Hello before I start, Eng is not my first language so my explanation can be rough sorry.
I am doing Crystal Violet biofilm assay for C. albicans but having trouble with washing process. Before I add CV I usually wash cell with PBS for 3-4times. I tried using multi channel pipette or inverting 96 well but some white colonies are still in the well so the results become inconsistent.
My questions are
1. How do I wash the 96plate with PBS?
2. Also do I have to elminate all the white colony that I can see?
This is how I do CV assay.
1. incubate strain in YPD for 24 h at 37 °C
2. Dilute Candida with RPMI
3. Add 100ul of Candida in the 96 well and incubate statically at 37 °C for 90 min. (for C. glabrata I incubate 4h)
4. Discard broth and wash PBS ( In this proccess I use multi channel pipette)
5. Add 100ul RPMI and 50ul of treatment material (I use CFS)
6. Incubate 24 h at 37 °C
7. Discard excessive broth and floating cell. ( I don't know how to effectively wash cell for clear results)
8. Fix biofilm with methanol for 15 min
9. Discard methanol and dry
10. Add 150ul of CV and dye for 20min
11. Wash excessive CV ( I put the plate the bowl of water to wash 3 times)
12. Dry and put 33% acetic acid
13. Measure with 570nm
I will be glad if you let me know how to wash the candida albicans and candida glabrata.
Thanks!
Washing during the crystal violet biofilm assay is a critical step and must be performed gently to avoid disrupting the biofilm. To wash a 96-well plate with PBS, it is recommended to use a multichannel pipette to add PBS (100–200 µL) gently along the side of each well while tilting the plate slightly. This reduces direct force on the biofilm. After adding PBS, carefully aspirate from the same side without touching the bottom of the well, or gently invert the plate and flick it onto absorbent paper. This process should be repeated 3–4 times.
The goal is to remove planktonic (non-adherent) cells and residual media without disturbing the adherent biofilm layer. It is not necessary to eliminate all visible white colonies, as these are often part of the mature biofilm structure. Attempting to remove them completely may result in detachment of the biofilm and inconsistent assay results. Maintaining a consistent, gentle washing technique is essential for reproducibility and reliable quantification in the assay.
Maisra Azhar Thank you for the specific method. I will try that. Also I have one more question. Some papers say to make more biofilm it needed to be incubated in shaking incubator, but others just incubate statistically. Which one is better? Again thank you for the answer!!
I would suggest that static incubation is generally preferred for C. albicans biofilm assays, especially when measuring surface-attached biomass using crystal violet staining. Shaking conditions may enhance nutrient distribution and biofilm dispersal in some systems, but they often interfere with the initial adhesion and structural stability of C. albicans biofilms. For reproducible and robust biofilm formation, static incubation at 37°C for 24–48 hours is typically more reliable.
Maisra Azhar Okay then I will use static incubation. Thank you for the answer it was really helpful I will try the method you recommeded. Again thank you for the kind and specific method recommendation. Have a nice day
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