Standardization of the methods of determining microorganism sensitivity to antibiotics. The effect of the size of the inoculate on the results of determining microorganism sensitivity to antibiotics and its standardization

The literature data and personal observations of the authors on the effect of the inoculate amount on the results of determination of microbial sensitivity to antibiotics by the methods of serial dilutions in the liquid and solid nutrient media and agar diffusion are discussed. It was shown that the inoculate of the density of 3.6.10(7) to 4.25.10(7) microbial bodies per 1 ml was optimal for the methods of agar diffusion and serial dilutions in agar. Recommendations for simplifying standardization and dilution of the inoculate are presented.     

Changes in microorganism sensitivity to antibiotics as affected by divalent metal ions

Ca+ and Mg+ in nutrient media significantly influence the results of antibiotic sensitivity determination in microorganisms. The data of the study are indicative of a necessity for the media standardization with respect to the content of bivalent metal ions. It is recommended that agar-agar manufactured by the South Sea Factory (tafuinsky) be used for preparation of nutrient media for determination of microbial sensitivity to antibiotics and Hottinger meat pancreatic digest as the nitrogen source providing the content of 120 mg per cent of amine nitrogen in the medium.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

Use of paper indicator discs for determining the minimal inhibitory concentration of antibiotics]

Sensitivity of clinical strains of Staphylococcus and some Enterobacteriaceae to a number of widely used antibiotics was compared simultaneously with the use of two methods, i. e. the agar diffusion method and the method of serial dilutions. Regularities in distribution of the staphylococcal strains according to their sensitivity to antibiotics, such as erythromycin, benzylpenicillin, levomycetin and others were also studied with respect to every year using indicator paper discs. Interrelation observed during the comparison of the microbial sensitivity with the use of the two assay methods provided elaboration of the criteria for classification of the strains as "resistant" or "sensitive". The differentiation boarder for these two groups was determined according to the principle of the assay error minimization. A necessity of using standard dry media for specification of individual characteristics of various drugs in estimation of the microbial sensitivity to them by the agar diffusion method is emphasized.     

Ways of improving the effectiveness of bacteriological diagnosis and treatment of nonspecific lung diseases

Isolation, identification and drug sensitivity assay of microorganisms from pathological materials of 177 patients with nonspecific diseases of the lungs, mainly pneumonia, were performed on blood and selective "chocolate" agars by using Baktofok-MK, a new dry nutrient basis developed by the authors. Blood and "chocolate" agars based on the Hottinger's hydrolysate were used as the control media. It was shown that with the quantitative procedure for inoculating the pathological material, the experimental media based on Baktofok-MK were much more sensitive to growth properties that the control media. That made it possible to detect larger numbers of etiologically important microbial species on the blood agar and to isolate clinical strains of Hemophilus spp. from a larger number of specimens on the "chocolate" agar.     

Development of a method for the quantitative evaluation of microorganism sensitivity to antibiotics utilizing disks. The determination of the minimal doxycycline concentration that depresses microorganism growth using disks containing this antibiotic]

A quantitative method for estimation of microbial sensitivity to doxycycline with the use of discs containing 10 gamma of the antibiotic was developed. The antibiotic concentrations in the agar were determined at a distance equal to the radius of the growth inhibition zone with the help of a curve of the dependence of the logarithm of the doxycycline concentration in agar at the period of the average critical time of the zone formation equal to 5 hours and the distance from the disc center. The antibiotic concentration in the agar at the zone edges was almost the same as the MIC of doxycycline against the test-cultures determined with the method of serial dilutions in agar.     

Nutrient media for the isolation and accumulation of Listeria

Dried culture media for the isolation and accumulation of Listeria from pathological material and foodstuffs have been developed. The media are suitable for use in bacteriological and sanitary-hygienic practice. The optimum nutrient base has been selected: dried broth (from sprat hydrolysate), produced by the Research and Manufacturing Amalgamation "Culture Media". The optimum concentrations of ingredients, stimulating the growth of Listeria and inhibiting the growth of associated microbes, have been experimentally established. The samples of died accumulation and isolation culture media ensuring the growth of L. monocytogenes, diluted 10(-6), after 24-hour incubation at 37 degrees C have been obtained. The possibility of using these media for the bacteriological diagnosis of listeriosis in pregnant women has been demonstrated.     

A nutrient medium for the isolation and cultivation of Campylobacter

Campylobacter agar, nutrient medium intended for the isolation of bacteria of the genus Campylobacter from clinical material, has been developed. The composition of the medium includes sprat hydrolysate, aerotolerant additive (ferric sulfate--oxide, sodium pyruvate, sodium pyrosulfite), sodium glutaminate, agar. The selective properties of the medium are ensured by introducing the mixture of antibiotics consisting of polymyxin B, rifampicin, amphotericin B, ristomycin. The balanced composition of Campylobacter agar ensures the aerotolerance of Campylobacter organisms and gives the optimal conditions for their growth when the inoculated material is cultivated in the atmosphere made up of the mixture of three gases (5% of oxygen, 10% of carbon dioxide, 85% of nitrogen), as well as under the conditions of a "candle vessel". The medium suppresses the development of the associative microflora diluted 10(-1). As shown in the trial of the quality of Campylobacter agar by the inoculation of material taken from patients with acute enteric infections, agricultural animals and monkeys, the medium has pronounced selective, properties with regard to extraneous microflora, while ensuring the isolation of Campylobacter on the level of the control medium.     

New immunofluorescent method for the rapid determination of microbial antibiotic sensitivity

A new procedure for rapid determination of the levels of antibiotic sensitivity in pathogenic microorganisms with the use of fluorescent antibodies is described. The procedure was developed with the use of a model of the vaccinal strains of Bacillus anthracis. It is based on determination of the microbial antibiotic resistance with the method of serial dilutions on solid media. Still, the medium with an antibiotic is inoculated instead of the pathogen with the native material subject to the analysis. The antibiotic effect on the microorganism is estimated with the method of fluorescent antibodies. The replica preparations obtained as a result of the pathogen growth in a mixed culture on nutrient media containing definite concentrations of the antibiotic are examined with the method of luminescence microscopy. The modification of the immunofluorescent procedure for rapid determination of the microbial sensitivity to antibiotics does not require obligatory isolation of the pathogen as a pure culture. This makes the procedure more economic with respect to the time necessary for the analysis. The following conditions for performing rapid analysis with respect to Bacillus anthracis are required: the minimal concentration of the pathogen in the specimen (2.10(5) spores/ml), preliminary thermal treatment of the specimen for destroying the spore microflora, additional cultivation for 6-8 hours at 37 degrees C. The presence of the accompanying sporulating microflora, i.e. common microorganisms present in the atmosphere, soil and open water bodies does not prevent the performance of the analysis.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Fermentative hydrolysate of mycelial waste of aminoglycoside antibiotics production as component of culture media used in biosynthesis of tobramycin]

New nutrient media for cultivation of the tobramycin-producing organism were developed. As an additional source of nitrogen the media contain fermentative hydrolysate of the mycelial waste of manufacture of aminoglycoside antibiotics (tobramycin and apramycin). The use of the media provided a 20 to 50% decrease of consumption of soybean meal, an essential food raw material, and design of a low-waste technology for biosynthesis of tobramycin.     

Determination of the sensitivity of bacteria to barium ions,a taxonomic marker of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Comparative efficacy of the determination of the sensitivity of bacterial cells to barium ions was evaluated on a synthetic nutrient medium, FMH agar, Mueller-Hinton agar, and AGV agar. The synthetic nutrient medium developed for this study contained L-proline and L-glutamine as the sole nitrogen and carbon source, which promoted growth of all Pseudomonas strains and ensured the minimal level of barium binding. The sensitivity of 80 strains belonging to 11 Pseudomonas species, including the type strains, as well as of 80 strains of 22 other bacterial species, was studied. The sensitivity of bacteria to barium ions was determined by using serial dilutions of barium chloride in the nutrient medium. The highest level of analytical sensitivity of pseudomonads to barium ions was determined on the synthetic nutrient medium: the minimal inhibitory concentration (MIC) values of barium chloride ranged from 0.5 to 6 g/L, the MIC₉₀ value was 2 g/L. At the same time, 86.1% of all strains of fluorescent Pseudomonas species produced fluorescein on the control BaCl₂-free synthetic nutrient medium. For representatives of other genera grown on all the studied nutrient media, the MIC values of barium chloride ranged from 20 to 50 g/L. The proposed method for determination of the sensitivity of bacteria to barium ions using the synthetic nutrient medium with 6 g/L of barium chloride as a criterion for the classification of barium-sensitive strains to the genus Pseudomonas is suitable for standardization.     

Short peptide induces an "uncultivable" microorganism to grow in vitro

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.     

Determination of the bacterial contamination of nutrient media for the biosynthesis of antibiotics

Contamination of separate components of nutrient media used in biosynthesis of antibiotics was tested. It was shown that corn steep liquor, corn and soybean meals were the most contaminated components. The results provided determination of the nutrient media contamination levels by the additive factor with respect to the percentage of the components. The safe period of 2-3 hours for the storage of the nutrient media was determined. After that period the media contamination levels markedly increased.     

Cultivation of Pseudomonas aeruginosa in media with chlorella fermentative hydrolysate as nutrient base

The specific features of the growth of P. aeruginosa in media prepared with the use of Chlorella fermentative hydrolysate as nutrient basis were studied. The accumulation of microbial biomass was shown to depend on increased content of amino nitrogen in the media, while the effectiveness of the pyocyanin formation was linked with decreased content of amino nitrogen and a rise in the amount of sodium chloride.     

A simple method for determining the sensitivity of micro-organisms to serial dilutions of antibiotics

Summary At this laboratory the routine sensitivity tests of micro-organisms against sulfathiazol and antibiotics have been performed in plastic dishes for half a year. The dishes are cleaned and sterilized in the same way as bacteriological glassware. The use of these dishes offers some advantages over other conventional dilution methods employing tubes or Petri dishes with solid media: the sensitivity to serial dilutions of one antibiotic may be measured in one and the same plate, and in this way a considerable amount of glassware and of space in the incubator may be saved; the readings are easily done and distinct. It has been demonstrated that sensitivity tests with two standard strains against sulfathiazol and various antibiotics may be reproduced with the same endpoints.     

Dry medium for isolating Clostridium perfringens

The possibility of using blood substitutes (hydrolysin, casein hydrolysate "tsolipk" and amino peptide) with expired shelf life as a culture medium for the isolation of Cl. perfringens has been studied. Dry culture medium based on these inedible products has been developed. To stimulate bacterial growth, fodder yeast extract has been used. The suppression of the growth of extraneous microflora is ensured by the use of antibiotics: polymyxin sulfate and mycerin sulfate. The recommended medium is not inferior in its quality (sensitivity, rapidity of growth, differentiating and inhibiting properties, etc.) to media based on meat and casein. The use of the newly developed medium is economically grounded and allows one to obtain standard results.     

Effect of the peptide and amino acid composition of nutrient bases on the parameters of microorganism growth

The parameters of the growth of three microbial strains cultivated in growth media prepared with the use of different nutrient bases, both locally produced and imported, have been studied. The characteristics of the processes of bacterial cultivation have been found to correlate with the peptide composition of hydrolysates used in the experiment. Nutrient bases containing a wide spectrum of peptides with different molecular weight have ensured the most favorable conditions for the growth of microorganisms belonging to the taxonomic groups under study.     

Virulence, viability and antibiotic sensitivity of the causative agent of plague growing on nutrient media at 28 and 37 degrees C

A comparative study of virulence, viability and antibiotic sensitivity of Y. pestis strains grown at 28 degrees C and 37 degrees C in yeast-casein medium, yeast medium with Hottinger's meat digest and yeast medium with protein hydrolysate obtained from sunflower seed groats has been made. These media have been found to be suitable for the prolonged cultivation of Y. pestis at 28 degrees C and 37 degrees C, for the determination of its sensitivity to antibiotics, as well as for the preservation of Y. pestis cultures.