Evaluation of a non-submerged cultivation assay combined to ESEM imaging for analysis of biofilms formed by dairy-associated sporeforming bacteria

Abstract

In the dairy industry, the biofilms formed by spore forming bacteria are not well characterized. Microscopic methods are crucial for the study of biofilm structural and architectural features. Here, a simple surface-associated non-submerged model combined to environmental scanning electron microscope (ESEM) imaging was used for the study of Bacillus cereus and Geobacillus spp. dairy biofilms. To evaluate the utility of this approach, non-submerged biofilms were compared to those developed in situ on stainless steel coupons introduced inside milk processing lines. Results reveal that both B. cereus and Geobacillus spp. are able to form specific biofilm characteristics on nonimmersed surfaces, notably an original dispersion style not previously described. Non-submerged biofilms in vitro are elaborate three-dimensional or extensive complex structures well resolved in ESEM and comparable to dairy biofilms in situ. The non-submerged surface-associated biofilm combined to ESEM imaging revealed a relevant model for the study of dairy biofilms.

Biofilm Disruption and Virulence Attenuation Effects of Essential Oil From Endemic Algerian Cistus munbyi (Cistaceae) Against Clinical Strains of Pseudomonas aeruginosa

“…After incubations coverslips were rinsed with distilled water to remove unbound planktonic cells and biofilms were fixed with 2.5% (v/v) glutaraldehyde in PBS for 5 h at room temperature. After washing, dehydration was done in ethanol with different concentrations (30, 50, 70, 90% and 100%) as described by Bose et al 2,56 Coverslips were dried, gold coated and viewed under field. 100 TM Hitachi environmental scanning electron microscope, at pressure in microscope chamber of 4 Torr.…”

“…Visualization of the inhibition of biofilm production in situ at three different time stages is based on a previously reported methodology with some modifications. 56,57 Briefly, 1% of overnight cultures of the tested pathogens (0.4 OD at 600 nm) were added in 24-well plates to 1 mL of fresh MH medium containing sterile glass coverslips in the absence and presence of different concentrations of C. munbyi EO. After an adequate incubation time (1 h for adhesion and 24 h for biofilm formation), the coverslips were rinsed with distilled water to remove planktonic cells and the biofilms formed were stained with a 0.1% crystal violet solution.…”

Microorganism Carrier-Surface Method as an Efficient Model for Microscopic Characterization of Biofilm Structure and Dispersion in Dairy Associated Spore-Forming Bacteria

“…Static batch models were also suitable for studying biofilm dispersal. The microorganism carrier-surface method previously described to test sanitizers' effectiveness [21], allows rapid biofilm dispersal in mesophilic and thermophilic dairy-associated spore forming bacteria [20]. The formation of non-submerged biofilms on open surfaces is a practical method that was also used in several works [22,23].…”

“…The formation of non-submerged biofilms on open surfaces is a practical method that was also used in several works [22,23]. The microorganism carrier-surface method combined to environmental scanning electron microscopy imaging (ESEM) or simply crystal violet staining revealed an efficient model for the investigation of biofilm structure and dispersion in dairy-associated spore forming bacteria [20]. Large biofilms that exceeded a minimum diameter of 40 μm, required for dispersion to occur in the way called seeding dispersal, previously described in flowing systems [24], are developed with this static batch model, by mesophilic and thermophilic spore forming bacteria (Fig.…”

“…[17] Assessment of biofilm biomass in a microtiter-based batch system [18] Cristal violet staining of ring biofilm in a glass tube [19] Microorganism carrier-surface method combined to Environmental scanning electron microscopy (ESEM) or crystal violet staining [20] Fig. 2.…”