State Research Centre of Applied Microbiology , Research and Production Department of the Gene and Engineering Preparations (s. Obolensk).

Research and Development Report on the Results of Experiments on Studying the “Biofon” Apparatus influence on the Pseudomonas aerughinosa and Staphylococcus aureas microorganisms

Period of conducting the experiments: May 17-28, 1998.

The purpose of this work has been the conducting of the experiments on studying the “Biofon” apparatuses influence on two microorganism cultures: Pseudomonas aerughinosa and Staphylococcus dureus.

To determine the “Biofon” apparatuses influence on the given microorganisms cultures the following experiments have been conducted.

To determine the cultures viability the calculation of the colony-forming units (CFU) of the microorganisms, processed and not processed by the apparatus was performed. For this purpose a daily suspension of the microorganisms, grown at 37 ° C in the LB broth (at continuous “swing”), was divided into two parts, one of which was processed by the apparatus, the other was used as a control. After titration (breeding) the produced suspension was used to seed cups with the LB agar for Ps.8 and St.6 breedings, the cups were placed into the thermostat at the temperature of 37 ° C. Registration for Ps. was performed in 20 hours and for St. – in 44 hours (degree of breeding registration time was determined before hand experimentaly). The obtained results showed that the processing by the apparatus decreased the number of colony-forming units for Ps. 10-15 times and for St. – by 20-25%. The processing by the apparatus also decreased the size of colonies, for Ps. – 3-4 times, for St. –1-2 times.

The suspension of microorganisms cells in the LB broth with an optical density of 0.2 was prepared to evaluate the time of doubling a number of microorganism cells (that characterizes growth of a biomass of suitable microorganism and therefore its physiologic activity) in a liquid medium. The optical density was measured for the suspension placed into 1-cm cuvet at the wave length of 540 nm by the Ultraspect-2 (LKB firm) spectrophotometer. The suspension with the initial optical density of 0.2 was placed by 25 ml into the bottles and was cultivated at the temperature of 37 ° C at continuous “swing”. Determination of the optical density was performed every hour in the selected aliquota (part of all suspension).

The obtained data showed, that time of the biomass doubling for Ps. processed by the apparatus was 2.15 – 2.20 h, not processed – 1.7 – 1.8 h. For St. – 2.5 – 3 h (experiment) and 2.3 – 2.44 h (control), respectively. In interpretation of data, obtained in the result of the experimen we proceeded from the fact, that increase of the optical density is directly proportional to the microorganism biomass increase.

To evaluate the membranes biological function the measurement of UF-absorbing matters leakage before and after processing by the “Biofon” apparatus were performed. It is known that at some sublethal actions for microorganisms (heating, starvation, low temperatures) the cells lose pool (definite part) of low-molecular matters, absorbing UF at 260 nm (aminoacids, low-molecular proteins, vitamins). The obtained suspension of the Ps. and St. Microorganisms was processed by the apparatus, then it was precipitated at 6000J on a centrifuge (Hitachi), UF absorbtion was determinated in a supernatant.

In the result of the experiment the following data were obtained: at processing by the apparatus the optical density increased for Ps. from 0.385 (control) to 2.388 (experiment); for St. – from 0.273 (control) to 0.423 (experiment) respectively. So, it was found that the leakage of the UF-absorbing matters after processing by the apparatus increased for Ps. approximately in 6 times, for St. – approximately by 60%. The optical density was determined by the Ultraspect 2 spectrophotometer (LKB Co.).

The base of the membranes barrier function evaluation method is the bromide ethidium fluorescence evaluation after its penetration inside the microorganism cell through the membrane. For this purposethe bromide ethidium was added to the microorganism suspension up to the concentration of 5x10^-6 mole, fluorescence was registered by the Hitachi-650 spectrofluoremeter. In the solutions the bromide ethidium luminescence quantum output is extremely low and therefore its luminescence intensity is poor. In binding the bromide ethidium with the nuclein acids a sharp increase of the quantum output is observed and the cell structures containing the nuclein acids obtain bright-crimsonred luminescence with maximum of radiation at the wave lengths of 590-610 nm.

Data, obtained in the result of the experiment, show that due to processing by the apparatus the fluorescence intensity for Ps. increases in respect to the control by 20-30% and for St. – by 10-12% respectively, which is the evidence of microorganism membranes barrier function disturbance.

Taking into account the fact, that the breathing process intensity is directly connected with the microorganism physiological activity, the determination of the given process is extremely important for evaluation of the microorganism state as a whole. The breathing intensity was determined by the color change of the methylene blue dye added into the microorganism suspension (decolorization of the suspension with the methylene blue adding is evidence of its reduction by the microorganism dehydrogenases i.e. about the breathing intensity of the given microorganism). Microorganism culture six-seven-hours old was used for experiments and for this purpose an inoculum was placed in the L.B. broth and it was grown on the "rocker" at the temperature of 37ºC up to the optical density of 2-2.5 (optical density was determied at the wave length of 540 nm by the Ultraspect 2 spectrophotometer (LKB firm) in the 1 cm cuvet). Then methylene blue was added to the microorganism suspension (in the concentration determined beforehand experimentally) up to the optical density of 1 at 650nm (the suspension of microorganisms without adding the methylene blue was taken for zero index in the given case). The optical density change was registered during 30 min – each 5 min. In a separate experiment glucose was added up to the final concentration of 0.5 to find out the influence of a medium depletion on the results. The obtained results show that Ps. intensivelly decolorizes a dye with glucose as well as without it; St. practically does not restore a dye without adding glucose. After processing the aliquota of microorganism suspension by the apparatus the breathing intensity of Ps. decreased by 40-50% and of ST. -–by 10-15% respectively.

To determine the specificity of the apparatus actions on the given microorganism cultures an experiment was performed for determing the apparatus influence on St., and number of colony-forming units for Ps. The obtained from the experiment data show, that the apparatus effect decreases (in the result of its influence) the number of the colony-forming units for Ps. 3 times and colony size 2-3 times.

Note: Significant difference between data on influence of the appropriate apparatus on Ps. and St. cultures may be explained by the insufficiently adequate medium and by the absence of the preliminary to define a time tests phase, that is more suitable for the experiments (at St. cultivation).

Senior research assistant of the "Bionix"

Research and Production Enterprise Kopitov E.B.

Senior research assistant of the State

Research Center of Applied Microbiology Baidus A.N.

Senior research assistant of the State

Research Center of Applied Microbiology Borisov N.B.

Laboratory assistant of the State

Research Center of Applied Microbiology Gerasimova A.K.