Use of paper discs in determining the biological activity of mycoheptin by the agar diffusion method]

Diffusion of mycoheptin from the discs into agar was studied. A procedure providing practical use of the discs for quantitative estimation of mycoheptin activity was developed. A possibility of using the discs in the assay of solutions containing significant amounts of an organic solvent was shown.     

A new method for determining the minimum inhibitory concentration of essential oils

A new microdilution method has been developed for determining the minimum inhibitory concentration (MIC) of oil-based compounds. The redox dye resazurin was used to determine the MIC of a sample of the essential oil of Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of 0·15% (w/v) agar as a stabilizer overcame the problem of adequate contact between the oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid version of the assay was also developed for use as a screening method. A comparison of visual and photometric reading of the microtitre plates showed that results could be assessed without instrumentation; moreover, if the rapid assay format was used, rigorous asepsis was not necessary. Accuracy of the resazurin method was confirmed by plate counting from microwells and MIC values were compared with results obtained using an agar dilution assay. The MIC results obtained by the resazurin method were slightly lower than those obtained by agar dilution.     

Stable paper indicator systems for rapid identification of microorganisms]

The authors prepared under experimental-industrial conditions paper indicator systems for the express identification of microbes, including carbohydrate discs (by the method of Nikitin et al.), and newly worked out types for the determination of the activity of cytochromoxidase, urease, indol formation, and indicator amino acid decarboxylases (lysin, ornithin, arginine). The use of paper indicator discs proved to be expedient for rationalization of laboratory diagnosis of bacterial intestinal infections.     

Use of serologic reactions for determining the concentration of the determinants of an antigen and its valence]

By the serological tests with the erythrocytic diagnostic agents it is possible to estimate the concentration of the determinants in the antigenic preparation under study. For this purpose it is necessary to have at one's disposal the data on the molecular weight and concentration of the reference antigen, valence of erythrocytes loaded with the antigen or antibodies, and antibody heterogeneity index. If these values are known, then one can calculate the concentration of antigenic determinants in terms divided by the valence of the reference antigen. With the aid of pure antibodies preparation obtained with the immunosorption procedure it is possible to measure the determinant concentration of the antigen tested and the valence of the reference antigen. For the capsular antigen of plaque bacilli the valence was estimated by two independent methods and in both cases it was close to 10. The valence of the reference capsular antigen permits to calculate the concentration of the antibody molecules in the hyperimmune sera without isolation of pure antibodies.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Use of immunosorbents for determining antibodies to histaglobulin]

A determination was made of antibodies to histaglobulin and gamma-globulin with the aid of immunosrobents in 178 sera of children suffering from allergic diseases, during the histaglobulin therapy. Results of investigations showed antibodies to histaglobulins to be absent in children untreated by this preparation. But they appeared in 57% of cases after the first course of histaglobulin treatment, and their incidence and their average level increased with an increase in the number of the courses of treatment carried out. gamma-Globulin antibodies were found at the initial condition in 42% of cases; this percentage rose to 68 after the first course of histoglobulin treatment. The authors believe that determination of histaglobulin antibodies during the treatment with this preparation could serve as an auxiliary immunological criterion of the efficacy of histaglobulin therapy.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

Method of membrane filtration for determining the sterility and microbial contamination of antibiotics]

During membrane filtration antibiotics belonging to different chemical groups are strictly absorbed on the filters. When the filters are put into liquid thioglycol medium, the residual amounts of the antibiotics on the filters did not prevent the growth of sensitive microflora experimentally added to the drug. When the filter was put onto solid nutrient medium, only resistant forms of the microbes grew as a rule on its surface, the amount of the grown microbes being 26--43 per cent of the added one. The sensitive microbes grew only in the amount of 0.3--1.3 per cent. Subsequently the residues of the antibiotic adsorbed on the filter inhibited the growth of the sensitive and partially resistant microflora.     

Development of a method for the quantitative assessment of microorganism sensitivity to antibiotics using discs. The study of different nutrient media for determining microorganism sensitivity to antibiotics

Five nutrient media used for determination of microbial sensitivity to antibiotics, i.e. beaf-peptone agar, Hottinger pancreatic beaf infusion agar, sprat hydrolysate nutrient agar of the Dagestan Research Institute of Nutrient Media, Muller-Chintone agar from Bulgaria and "Oxoid" agar for determination of microbial sensitivity were studied comparatively. The media were compared with respect to the growth density with the use of different test-cultures and the clearance of the inhibition growth zones around the discs containing different antibiotics. The best results were obtained with the use of sprat hydrolysate nutrient agar. Further studies on the medium standardization are necessary.     

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae

Aim:  In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MICs) of nine antibacterial agents (enrofloxacin, ciprofloxacin, oxytetracycline, chloramphenicol, tylosin, lincomycin, gentamycin, spectinomycin and streptomycin) against M. hyopneumoniae .
Methods and Results:  Flow cytometry was able to detect Mycoplasma hyopneumoniae inhibition at 12 h postincubation, whereas the results obtained by the traditional method were only obtained at 48 h, when a visible change in the medium had occurred. At 48 h, both methods gave the same result for eight antibacterial agents, whereas flow cytometry gave slightly higher MIC values for one antibacterial agent (tylosin). This was attributed to the fact that the M. hyopneumoniae growth that had occurred in those tubes was not enough to visibly change the colour of the medium. A good relationship was found between the flow cytometry and the traditional method.
Conclusion:  Flow cytometry was found to be a good method for the determination of antimicrobial MICs in M. hyopneumoniae .
Significance and Impact of the Study:  The flow cytometric method allows the determination of the response of M. hyopneumoniae to each of the antibacterial agents in near real time, and has potential for the identification and study of resistant subpopulations.     

Utilization of automatic systems for determining the biological activity of antibiotics by the agar diffusion method]

The Automatic Zone Analyzer AR-140 of Millipor intertech., inc. is an apparatus used for measurement of inhibition growth zones of test-microbes and estimation of antibiotic activity. The analyzer provides automatic registration of the data with high levels of accuracy and at least 99% reproducibility. The analyzer gives an effect in saving time as compared to the manual method by 5-7 times for the single-dose procedure and by 10-12 times for the three-dose procedure.     

Elaboration of a rapid method of determining the sensitivity of Brucella to antibiotics]

Optimal conditions were developed for determination of antibiotic sensitivity in Brucella by using enzyme immunoassay directly in the primary cultures of the material tested. The Brucella concentration in the material tested should be not lower than 1.10(6) microbial cells/ml and the time of culture incubation be 24 hours at 37 degrees C. The obligatory condition is to use a liquid medium, i.e. the Albimi broth with 1% glucose. To inhibit the foreign microflora it is recommended to use polymyxin B and amphoglucamine in a concentration of 3 microgram/ml. The use of enzyme immunoassay was shown that it was possible to determine the antibiotic sensitivity of Brucella in practice.     

Use of paper indicator systems for the rapid identification of Corynebacterium diphtheriae, Neisseria meningitidis and the genus Staphylococcus

The possibility of using paper indicator discs with glucose, saccharose, lactose and urea, manufactured at the Gorky Research Institute of Epidemiology and Microbiology, has been studied and the methods for the preparation of indicator discs with fructose, maltose, starch and sodium phenolphthalein-phosphate for the biochemical identification of C. diphtheriae, N. meningitidis and staphylococci have been proposed. The study of 395 C. diphtheriae strains, 98 N. meningitidis strains and 328 staphylococcal strains has shown that paper indicator systems are highly sensitive and specific, which makes it possible to recommend their introduction in laboratory practice for the rapid identification of the above-mentioned organisms.     

Use of antibiotics in selecting a nystatin producer]

The lethal and mutagenic effect of streptomycin and nystatin on Act. noursei, strain 408 producing nystatin was studied. The survival of the spores of strain 408 on the medium with streptomycin decreased with an increase in the antibiotic concentration. Streptomycin had a selective effect on the nystatin-producing organism decreasing the frequency of morphologically changed and low active variants and revealing highly active and antibiotic stable variants. The survival of the spores of strain 408 on the medium with nystatin (20,000 units/ml) amounted to 35 per cent. Nystatin had an inhibitory effect on the organism producing it which was evident from delayed growth and significant modification variation of the colonies, as well as from a marked increase in the number of the variants characterized by low antibiotic production.