Hello,
I'd like to include some background for the problem I'm having. I'm working with Candida glabrata isolates in 96 well plates. My plate conditions are: 1*10^6 cells/mL, 5%CO2, 24h incubation and RPMI 1640. Media was made fresh on Monday and cells were counted and seeded on Thursday.
For two weeks I've had spotty growth in my biofilms, instead of a nice clean sheet across the bottom of the well I see several small dots like colonies. This is consistent across all of my wells except for my negative control. Does anyone have any suggestions on how to prevent this from happening in the future?
Nigel Gale
Cell preparation and seeding: Make sure cells are well suspended and evenly distributed when seeded. Clumping of cells during seeding can result in inconsistent growth patterns. Surface of the wells: The surface of the well plate can significantly impact biofilm formation. If possible, try using a different brand or type of plate. Some researchers have found better biofilm formation with polystyrene plates, for example. Media: Although you mentioned that your media was fresh, it may still be worth investigating. Make sure the media is properly prepared, with the correct pH and nutrient content. If the media is not properly sterilized, it could potentially contain contaminants that interfere with biofilm formation. Incubation conditions: Although you've mentioned specific incubation conditions, slight variations in temperature, CO2 levels, and humidity within the incubator can affect biofilm formation. Ensuring consistent incubation conditions is critical. Strain variation: Not all strains of C. glabrata form biofilms equally well. You might want to confirm the identity and characteristics of your strain. Sub-MIC of Antifungal drugs: If you are using antifungal drugs at sub-MIC levels, they may cause a stress response in Candida spp., leading to the enhancement of biofilm formation and the increase in resistance to antifungal drugs.
Here are several potential reasons for your inconsistent biofilm formation:
Here are a few suggestions to improve the uniformity of your biofilms:
- Enhancing agitation: Providing a gentle agitation during the initial stages of biofilm growth can help cells to distribute evenly across the bottom of the well, leading to more uniform biofilm formation.
- Using conditioning film: Prior to inoculation, coating the well surfaces with a conditioning film (like serum, or specific proteins like fibronectin) might promote uniform biofilm formation.
- Optimizing seeding density: Too high or too low initial seeding densities might lead to the inconsistent formation of biofilms. It might be worthwhile to experiment with different densities to identify an optimal range for your specific strain and conditions.
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