Liquid culture & strain storage
A single colony inoculated into 2–5 mL of medium with the appropriate antibiotic and shaken 12–16 h at 37 °C (or 30 °C for difficult plasmids), giving a dense, pure, plasmid-bearing culture for a prep, an experiment or a stock.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Overnight cultures
A single colony inoculated into 2–5 mL of medium with the appropriate antibiotic and shaken 12–16 h at 37 °C (or 30 °C for difficult plasmids), giving a dense, pure, plasmid-bearing culture for a prep, an experiment or a stock.
How it differs from the standard method
- Pick one isolated colony from a fresh plate; avoid sweeping old plates with satellites.
- Keep the culture to about a fifth of the vessel's volume with a vented cap, shaking at 200–250 rpm.
- Always include the antibiotic; carbenicillin holds selection longer than ampicillin in dense cultures.
- Shake a no-cell tube of the same medium alongside as a sterility control.
1The overnight: a dense culture from one cell
Almost every bacterial experiment starts from a liquid culture: a few millilitres of medium inoculated from a single colony and shaken overnight, so that by morning one clone has become several billion cells. Three things decide whether that culture is what you think it is.
2Keeping the plasmid
Every division gives a small chance of a daughter cell without the plasmid, and plasmid-free cells often grow slightly faster because they no longer carry its cost. Without an antibiotic they take over within a few dozen generations.
Overnight
Early stationary: the highest yield of healthy cells and the best plasmid preps.
Late
Usable, but cells are starving; plasmid yield and quality start to fall.
Declining
Lysis, nucleases, plasmid loss on ampicillin. Start again from a colony.
3Glycerol stocks: the long-term archive
Plates last weeks and cultures last days, but a strain mixed with glycerol and frozen at −80 °C lasts for many years. Glycerol enters the cells and lowers the amount of ice and the salt concentration they meet as the water freezes; without it, most cells die in the freeze.
- Sterility control
- A tube of the same medium and antibiotic with no cells, incubated beside the culture. If it grows, the medium or the antibiotic stock is contaminated, and so is the culture.
- Plasmid retention
- The fraction of cells in a culture still carrying the plasmid, scored by plating on non-selective medium and patching colonies onto selective medium.
- Strain database
- The lab's record of every banked strain: number, genotype, plasmid, who made it and when, and where each vial is stored.
Key terms
Practise these as flashcards →A small liquid culture, typically 2–5 mL, grown 12–16 h from a single colony to early stationary phase.
An antibiotic or other condition in the medium that only plasmid- or marker-carrying cells survive.
Small plasmid-free colonies growing where a resistant colony has destroyed the ampicillin around it.
A culture mixed with glycerol to 15–25% final and frozen at −80 °C for long-term storage.
A solute (glycerol, DMSO) that reduces ice and salt damage to cells during freezing.
Warming a frozen stock and freezing it again; each cycle kills a large part of the cells.
When to use it
- Growing cells for a plasmid miniprep or midiprep, a protein expression culture, competent cells, or any experiment that needs a known, dense, pure culture.
- Starting a growth curve or a sub-culture with a reproducible inoculum.
- Banking every new strain or verified construct as soon as it is confirmed, before anything else is done with it.
- Recovering a strain from the archive for a new experiment, by scraping the frozen stock and streaking it out.
Compared with alternatives
The step before an overnight, and the step after reviving a stock: a single isolated colony is what makes the culture one clone.
Follows a culture through its phases in detail; an overnight is the same growth stopped at early stationary phase.
The mammalian-cell equivalent of a glycerol stock, with DMSO, controlled cooling to −80 °C and storage in liquid nitrogen.
The usual destination of an overnight; preps from cultures that have lost the plasmid or gone into decline give poor yields.
A strain collection is only useful if every vial can be found and its genotype trusted.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A single colony inoculated into 2–5 mL of medium with the appropriate antibiotic and shaken 12–16 h at 37 °C (or 30 °C for difficult plasmids), giving a dense, pure, plasmid-bearing culture for a prep, an experiment or a stock.
- Pick one isolated colony from a fresh plate; avoid sweeping old plates with satellites.
- Keep the culture to about a fifth of the vessel's volume with a vented cap, shaking at 200–250 rpm.
- Always include the antibiotic; carbenicillin holds selection longer than ampicillin in dense cultures.
- Shake a no-cell tube of the same medium alongside as a sterility control.