Plating & streaking
A loop drags cells across the agar in three or four sections, sterilised between them, so the last sections hold single colonies. The everyday way to purify a strain, revive a stock and see whether a culture is mixed.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Streak plate
A loop drags cells across the agar in three or four sections, sterilised between them, so the last sections hold single colonies. The everyday way to purify a strain, revive a stock and see whether a culture is mixed.
How it differs from the standard method
- Start light: a touch of a colony or one loopful of broth. A heavy scrape cannot be diluted out in four sections.
- Flame and cool the loop (or take a fresh disposable loop) before each section; make two or three passes into the previous one, no more.
- Read at 16–24 h for E. coli; slower organisms need longer. Pick from the last sections, where colonies have clear agar round them.
- Purity is earned: re-streak a single colony once more before you trust it.
1One cell, one colony
Plating rests on a simple fact: a single viable bacterium or yeast cell left alone on nutrient agar divides where it sits, and after a night at the right temperature its descendants form a visible heap of perhaps a hundred million cells. If the cells that went onto the plate were far enough apart, each heap came from one cell and is a clone.
2Why a streak works
In a quadrant streak the loop lays down a heavy line of cells in the first section, is sterilised, and then makes two or three passes back through the end of that section before streaking the next. Each pass picks up only a small share of the cells it crosses, so every section starts from roughly a hundredth of the cells of the one before.
Isolation
Dilutes cells across the plate with a loop. Fast, needs no pipette, and the standard way to purify a strain or check a culture. Not quantitative.
Even surface colonies
A measured volume of a dilution spread over the surface. Colonies are all on top, easy to pick, and their number reflects the dilution.
Colonies in the agar
A dilution mixed with molten agar at 45–50 °C. Takes up to 1 mL and suits organisms that prefer less oxygen, but most colonies are embedded and small.
Same pattern, other media
A velvet carries the colony pattern of a master plate onto several others, so you can see which colonies fail on a selective medium.
3Spreading, pouring and the heat problem
A spread plate is only as even as the spreading. The liquid has to be pushed over the whole surface until it soaks in; a plate that cannot absorb it, because it is wet or because more than about 200 µL went onto a 90 mm dish, lets the liquid run and carry the cells to the edge.
4Replica plating
Joshua and Esther Lederberg's replica method presses a master plate onto sterile velveteen, whose fibres pick up cells from every colony in place, and then presses that velvet onto fresh plates. Colonies that grow on complete medium but not on minimal medium cannot make some nutrient for themselves: they are auxotrophs.
- Isolated colony
- A colony with clear agar all round it, so a loop or toothpick touches nothing else. The only colony you should pick as a clone.
- Confluent growth
- Colonies merged into a continuous film. Nothing in it is a clone and nothing can be counted.
- Auxotroph
- A mutant that cannot make a nutrient (an amino acid, a vitamin, a base) and grows only when it is supplied. Its parent, which can, is a prototroph.
Key terms
Practise these as flashcards →A streak in three or four sections, sterilising the loop between them, so each section is a further dilution of the one before.
Whatever gave rise to one colony: a single cell, or a chain or clump that could not be separated. Plates count CFU, not cells.
A known volume (usually 50–200 µL) of a dilution spread over the surface of a dried plate with a sterile spreader or glass beads.
Up to 1 mL of a dilution mixed with about 20 mL of molten agar cooled to 45–50 °C in an empty dish, swirled and allowed to set.
Transferring the pattern of colonies on a master plate to other plates with sterile velvet, to find colonies that fail to grow under a selective condition.
Small colonies growing in the zone around a large colony where it has used up or destroyed an inhibitor; common round ampicillin-resistant colonies on old plates.
When to use it
- Purifying a strain or checking that a culture is pure before it goes into an experiment or a freezer stock: streak for single colonies.
- Recovering a strain from a glycerol stock, a stab or someone else's plate: streak, then pick one isolated colony.
- Getting evenly spaced colonies from a known dilution for picking, screening or counting: spread a plate, or pour one for organisms that grow better inside the agar.
- Screening many colonies at once for a phenotype on a different medium, such as auxotrophy, antibiotic sensitivity or temperature sensitivity: replica plating.
Compared with alternatives
Uses the same spread and pour plates, but the point is the number: a dilution series, countable plates in the 30–300 range and a titre in CFU/mL.
Changes what is in the agar rather than how the cells go on: selective media let only some organisms grow, differential media make different ones look different.
Where replica plating comes from and where it is still used: finding, mapping and complementing mutants.
The step after a streak: a single isolated colony goes into a liquid culture, and from there into a glycerol stock.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A loop drags cells across the agar in three or four sections, sterilised between them, so the last sections hold single colonies. The everyday way to purify a strain, revive a stock and see whether a culture is mixed.
- Start light: a touch of a colony or one loopful of broth. A heavy scrape cannot be diluted out in four sections.
- Flame and cool the loop (or take a fresh disposable loop) before each section; make two or three passes into the previous one, no more.
- Read at 16–24 h for E. coli; slower organisms need longer. Pick from the last sections, where colonies have clear agar round them.
- Purity is earned: re-streak a single colony once more before you trust it.