A new approach for the study of the morphological structure of microbal biofilms.

Microbial biofilms are heterogeneous, moving and constantly changing communities of microorganisms, often of various taxons. Approaches to study and assessment anti-biofilm drugs widely available today do not adequately assess their effects, while the results of studying the interaction of drugs with components of the film composition can provide them the right choice. The aim of investigation was to test a new method of morphological evaluation of biofilms. To form biofilms, we used an approach when the slide was placed at an angle of 30o-45o relatively to the Petri dish, and a suspension of test strains S. epidermidis in peptone broth was poured into the space between the Petri dish and the slide. A sterile cotton swab moistened with distilled water was placed next to the glass slide to create optimal humidity. The system was placed in a thermostat for 24 hours. The formed films were examined under a microscope using the DCM 310 video eyepiece and the Scope photo x86,3.1.312 program that allowed to conduct a complete morphometric study of the film: select layers, channels, cavities and make measurements, and then save the results on electronic media in jpg file format. Microscopy of the stained slides revealed that the biofilm has a layered structure. In each image obtained using a video eyepiece, it was possible to differentiate 4 layers. From the border of the two media to the inside: the fragmentation layer, the dense layer, the matrix substance layer, and the last one - the persistence layer. Channels of different diameters (from 10 to 24 microns) are observed across the entire thickness of the biofilm. Thus, used approach allows us to visualize and evaluate the structure of microbial biofilm, measure the thickness of layers and channel diameters. In addition, this method can be used to study the effect of antimicrobial drugs on bacterial films.