Koch's Method Research Articles

Isolation and Characterization of Pathogenic Mold Causing Potato Tuber Rot Disease

The potato plant (Solanum tuberosum L.) is a perennial crop that contains high amounts of carbohydrates, minerals, and vitamins in its tubers, making it a carbohydrate-rich alternative food to rice or corn. One of the diseases that often appear on potato plants is potato rot caused by pathogenic molds. This study aims to isolate and characterize pathogenic fungi that cause blight on potato tubers based on Koch's Postulates. Potato pathogenic molds were isolated from potato tubers that had been rotted, then the molds were grown on PDA and purified twice. The purified isolates were then inoculated onto 30 healthy potato tubers and incubated for 7 days. Healthy potato tubers experienced the same symptoms as potato tuber rot. The isolated pathogenic fungi were then characterized so that the pathogenic fungi of Phytophthora infestans were obtained. In this study, Koch's Postulate method was successfully applied to isolate and characterize the pathogenic fungi that cause potato tuber rot. The pathogenic fungi caused a potato tuber rot disease incidence value of 100% with a disease severity value of 60.7%. The two factors that determine the disease incidence and disease severity values are internal factors (genes and traits of the pathogen) and external factors (environment).

Chocolate with probiotic microcapsules is a new product of functional nutrition

Probiotics can help improve digestion, strengthen the immune system and even fight diseases. But until now, there was no easy way to introduce enough probiotics into our body. Microencapsulated probiotics offer a solution to this problem. They are small enough to be easily digested, and are protected from the aggressive environment of the stomach. Chocolate represents a ubiquitous and popular consumable, yet probiotics within food matrices necessitate strategies for stress mitigation and enhancement of viability. One such method involves the encapsulation of probiotic cells within alginate microspheres, amenable for incorporation into chocolate matrices. In this regard, the aim of study is to explore the effect of microencapsulation on the survival of probiotics in chocolate. Conventional microbiological methods were used: growing bacteria in liquid and solid media, determining the titer of bacteria using the Koch method. The results demonstrated the successful production of chocolate containing microcapsules of probiotics with favourable organoleptic properties. Enhanced viability preservation of Lactobacillus rhamnosus GG in microcapsules compared to free cells was observed.

EFFECT OF MAGNETIC FIELD AND ENVIRONMENTAL PH ON THE ADSORPTION EFFICIENCY OF CELLS OF THE GENUS LACTOBACILLUS

Subject. Adsorption of probiotic cultures Lactobacillus acidophilus, namely strains 27 and Narine, and Lactobacillus plantarum, on high-dispersed silica. Purpose. The purpose of this study was to investigate the influence of a magnetic field with intensities of 0.03 and 0.09 T and the pH of the medium on the efficiency of cell adsorption of Lactobacillus probiotic strains. Method. Microbiological and biotechnological methods were applied in this research. Hydrogels based on high-dispersed silica "Enterosgel" and "Toxin.NET" were used as adsorbents. Neodymium magnets with intensities of 0.03 T and 0.09 T were employed to create the magnetic field. The efficiency of adsorption was evaluated by the cell survival of probiotic cultures after adsorption on hydrogel preparations under the influence of the magnetic field and in the case of pH adjustment by adding acid or alkali. The functional properties of the obtained composite preparations were assessed by the time of clot formation after milk fermentation and its acidity. Results. The research results demonstrated that immobilized cells of Lactobacillus acidophilus on the hydrogel of methylsilicic acid had enhanced protection not only against harsh conditions of the gastrointestinal tract (low pH and bile influence) but also against magnetic fields with intensities of 0.03 T and 0.09 T, which had an overall negative impact on native (non-immobilized) cells. The functional activity of cells immobilized in this way on the hydrogel based on high-dispersed silica "Enterosgel," evaluated by acidity and clot formation time, was only slightly reduced: up to 4,3-11,3%. This confirms the assumption that the assessment of cell survival by the Koch's cup method does not fully correspond to the real state of the cells. It was also determined that conducting adsorption in the pH range below 4 is impractical, and the optimal conditions for L. plantarum adsorption were in the pH range of 6 to 7. Scope of results. The research findings can be utilized for the development of complex probiotic preparations based on high-dispersed silica, which may be appropriate for preserving the functionality of probiotic cells used for starter cultures or in the production of dietary supplements containing beneficial microorganisms.

Protection of building materials from biodeterioration

Introduction. The foci of biological damage can lead to destruction of building materials. The paper presents the results of an examination of samples of brickwork and cladding of an industrial building with traces of biological damage.
 Materials and methods. The contamination of materials was identified using the Koch method. The effectiveness of biocides was evaluated using the disk diffusion method, namely, its modification applied to identify the fungicidal activity by adding antiseptics to samples of damaged building materials, identifying the number of viable microorganisms in the material, processing damaged building structures and monitoring the microorganisms on their surfaces.
 Results. The number of viable microorganisms in the studied samples of building materials exceeded the acceptable value, which evidences the process of biodegradation. The resistance of isolated strains of fungi to biocides, that have various derivatives of isothiazolone as the main active ingredient, was determined.
 Conclusions. Sanatex, Macrosept, Rocima GT, Rocima 243, Neomid 440 Eco can be recommended for the disinfection treatment of biologically damaged building materials.

Разработка технологии производства биомассы Bacillus megaterium кормового назначения

Introduction. Obtaining protein and vitamin fodder is one of the urgent tasks that modern industrial biotechnology has to solve. Another task is a search of novel medium compositions for microbial fermentation that can lower production costs. Russian food industry produces a significant amount of sunflower seed processing byproducts every year. Sunflower meal is a promising source of sunflower protein isolate. The research objective was to develop a new technology for the production of Bacillus megaterium bacterial biomass for fodder purposes.
 Study objects and methods. The research featured a sunflower protein isolate, an enzyme complex Protex 7L, and a B. megaterium strain (VKPM B-3750). The carbohydrate content was determined using a modified Bertrand method. Amine nitrogen was studied using formol titration, the number of viable cells – by the Koch method, the content of amino acids – by capillary electrophoresis. 
 Results and discussion. When processed with enzyme complexes, sunflower protein can be an alternative source of nitrogen for industrial fermentation. The study featured amino acid of sunflower protein isolate and enzymatic hydrolyzate obtained using Protex 7L. A comparative analysis of the content of amino acids in the hydrolyzate and the protein isolate showed that enzymatic hydrolysis can significantly increase the content of free amino acids in the medium available for microbial accumulation. The research proved that sunflower protein enzymatic hydrolyzate obtained using Protex 7L can be used to cultivate strains of B vitamins producers. 
 Conclusion. Sunflower protein enzymatic hydrolyzate can be used as a nitrogen source for B vitamins producer fermentation and as an alternative to expensive meat peptone. The research involved technical and economic assessment of the B. megaterium fermentation on enzymatic hydrolysates of sunflower protein at a production capacity of 100 kg per year. The cost of the protein-vitamin supplement was calculated as 413 rubles per kg, while the market price could reach 826 rubles per kg. The payback period for capital expenditures was estimated at 1.5 years. Thus, replacing commercial meat peptone with sunflower protein enzymatic hydrolyzate obtained with Protex 7L reduced the cost of 1 kg of feed additive by three times without affecting B. megaterium. Overproduction of B vitamins by the B. megaterium strain on a medium containing sunflower protein hydrolyzate requires optimization of fermentation conditions.

The biotechnological studies when developing lozenges with probiotics

Aim. To develop lozenges with probiotic components for the treatment and prevention of ENT diseases and analyze their effectiveness.Materials and methods. The study objects were lozenges based on xylitol and sorbitol of two compositions – based on live probiotic cultures and their metabolites. The following methods of control of the sample efficiency werechosen: determination of the number of viable lactobacilli in the study of their combined use with the drug components and the number of viable bacteria in finished oral probiotics by the Koch method (direct seeding on cups), as well as the method of diffusion into agar with the growth inhibition zone determination.Results and discussion. The studies of the properties of probiotic strains of L. fermentum 90-TC and L plantarum 8P-A3 have shown the prospects for their use in oral probiotics. It has been also proposed to use metabolitesof these strains as active components, which have a rather high antimicrobial potential to opportunistic pathogens that cause infectious ENT diseases. The oral probiotics in the form of lozenges of two compositions – based on live lactobacilli and their metabolites – have shown high efficiency, bacterial survival and antimicrobial properties, respectively.Conclusions. During the complex of the theoretical, technological and biotechnological studies, lozenges with a probiotic of two compositions (based on viable probiotic cultures and their metabolites) have been developed. After further research they can be recommended as a biologically active additive to support the oral microflora, stimulate the own immunity and as a therapeutic and prophylactic agent in infectious diseases of the oral cavity for use in both adults and children.

Formation of biocomplexes based on the bacteria Pseudomonas fluorescens and substances of a stimulating nature to limit the development of harmful organisms in potatoes

Goal. Selection of combinations of biocomplexes based on Pseudomonas fluorenscens bacteria with stimulant preparations based on various derivatives of ammonium salts of dihydropyrimidine and study of their effectiveness.
 Methods. Biotechnological methods for the study of bacteria Pseudomonas fluorenscens strain AR-33. The concentration of viable bacteria (CFU/cm3) was determined by the Koch method. Accounts were performed according to generally accepted methods using experimental methods in phytopathology and plant protection. Determined the effectiveness of drugs at different rates of consumption against fungal diseases.
 Results. Derivatives of ammonium salts of dihydropyrimidine did not show a toxic effect on reducing the concentration of viable cells of strain AR-33 bacteria Pseudomonas fluorescens. The best indicators of the weight of 100 seeds and the number of beans in soybean plants showed a combination: Planriz, v.s. (bacteria of strain AR-33 Pseudomonas fluorescens, 3 ќ 10 9 CFU/cm3) (5 l/ha) + 0.1% solution of ximedon + 0.2% solution of succinic acid + 2 ml of DMAE + 2 ml of DMSO. The use of all combinations of biocomplexes showed the effectiveness of drugs against diseases in the range of 59.31—69.63%. With the use of biocomplexes, due to the fungicidal, immunoprotective and stimulating effect, a yield increase of 1.15—1.7 times relative to the control was recorded. The best yield on potatoes (3.4 t/ ha) was provided by the combination Planriz, v.s. (5 l/ha) + 0.1% solution of ximedon + 0.2% solution of succinic acid + 2 ml of DMAE + 2 ml of DMSO. The effectiveness of the drug against late blight was 79.1%.
 Conclusions. The use of stimulants and excipients DMAE and DMSO as substances that affect various transmembrane functions, provided an increase in the effectiveness of drugs by 8—14% relative to combinations without their use.

Identifikasi Cendawan Patogen Penyebab Penyakit pada Daun Jabon Merah (Anthocephalus macrophyllus (Roxb.) Havil)

Red Jabon (Anthocephalus macrophyllus (Roxb.) Havil) is a forest plant that can replace sengon (Falcataria moluccana) because it has advantages. The demand for red jabon wood is increasing, but the cultivation of red jabon is still constrained by the attack of pathogens that cause leaf disease. The attack of these pathogens can cause damage and death of seedlings so that it can be economically detrimental. The aim of this study was to identify the pathogens that cause disease in red Jabon. The method used is the Postulate Koch method, which starts from sampling red leaves with leaf disease symptoms, isolation, inoculation and analysis of disease incidence, re-isolation, and identification. Based on the identification results found as many as 2 pathogenic fungi that cause leaf spot disease, namely Pestalotia sp. and Rhizoctonia sp. In addition, 6 pathogenic fungi were found that cause blight, namely Lasiodiplodia theobromae, Fusarium sp., Colletrotrichum sp., Marssonina sp., Gloeosporium sp. 1, Gloeosporium sp. 2.
 Keywords: identification, percentage of disease incidence, Postulat Koch, red jabon

Microbial Contamination In The Outdoor Air At Some Garbage Collecting Places And Waste Transfering Stations

This study was conducted to provide a preliminary assessment on the microbial pollution in the air at some garbage collecting places and waste transferring stations at Ho Chi Minh City. The airborne microbial pollution is becoming more and more important, so the research was carried out to build long-term practical solutions that directly or indirectly protect the environment and ensure the human health. We performed sampling at 3 locations and quantitative analysis - identification for bacteria and fungi that existed in the outdoor air at the collected places of some districts. Samples were collected from 03–06/2019 according to Koch's deposition method. Sampling plate was placed at a height of 1.5 meters above the ground, and the sampling time was 15 minutes. The dish after collecting the sample was placed in the incubator at 37 +/- 1 oC in 24–48 h for bacteria and 25 +/- 1oC in 72–120 h for fungi. The study results showed that the density of bacteria and fungi ranged between 6,408.1–14,599.9 CFU/m3 and 733.6–2.497.6 CFU/m3. In particular, the density of bacteria tended to increase from the morning to the afternoon and decreased slightly in the evening, but the density of fungi tended to increase from the morning to the evening. All strains of microorganisms in the outdoor air were influenced by human activities and environmental factors. Dominant microorganisms were identified including Bacillus pseudomycoides, Bacillus pumilus, Arthrobacter cretinolyticus, Staphylococcus kloossi, Bacillus sp. and the three common fungi including Cunninghamella sp., Aspergillus flavus and Aspergillus brasiliensis. All these microbes are associated with a number of human diseases.

Influence of the Premises Dustiness Degree in an Educational Institution on the Choice of the Appropriate Air Filter Type

Students and teachers spend about 70% of the day in educational institutions. Therefore, recently in the world studies of the effect of air quality on human well-being and human performance have been conducted more often. The high content of suspended particles in the indoor air can lead to the development of allergies, and also serves as a factor of the transmission of respiratory viral infections. The purpose of this study was to determine the degree of dust in the indoor air in the classrooms. For this purpose, the photoelectric method of registration of aerosol particles and the Koch method were used. The results were evaluated according to two requirements for the amount of suspended particles in the air of the educational institution. Depending on the amount of suspended particles and their size, recommendations were given for filters to reduce the dust content of the internal air.

The experimental study of diquat on the half-Lethal dose and pothological injuny of related organs in wistor rats

Objective: To explore the acute toxicity of Diquat in mice and to calculate the median lethal dose (LD(50)) of Diquat to rats and observe the pathological changes of tissues and organs in rats with different concentrations of Diquat. Methods: Diquat solution of 50 mg/kg was prepared freshly with 1 000 mg of Diquat and dilute the solution with water to a total of 20 ml. A total of 99 healthy adult male Wistar rats were randomly divided into part one, part two and control groups. In the first part, 36 rats were randomly divided into 4 groups: 100 mg/kg group, 200 mg/kg group, 300 mg/kg group and 400 mg/kg group, which were treated with 100 mg/kg, 200 mg/kg, 300 mg/kg and 400 mg/kg of Diquat solution by gavage, respectively. The death and symptoms of poisoning after intragastric administration were recorded, and the maximum tolerated dose and absolute lethal dose were measured. In the second part, 54 rats were randomly divided into 6 groups: 200 mg/kg group, 220 mg/kg group, 240 mg/kg group, 260 mg/kg、280 mg/kg group and 300 mg/kg group, whichwere treated with 200 mg/kg, 220 mg/kg, 240 mg/kg, 260 mg/kg, 280 mg/kg and 300 mg/kg of Diquat solution by gavage, respectively. The survival of rats in different concentration of Diquat was observed and the LD(50) was calculated by Excel processing the formula of Koch's method. The control group were given equal volume water under the same experimental conditions. And moreover, the lungs, kidneys, hearts, livers, and brain tissues were collected and fixed by formaldehyde, embedded by paraffin, and sectioned for histopathological light microscopy. Results: The maximum tolerated dose was 240 mg/kg and the absolute lethal dose was 300 mg/kg. The LD(50) of Diquat for Rats was 280.58 mg/kg. The high-dose group had significantly more organ damage than the low-dose group after diquat poisoning. Conclusion: The determination of the half-lethal dose of diquat, at the same time observed multiple organs damaged in rats after the diquat quickly poisoned. Kidneys, lungs and heart might be the main organ which was heavily damaged. With the extension of observation time, the organ damage of rats exposed to small doses gradually stabilized.

Antibacterial activity of thin films TiO2 doped with Ag and Cu on Gracilicutes and Firmicutes bacteria

This article aims to explore the antibacterial activity of thin films of TiO2 doped with Ag and Cu using two types of Gram-negative and Gram-positive test bacteria with clinical significance (Gracilicutes and Firmcutes bacteria). The thin films (thickness of about 60 nm) were deposited on glass substrates by radio frequency magnetron co-sputtering (r.f. power of 50 W) of TiO2 target with Ag and Cu pieces on its surface in an Ar atmosphere (0.8 Pa) without heating during the deposition. The total surface area of the Ag was 60 mm2 and that of the Cu was 100 mm2. Bacillus cereus, Staphylococcus epidermidis, Salmonella enterica, Escherichia coli and Pseudomonas sp. were used as test strains. The antibacterial actvity of the films was evaluated by the classical Koch's method and optical density measurements. The bactericidal effect was established at different time points between 30 min and 90 min for Pseudomonas sp. and S. enterica. The Firmicutes bacteria B. cereus and S. epidermidis were killed at the 4th and 8th hour of the treatment, respectively. The effect on E. coli was bacteriostatic until the 10th hour. The results were confirmed by assessment of the bacterial dehydrogenase activity. The studied thin films of TiO2 co-doped with Ag and Cu have a potential for application as antibacterial coatings.

Validation of a New Web-Based System for Point-of-Care Implant Documentation in Total Knee Arthroplasty.

Implant documentation in total knee arthroplasty (TKA) must be both accurate and comprehensive. A new system enables surgeons to complete a secure, web-based operative report for each surgery. This study evaluated implant documentation using this system and hypothesized that it would be as reliable as the current standard. This was a retrospective study of all primary and revision TKA performed at a single academic institution from January 1, 2015 to March 31, 2015, by eight adult reconstruction surgeons who used the web-based system. Electronic medical records and implant logbooks were chosen as a composite standard reference for implant documentation. Cohen's kappa statistic and Pearson's correlation coefficients were used to determine agreement between the system and the standard reference. Mean kappa value for entire system was 0.916 ± 0.152 (p = 0.015) indicating "almost perfect" agreement (as per Landis and Koch's method) with standard reference. Pearson's correlation coefficient was 0.926 ± 0.147 (p = 0.021) further corroborating this excellent agreement. There was "substantial" or "fair" agreement for items pertaining to cement and augments. Overall, sensitivity was 0.98 (95% confidence interval: 0.71-0.98) and specificity 0.93 (95% confidence interval: 0.53-0.98) indicating that the system was very effective at documenting whether or not an implant was placed. The system exhibited significantly greater sensitivity than specificity (p = 0.027). This study demonstrated excellent performance of this novel system in point-of-care TKA implant documentation. Further research is needed to validate its use in arthroplasty of other joints and its potential for documentation of biomedical device implantation in other fields such as cardiothoracic surgery and gastroenterology.

SUPRESSION OF BIOFILMS OF OPPORTUNISTIC BACTERIA IN URINARY CATHETERS

Aim. Study regularities of development of bacterial biofilms in opportunistic bacteria in urinary catheters, as well as a possibility of their suppression by an officinal preparation of plant origin - Kanefron and nitrofuran class preparation - Furamag. Materials and methods. Escherichia coli Ml7 and Staphylococcus aureus 193 cultures were cultivated as biofilms on an inner surface of Foley latex catheter. Effect of Kanefron and Furamag on the ability of these strain to form biofilms was evaluated, as well as suppression of already developed biofilm of E. coli M17 and S. aureus 193 on urine catheters. Determination of bactericidal effect of preparations during incubation of microorganisms on the surface of urine caterers was carried out by Koch method. Morphologic and i ultrastructure changes in bacterial cells and biofilms were studied by transmission and scanning j electron microscopy. Results. A pronounced bactericidal effect of Kanefron and Furamag on the formed bacterial biofilms was detected. Inhibiting effect on growth and development of biofilms of the strains was noted. Destructive changes in cells and biofilms during the effect of the studied preparations are shown on electron microscopy level. Conclusion. The detected bactericidal effect of officinal preparation of plant origin - Kanefron and nitrofuran class preparation - Furamag on opportunistic bacteria on Foley urine catheters allows not only to suppress biofilm formation process of E. coli M17 и S. aureus 193 compared with classic antiseptic methods, but also effectively destroy already formed biofilms that must prevent development of catheter-associated infections and result in enhancement of recuperation of patients with diseases of the urinary tract.

Antimicrobial effect of TiO2 doped with Ag and Cu on Escherichia coli and Pseudomonas putida

Antimicrobial effect of TiO2 doped with Ag and Cu on Gram-negative bacteria Escherichia coli and Pseudomonas putida is studied. The thin films are deposited on glass substrates without heating during the deposition by r.f. magnetron co-sputtering of TiO2 target and pieces of Ag and Cu. The studied films, thickness about 65 nm, were as deposited and annealed (5200C, 4h, N2+5%H2, 4Pa). The as deposited thin films TiO2:Ag:Cu have band gap energy of 3.56 eV little higher than the band gap of crystalline anatase TiO2 which can be explained with the quantum effect of the granular structure of r.f. magnetron sputtered films. The annealed samples have band gap of 2.52 eV due to formation of donor levels from Ag and Cu atoms near the bottom of the conduction band. The toxic effect was determined through the classical Koch's method and the optical density measurements at λ=610 nm. The as deposited TiO2:Ag:Cu thin films demonstrate stronger inhibition effect - bactericidal for P. putida and bacteriostatic for E. coli (up to the 6th hour) in comparison with the annealed samples. The both methods of study show the same trends of the bacterial growth independently of their different sensitivity which confirms the observed effect.

Антимікробна дія по верхнево-активних речовин Nocardia vaccinii ІМВ В-7405

To study the effect of Nocardia vaccinii IMV B-7405 surfactants on some bacteria (including pathogens of genera Proteus, Staphylococcus, Enterobacter), yeast of Candida species and fungi (Aspergillus niger R-3, Fusarium culmorum T-7). The antimi- crobial properties of surfactant were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). It is shown that the antimicrobial properties of N. vaccinii IMV B-7405 surfactant depended on the degree of purification (supernatant, solution of surfactant), concentration and exposure. Survival of Escherichia coli IEM-1 and Bacillus subtilis BT-2 (both vegetative cells and spores) after treatment for 1-2 hours with surfactants solution and the supernatant (the surfactant concentration 21 µg/ml) was 3-28%. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants on studied bacteria, yeast and micromycetes were 11.5-85.0; 11.5-22.5 and 165.0-325.0 µ/ml respectively. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants are comparable to those of the known microbial surfactants. The possibility of using the supernatant of culture liquid as an effective antimicrobial agent noticeably simplifies and reduces the cost of the technology of its obtaining.

Analysis of the Fractal Koch Method in Computer Games Development

Lately according to the software and hardware progress, the development of the computer games has been so ‎much. As the development of the computer games is one of the most favorite projects of computer sciences. A requirement ‎for development of the computer games is the computer graphics programming science and the mathematical calculations. A ‎calculation method is fractal Koch which uses the selfsimilarity specifications of itself. Formation of fractals takes place by the ‎repetitions, so it is possible to transform a simple geometric shape to a more complex one and use the fractal capabilities to ‎develop and produce the computer games. So, we have tried to use the OpenGL graphic functions in VC++6.0 and review the ‎application of fractal Koch in development of the computer games.‎

Мікробоценози стічних вод Львова на різних етапах очищення

The aim of this work was to investigate some physiological groups of microorganisms which are components of wastewater microbiocenoses. Microorganisms were grown in Petri dishes containing 20–30 ml agar selective medium and in 25 ml tubes at a temperature +30 ºC. The selective media were: wort agar for microscopic fungi and yeasts, Hutchinson medium for the cellulose-destroying microorganisms, starch-ammonium medium for microorganisms that can utilize mineral nitrogen forms, Postgate B medium for sulfate-reducing bacteria, Vinogradsky medium for nitrifying bacteria, Ashby medium for the nitrogen-fixing bacteria, Chapek medium for the actinomycetes. 1 mM Cr (VI) (104 mg/l) in the form of К2Cr2О7 was added to the medium. The number of colonies was determined by the Koch method. We studied wastewater microbocenoses of Lviv city at various stages of purification. We showed that the quantitative and qualitative composition of microorganisms differed significantly in primary and secondary clarifiers, the aerotank and sludge at different stages of sewage treatment. In the initial stages of purification, in the primary sump, bacteria that reached the treatment plant with sewage were found. Nitrifying bacteria (7.1 × 106colony forming units (CFU)/ml), nitrogen-fixing bacteria (9.0 × 106CFU/ml), and fungi (3.4 × 106 CFU/ml) dominated. The qualitative composition of microorganisms in primary clarifiers and the aerotank was similar, but their number in the aerotank was significantly higher than in the primary sump: 1.5 × 107 CFU/ml of nitrifying bacteria, 1.4 × 107CFU/ml of nitrogen-fixing bacteria, 6.7 × 106CFU/ml of fungi. The ratio of different physiological groups of microorganisms in the active sludge changed significantly. The predominant microorganisms were those that assimilate mineral forms of nitrogen (65%), their number was 1.6 × 108CFU/ml. In the secondary clarifier, the largest group was cellulose-destroying microorganisms (6.0 × 105CFU/ml). However, their numbers in the secondary sump were lower compared to their numbers in the aerotank and sludge (1.5–3.9 × 106CFU/ml). Among the representatives of various physiological groups of bacteria a significant number of chromium-resistant strains was detected. The largest number of chromium-resistant strains was detected in the active sludge and aerotank, which is probably due to the recirculation of microorganisms in the wastewater treatment. The highest percentage of Cr (VI) resistant microorganisms was among sulphate-reducing bacteria. An increase in the percentage of chromium-resistant microorganisms occurred together with the lowering of the total number of microorganisms of a certain physiological group. These microorganisms could prove useful for the development of biotechnological methods wastewater treatment to eliminate chromium compounds, which are highly toxic to living organisms.

Nonparametric Tests for 2×2 Cross-Over Design

A <TEX>$2{\times}2$</TEX> Cross-over design is widely used in clinical trials for comparison studies of two kinds of drugs or medical treatments. This design has many statistical methods such as Hills-Armitage's (1979) method or Koch's (1972) method. In this paper, we propose a nonparametric test for <TEX>$2{\times}2$</TEX> Cross-over design based on a two-sample test suggested by Baumgartner et al. (1998). In addition, a Monte Carlo simulation study is adapted to compare the power of the proposed methods with those of previous methods.

Results of one-year investigations of the contamination of dairy cattle feed and raw milk with moulds and mycotoxins

The contamination of 98 dairy cattle feed samples with moulds and mycotoxins (AB1, OTA, ZEA) was investigated. The contamination of 80 raw milk samples with aflatoxin M1 was also tested. Determination of total viable counts per 1g of feed was performed using Koch's standard method. The content of AB1, OTA and ZEA in feed, as well as aflatoxin M1 in raw milk samples, was determined by VICAM fluorimetric method. Results showed that 91% of all tested samples was contaminated with moulds, and total viable mould count per gram ranged from 10 (corn silage - spring) to 4.9 x 106 per gram (dried alfalfa - winter). The isolated moulds belong to 20 genera and 72 species. Results of mycotoxicological investigations showed that 42 samples of total 98 tested, were contaminated with mycotoxins. The most frequent was OTA (24 samples, 20.00 - 210.00 ?g/kg) followed with ZEA (12 samples, 250.00 - 980.00 ?g/kg) and AB1 (3 samples, traces - 18.50 ?g/kg). Aflatoxin M1 in raw milk samples wasn?t found.