P1000
For 1 mL aliquots -
Spectrophotometer
Measurement
Specs: 600 nm capable, ≥1 mL cuvette or microvolume
Any instrument reading absorbance at 600 nm works -
Biosafety cabinet
Safety
Specs: Class II Type A2 or equivalent
For GMO sampling
-
Superior Broth
(Athena Enzyme Systems)
0105Growth media
Qty: 1 mL per blank
Must match the culture's media for accurate blanking
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
wavelength_nm |
number | — |
600
|
nanometer (UO:0000018) | Measurement wavelength. 600 nm standard for bacterial cell density. Other wavelengths (e.g. 595 nm) give nearly identical results within 1–2%. |
path_length_cm |
number | — |
1.0
|
— | Optical path length of the cuvette. 1.0 cm standard for disposable plastic cuvettes. Microvolume instruments (NanoDrop-style) use a different path length set automatically. |
dilution_factor |
number | — |
1
|
— | Dilution applied to the culture before measurement. 1 = undiluted. Use 10 for OD > 1; 100 for OD > 5. Multiply the instrument reading by this factor for the actual culture OD. |
reading_temperature_c |
number | — |
22
|
degree Celsius (UO:0000027) | Room temperature at the time of reading. Culture freshly removed from 37°C incubator reads slightly different than culture equilibrated to room temperature. |
Procedure Steps (Version 0.1.1)
Turn on the spectrophotometer. Allow 5 minutes for the lamp to warm up and stabilise.
Set the wavelength to 600 nm. Select absorbance (or OD) mode. Confirm path length is set to 1.0 cm (the standard for 1 mL disposable cuvettes).
Retrieve your culture from the incubator. Observe it visually: uniform cloudiness is normal log-phase growth; surface films, flakes, or pigmented spots indicate contamination (do not proceed — discard and restart).
Mix the culture thoroughly by gentle inversion (5–6 times) or careful swirl. Cells can sediment within minutes, giving misleadingly low readings.
In the biosafety cabinet, take a 1 mL aliquot of the culture using a sterile filter tip.
Transfer the aliquot to a clean 1 mL cuvette. Wipe the outside of the cuvette with a lint-free wipe to remove any fingerprints or drops (these scatter light and distort readings).
If this is the first measurement of the session, first take a blank reading: fill a fresh cuvette with 1 mL of sterile growth media (the same composition as your culture), wipe it, insert with clear sides facing the light path, and zero the instrument per its manual.
Insert your culture cuvette with clear sides (not frosted/stippled sides) facing the light path. Different cuvettes have different orientations — the instrument manual specifies the correct direction.
Take the reading. Record the OD600 value.
If the reading is above 1.0: remove the cuvette, dilute 10 µL of your culture into 990 µL of blank media (1:100 dilution using a clean cuvette), take a new reading, and multiply the result by 100. For readings in the 1.0–2.0 range a 1:10 dilution is fine and gives more accuracy than no dilution.
Record the OD600 reading on the Process record in LibreBiotech, along with the time of measurement. If this measurement gates the next step (e.g. induction in procedure 59), proceed immediately — OD600 can change meaningfully in 5–10 minutes during log phase.
Clean up: discard disposable cuvettes to autoclave waste. If using reusable glass cuvettes, rinse with distilled water, then 70% ethanol, then allow to air-dry inverted.
Expected outcome. An OD600 reading (dimensionless, typically 0.0–2.0). For E. coli grown in rich media at 37°C from 1:200 dilution of an overnight, expect:
- 0–30 min: OD600 <0.1 (lag phase, barely detectable)
- 1–2 h: OD600 0.1–0.7 (log phase, active division)
- 2–4 h: OD600 0.7–2.0 (late log, approaching stationary)
- 4+ h: plateau (stationary phase)
When OD600 > 1.0. Readings above 1.0 are typically outside the linear range of most spectrophotometers. Dilute 1:10 in blank media, remeasure, and multiply the reading by 10. For readings above 5.0, dilute 1:100.
Recording. Every induction-gated protocol (like procedure 59) should record the OD600 at induction time on the Process record. This is a tunable parameter (harvest_od600) whose actual value impacts downstream yield and is important for batch-to-batch consistency.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| Reading of 0.0 on culture | Forgot to blank with correct media | Re-blank and remeasure |
| Negative reading | Blanked against a denser reference than the sample | Re-blank with sterile media, not used culture |
| Reading doesn't increase over time | Culture not growing (stuck, contaminated, or wrong temp) | Verify incubator temp, check for contamination visually, verify antibiotic was added |
| Highly variable readings between measurements | Culture sedimenting between readings | Mix thoroughly (invert or gentle swirl) before each sample |
| Reading much higher than expected | Cell debris or clumping from old culture | Dilute and remeasure; cloudy media / clumping is a sign of contamination or overgrowth |
References
- Standard spectrophotometry practice; see standard molecular biology references (e.g. Green & Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed, CSHL Press 2012) and instrument manuals. book