DOI: 10.36871 / vet.san.hyg.ecol.201801008
UDC 619: 579

Authors

A. B. KONONENKO, CAND. BIOL. SCIENCES HEAD OF THE LABORATORY
I. B. PAVLOVA, DR. BIOL. SCIENCE, PROFESSOR, CHIEF SCIENTIFIC EMPLOYEE
D.A. BANNIKOVA, CAND. BET. SCIENCE LEADING A SCIENTIFIC EMPLOYEE
S.V. BRITOVA, CAND. BET. SCIENCE, SENIOR SCIENTIFIC EMPLOYEE
E.P. SAVINOVA, SCIENTIFIC EMPLOYEE
D.N. NABIULLINA, RESEARCH Fellow of the Federal State Budgetary Scientific Institution "All-Russian Research Institute of Veterinary Sanitation, Hygiene and Ecology"

Abstract

To study the process of biofilm formation, microorganisms were cultured in 96-well plates, in meat-peptone broth, stained with 0,1% crystal violet solution for 10 ... 15 min, after which the unbound dye was washed off. Quantitative registration of the bound dye was carried out by spectrophotometry at a wavelength of 490 nm. A technique for preparing bacterial preparations for light and scanning electron microscopy on cover glasses immersed in Petri dishes with a liquid nutrient medium is proposed. A suspension of bacteria at a concentration of 105 mc / ml in a volume of 5 ml was shaken on a Vortex apparatus and added to Petri dishes with 20 ml of meat-peptone broth.
Sterile fat-free coverslips were placed on sterile slides and immersed in liquid nutrient medium in Petri dishes.
The material was incubated for 18 ... 24 h at 37 ° C. Then the cover slips were removed with tweezers and some of them were stained with 1% aqueous solution of methylene blue (for light microscopy), and some were placed in Petri dishes with filters laid on the bottom (for electron microscopy). In order to preserve the natural architectonics, the latter were fixed in vivo in vapors of 25% glutaraldehyde for 3 ... 5 hours. For contrasting preparations, vapors of 2 ... 4% solution of osmic acid were used for 2 ... 3 minutes. After treatment with osmic acid vapor, biofilms with incorporated bacteria acquired a yellowish or brown color. The obtained preparations after dehydration with propylene oxide vapor and deposition with gold ions were examined in a scanning electron microscope (SEM). The technique makes it possible to study the phases of biofilm development and obtain objective data on the morphology of populations of pathogenic and opportunistic bacteria without disturbing the natural architectonics.
It was shown that the intensity of biofilm formation by pathogenic microorganisms, such as Salmonella, Yersinia, Staphylococcus aureus, was slightly higher than that of non-pathogenic: Escherichia, Proteus, cytrobacter, entero-bacterium.

Keywords

biofilm formation, morphology of bacterial populations, bacterial resistance.