Jun 10, 2026
Biofilm inhibition assay
- Marbio 1
- 1UiT The Arctic University of Norway
- Marbio
Protocol Citation: Marbio 2026. Biofilm inhibition assay . protocols.io https://dx.doi.org/10.17504/protocols.io.261geq62jg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 10, 2026
Last Modified: June 10, 2026
Protocol Integer ID: 318830
Keywords: Biofilm inhibition assay, Staphylococcus epidermidis, Staphylococcus haemolyticus, Crystal violet , natrual product, biodiscovery, bioprospecting, bioactivity screening, biofilm formation by staphylococcus epidermidi, biofilm inhibition, biofilm formation, biofilm, staphylococcus epidermidi, staphylococcus haemolyticus, purpose of the assay, assay
Abstract
The purpose of the assay is to evaluate whether samples or fractions inhibit the biofilm formation by Staphylococcus epidermidis. Growth on the surface of polystyrene plates as an indicator of biofilm formation. Cultures of S. epidermidis are diluted in fresh medium supplemented with glucose to induce the ica-operon. The culture is inoculated into microtiter plates and incubated overnight, after which biofilm formation is quantified by staining with crystal violet. Staphylococcus haemolyticus, which does not form biofilm, is used as a negative control.
Guidelines
All bacteria used in this assay belong to biosafety levels 1 and 2 and can be handled on the bench. However, we work in the hood to reduce the risk of contamination. Waste from this assay must be autoclaved before being transferred to waste bins marked with risk symbols. Glassware used in this assay should be autoclaved before being washed.
Materials
**Equipment**
Falcon tubes
Microtiter plates (VWR or Nunc, 734-2097)
Multichannel pipette
Inoculation loops
Autoclaved 100 ml Erlenmeyer flasks
Shake incubator
Tecan® Spark
**Media/reagents**
Autoclaved MQ-H₂O
Tryptic soy broth (Merk, 1.05459)
Blood agar plates
Crystal violet (Merck, 1.15940)
Glucose (Sigma, D9434)
70 % ethanol (VWR Chemicals, 20824.365)
**Bacteria**
Staphylococcus epidermidis (RP62A 42-77) ATCC 35984
Staphylococcus haemolyticus (clinical isolate 8-7A)
Safety warnings
Wear a dust mask, gloves, lab coat and safety goggles when preparing the crystal violet solution. Wear nitrile gloves and labcoat when working with crystal violet solution. Wear a dust mask in addition to nitrile gloves and labcoat when preparing crystal violet solution from its powder form.
Preparation of bacteria
Transfer a scoop of bacteria from the freezing stock to a blood agar plate using an inoculation loop. [Staphylococcus epidermidis (RP62A 42-77) ATCC35984; Staphylococcus haemolyticus (clinical isolate 8-7A)]
Incubate the blood agar plate over night at 37 °C.
Preparation of samples
Samples are diluted in ddH₂O with a maximum of 0.25% DMSO (final concentration in the assay).
Preparation of growth media
Prepare Tryptic soy broth (TSB) according to the instructions.
On day 2 the bacteria are diluted in TSB containing 1% glucose.
Preparation of crystal violet solution, 0.1%
Add 1 g of crystal violet to 1000 ml of MQ-H₂O. Wear a dust mask, gloves, lab coat and safety goggles when preparing this solution.
1. screening
Day 1
Transfer a scoop of bacteria from the blood agar to 5 ml of Tryptic soy broth (TSB). Incubate at 37 °C over night on a shaker.
Day 2
1. Transfer 50 µl of the samples in triplicates to microtiter plates (figure 1). FLASH-fractions are tested at a final concentration of 50 µg/ml.
2. Dilute the overnight cultures of S. epidermidis and S. haemolyticus (adjust the OD600nm to 0.3) 1:100 in TSB with 1% glucose.
Transfer bacteria and controls to the microtiter plates as shown below.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| 1 | 1 | 1 | 9 | 9 | 9 | 17 | 17 | 17 | Positive control (S. epidermidis + ddH2O) | Negtive control (S. haemolyticus + ddH2O) | Medium blank (TSB + ddH2O) | |
| 2 | 2 | 2 | 10 | 10 | 10 | 18 | 18 | 18 | ||||
| 3 | 3 | 3 | 11 | 11 | 11 | 19 | 19 | 19 | ||||
| 4 | 4 | 4 | 12 | 12 | 12 | 20 | 20 | 20 | ||||
| 5 | 5 | 5 | 13 | 13 | 13 | 21 | 21 | 21 | ||||
| 6 | 6 | 6 | 14 | 14 | 14 | 22 | 22 | 22 | ||||
| 7 | 7 | 7 | 15 | 15 | 15 | 23 | 23 | 23 | ||||
| 8 | 8 | 8 | 16 | 16 | 16 | 24 | 24 | 24 |
Figure 1: Layout of the microtiter plates.
3. Incubate the microtiter plates at 37 °C overnight.
Day 3
1. Before staining the Biofilm, measure OD600 of test plates and mark all wells with inhibited planktonic growth (to determine whether lack of biofilm formation is due to inhibited planktonic growth).
2. Remove the bacteria suspension from the plates using absorbent paper.
3. Gently wash the wells with tap water 2-3 times.
4. Fix the biofilm at 65 °C for 1 h.
5. Add 70 µl of 0.1% crystal violet solution to the wells and incubate at 65°C for 10 min.
6. Remove the crystal violet solution using absorbent paper.
7. Wash the wells with tap water 2-3 times until all unbound crystal violet is removed.
8. Dry the plates at 65 °C for 1 h until all the water has evaporated.
9. Add 70 µl 70% ethanol to each of the wells and incubate on a shaker for 5-10 min and then measure absorption at 570nm using a Tecan Spark (double check the type of plate).
Control
Negative control: 50 µl S. haemolyticus + 50 µl autoclaved MQ-H₂O
Positive control: 50 µl S. epidermidis + 50 µl autoclaved MQ-H₂O
Medium blank: 50 µl TSB + 50 µl autoclaved MQ-H₂O
Results
Make a note of every active fraction. Samples that inhibit biofilm formation will result in wells with little or no violet coloration.
Do not use the results if there is visible growth in the negative control or no growth in the positive control.
Active extracts: percent biofilm formation below 30% of growth control
Questionable: percent biofilm formation between 30 - 40% of growth control
Inactive extracts: percent biofilm formation above 40%
Protocol references
1. O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp. 2011 Jan 30;(47):2437. doi: 10.3791/2437. https://pmc.ncbi.nlm.nih.gov/articles/PMC3182663/#sec1
2. Igumnova EM, Mishchenko E, Haug T, Blencke HM, Sollid JUE, Fredheim EGA, Lauksund S, Stensvåg K, Strøm MB. Amphipathic sulfonamidobenzamides mimicking small antimicrobial marine natural products; investigation of antibacterial and anti-biofilm activity against antibiotic resistant clinical isolates. Bioorg Med Chem. 2018 Sep 15;26(17):4930-4941. doi: 10.1016/j.bmc.2018.08.032. Epub 2018 Aug 28. PMID: 30185388. https://www.sciencedirect.com/science/article/pii/S0968089618308976?via%3Dihub#s0050
3. Sathish Rajamani, Reuel Sandy, Krishna Kota, Linnea Lundh, Glenn Gomba, Katlin Recabo, Allen Duplantier, Rekha G. Panchal, Robust biofilm assay for quantification and high throughput screening applications, Journal of Microbiological Methods, Volume 159, 2019, Pages 179-185, ISSN 0167-7012, https://doi.org/10.1016/j.mimet.2019.02.018. (https://www.sciencedirect.com/science/article/pii/S0167701219300296)