Biofilm assays
Quantify how well microbes form biofilms on surfaces.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Measuring what stays stuck
Most bacteria spend most of their lives attached to a surface inside a matrix they secrete, not swimming free in broth. A biofilm assay asks how much of a culture ends up in that state.
2What the well is actually doing
- Hours 0–2: cells sediment and attach reversibly. Staining now measures settling, not biofilm.
- Hours 2–8: attachment becomes irreversible, cells divide on the surface and matrix genes come on.
- Hours 8–24: biomass accumulates fastest; this is where a crystal violet assay is normally read.
- After 24–48 h: growth at the surface slows as the medium is exhausted, and parts of the biofilm detach. In a static well the detached material is poured away, so the reading can fall while the biofilm is still mature.
3The three controls that make it a measurement
Medium only
Carried through growth, washing, staining and destaining. It measures the dye the plastic holds, and it is the zero every other well is read against.
Non-adherent strain
A mutant known not to adhere should read near the blank. If it does not, you are measuring carried-over planktonic cells, not a biofilm.
Known strong former
Separates a bad day (wrong plastic, over-washing, old dye) from a strain that genuinely makes little biofilm.
Planktonic OD600
Read before the plate is poured off. Dividing stain by growth separates 'does not adhere' from 'does not grow', which is the commonest confusion in a mutant screen.
4Why the numbers scatter, and what to do about it
This assay has a reputation for poor reproducibility, and it is earned: a few per cent of the biofilm comes off with every wash, a jet from a wash bottle takes far more and takes a different amount from each well, outer wells lose volume to evaporation and read high, and the dye sits as a ring on the wall where a plate reader's beam may or may not pass through it. None of that is mysterious, and all of it is controllable.
- Biofilm
- A surface-attached community of cells in a self-produced matrix of polysaccharide, protein and extracellular DNA, with a physiology different from that of the same cells in broth.
- Planktonic
- Free-swimming or suspended cells: what is poured away before staining, and what has to be measured separately as OD600 if the biofilm number is to be normalised.
- Biofilm index
- Stain divided by growth (A595/OD600) for the same well, so that a growth defect is not reported as an adhesion defect.
- Minimum biofilm eradication concentration
- The drug concentration needed to kill an established biofilm, typically orders of magnitude above the MIC for the same organism in broth.
Key terms
Practise these as flashcards →A cationic triarylmethane dye used at 0.1%; binds negatively charged surfaces, so it stains cells and matrix without distinguishing them.
Growth without shaking, so cells can settle and attach. Shaking the plate converts the assay into a (badly aerated) broth culture.
Dissolving the bound dye in 30% acetic acid (or ethanol) so the absorbance is proportional to the dye rather than to where it sits in the well.
Systematically different readings from the outer ring of a microplate, from evaporation and thermal gradients. Avoided by filling the outer wells with medium.
Many organisms build their heaviest biofilm at the meniscus rather than on the bottom, which is why the stain is read as a solution, not through the well.
Safranin and alcian blue stain biomass similarly; resazurin and XTT report metabolic activity; a CFU count after sonication reports viable cells.
When to use it
- Screening a mutant library, a strain collection or a panel of media for differences in surface attachment, where throughput matters more than resolution.
- Comparing a wild type with a defined mutant and a complemented strain on the same plate, as a first phenotype before any microscopy.
- Testing whether a compound, surface coating or medium component prevents biofilm formation, with the planktonic MIC measured alongside.
Compared with alternatives
Scrape or sonicate the biofilm and plate it when you need viable cells rather than biomass: the two diverge completely after an antimicrobial treatment.
Run a growth curve first: a strain that grows poorly will stain poorly, and only the planktonic curve can tell you which you are looking at.
An MIC is measured on planktonic cells; biofilms survive concentrations far above it, so susceptibility has to be tested on the attached population.
Use confocal imaging of a stained or fluorescent biofilm when thickness, architecture or the distribution of live cells is the question, which absorbance cannot address.
The read itself is a plate-reader absorbance measurement, with the same linearity and path-length limits as any other.