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Page 36


Volume 2

Journal of Microbiology and Biotechnology Reports

Microbial Biotechnology 2018

September 17-18, 2018

Microbial Biotechnology & Vaccine Design

September 17-18, 2018 Lisbon, Portugal



World Congress on

Optimization of confocal microscopy for visualization and quantification of yeast biofilm

Smolova Jana


, Nemeckova I


and Turonova H



Dairy Research Institute, Czech Republic


University of Chemistry and Technology, Czech Republic

Introduction & Aim:

Due to the resistance of biofilm to conventional cleaning processes, an industrial biofilm, especially in

dairy industry, could cause extensive problems in final products in case of insufficient sanitation process. Our work focuses on

visualization and quantification of yeasts biofilm using confocal microscopy. Commercial dying kits are optimized for pathogenic

bacteria; therefore, the dying protocol for yeasts must have been created. The yeasts were isolated as industrial contaminants

from cheese and saline solution and were identified as Yarrowia lipolytica and Debaryomyces hansenii using MALDI-TOF.

Material & Methods:

µCLEAR® Chimney well plate was used for yeasts cultivation and the cultivation time, washing, dying and

visualization of yeasts biofilm was optimized. LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit was used for dying and

the biofilm visualization was performed by a confocal laser scanning microscopy with a rotating disc, a ten-fold magnification and a 1

μm z-step. For the living, dead and damaged cells, biomass volume and total biofilm structure were determined using Imaris software.


The cultivation time was set to 24 hours with fresh broth well washing after 2 hours which better corresponds with real

conditions. While living cells predominated in D. hansenii biofilm, the damaged cells prevailed in the

Y. lipolytica

biofilm, which may

have been related to the different aging rates of the biofilm of both strains. The dying time was shortened to 60 minutes to reduce the

toxic effect of propidium iodide.


New approaches for visualization and quantification of biofilm are needed for better understanding of the biofilm

forming and persistence. We have shown that visualization of yeast biofilm is one of the ways to study these biofilms. These findings

can lead to monitoring the effectiveness of sanitation solutions and to designing better cleaning processes.


Jana Smolova is a PhD candidate working under many research works related to microbiology and biotechnology. She has been working on biofilms with different

methods and techniques to get the best results with the latest development of biofilms. She has interest in biotechnological experiments based on microbes.

Jana Smolova et al., J Microbio and Biotech Rept 2018, Volume 2