Comparative study of the phospholipase activity of pathogenic and saprophytic Leptospira cultured on a serum-lecithin agar

The phospholipase activity of leptospires cultivated on serum-lecithin agar has been studied. Two zones of changes in the medium have been found to appear around the colonies of saprophytic Leptospira strains: transparent (5.25 +/- 2.09 mm wide) and turbid (6.90 +/- +/- 1.46 mm wide), which is linked with the production of phospholipases A and C. Only a single clear zone is formed around the colonies of pathogenic strains due to the production of phospholipase A. At the same time virulent Leptospira strains show greater phospholipase activity (the zones are 6.0 +/- 1.2 mm wide) than avirulent strains (the zones are 1.6 +/- +/- 0.04 mm wide).     

A novel combination of selective agents for isolation of Leptospira species

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10(1) cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.     

Growth of Pathogenic Leptospira in Chemically Defined Media

A protein-free chemically defined medium for cultivation of pathogenic Leptospira was developed. The medium permitted continued serial subculturing of 9 serogroups (52 strains) of the 12 serogroups (61 strains) tested. Growth was initiated from small inocula, and the growth rate and maximal cell yields were similar to those on serum-containing media. The nutritional requirements of serogroups L. canicola, L. pomona, and L. grippotyphosa were studied in a basal medium composed of inorganic salts, a fatty acid, vitamin B(12), and thiamine. All strains tested utilized ammonium chloride as the sole nitrogen source. A fatty acid, vitamin B(12), and ferrous ions were essential. Growth was stimulated by thiamine, potassium, and calcium ions.     

Hemolytic Activity of Leptospira interrogans Serovar canicola Cultured in Protein-Free Medium

Hemolytic activity of the culture supernatant of Leptospira interrogans serovar canicola strain Moulton grown in protein-free medium was demonstrated. The activity began to appear in the late logarithmic phase of growth of the organism and reached a plateau after 2 weeks of cultivation. It was inactivated by the addition of dipotassium ethylenediaminetetraacetate, but was effectively restored by Mg²⁺. Hemolysis by the culture supernatant was stimulated by “hot-cold” incubation. Sheep erythrocytes treated with the culture supernatant of the organism were transformed into spherocytes, in which invagination was observed. A hemolysin inhibitor in rabbit serum was found to be in the chloroform-methanol soluble fraction of the serum. The hemolysin of Leptospira may be phospholipase C.     

Computer-controlled maintenance of the dissolved oxygen level in a medium during controlled cultivation of Neisseria meningitidis

A new method for controlling the processes of N. meningitidis cultivation by the content of oxygen dissolved in the medium with the use of a computer, including the software, technical and methodological provision of the computer-operated control system, has been developed. The processes of the cultivation of N. meningitidis, groups A and B, under fully controlled conditions have been realized. The computer-operated control of the content of oxygen in the medium, the most important parameter of the process, has been shown to be essentially more effective than manual control. Information, accumulated in the course of the experiment, may be used in future for correction of mathematical models and optimization of the process. This will by necessary for obtaining biomass with predetermined properties in the development of new diagnostic and vaccine preparations.     

钩端螺旋体在复方明胶培养基中长期传代培养的抗原性分析

本文报道了钩端螺旋体(简称钩体)在复方明胶培养基中经过三年的传代培养,用显微镜凝集试验、对流免疫电泳及SDS-聚丙烯酰胺凝肢电泳(PAGE)分析,同Korthof培养基的钩体抗原比较。结果证明:(1)钩体抗原的组成是非常复杂的,SDS-PAGE可显示20多条抗原带;(2)两种培养基所培养的钩体,未发现抗原性变异。说明钩体在复方明肢培养基中长期传代培养过程中,其抗原组成是相对稳定的。这对进一步探讨钩体长期在综合培养基中培养以后的抗原稳定性,具有一定的理论及实际意义。     

Morphology of a natural focus of Pomona leptospirosis in a steppe area

The natural focus of Leptospira pomona infection has been found to include areas of the forest-meadow flood plain where the circulation of leptospires is constantly maintained among small mammals (the nucleus of the focus). A high level of Leptospira carriership (17.7%) among the animals has been registered in the central part of the flood plain and near the terraces, these areas having the most favorable conditions for the development of epizootic leptospirosis.     

Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species "Leptospira licerasiae" serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, "Leptospira licerasiae" serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a titer of 1:100. LipL32, although not detectable by PCR, was detectable in "Leptospira licerasiae" serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against "Leptospira licerasiae" serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.     

Effect of the viable cell count on the growth parameters of Leptospira cultures

The influence of the amount of live cells on the growth characteristics of 41 Leptospira pathogenic strains belonging to 4 serogroups at different stages of growth has been studied. The study has revealed that under the conditions of batch cultivation the maximum concentration of pathogenic leptospires in the inoculum decreases the duration of the lag phase and determines the highest specific growth rate characterizing the individual features of leptospires in the serogroups under study.     

Cultivation of yeasts on hydrolysate nutrient medium obtained from the production of microcrystalline cellulose

Studies of the biomass production during a continuous cultivation of yeasts on a nutrient medium, prepared from a hydrolysate from the production of microcrystalline cellulose, have been carried out. A new strain of yeasts has been used. Its cultivation has been achieved without addition of biostimulators to the nutrient medium in spite of their absence in the initial hydrolysate. Practically a complete assimilation of sugars has been achieved at high dilution rates (D = 0.25 to 0.50 h⁺¹). The yield of biomass achieved is above 50% compared to the initial sugars and it contains 48.89% true protein. The results obtained offer the possibility of a complex utilization of the products of cellulose hydrolysis in the production of microcrystalline cellulose with a realization of a waste free technology.     

Isolation of the specific endonuclease BamH1 in the multicyclic cultivation of Bacillus amyloliquefaciens

The present article deals with the study of the multicyclic process of the cultivation of B. amyloliquefaciens in a liquid medium prepared from natural raw materials and with obtaining restrictase Bam H1 from the biomass with different microbiological characteristics. The selected medium has been found to be suitable for the realization of the multicyclic cultivation process on an industrial scale. The tendency towards a drop in the level of nonspecific nucleases simultaneously with a decrease in the specific growth rate has been revealed. A simplified scheme for the purification of the restricting endonuclease is proposed.     

Fatty acid composition of Leptospira lipids as a taxonomic criterion

The fatty-acid composition of microbial cells in 17 pathogenic and saprophytic Leptospira strains, comprising 14 serovars and 10 serogroups, has been studied. The strains under investigation have proved to fall into 3 groups differing by this characteristic. The group of saprophytic strains is characterized by a comparatively high level of myristic acid and, consequently, by the ratio of saturated and unsaturated fatty acids with 14 carbon atoms approaching 1:1; besides, it is also characterized by a lower, in comparison with the pathogenic Leptospira strains belonging to the serogroups Icterohaemorrhagiae, Canicola, Ballum has a higher level of unsaturated fatty acids. The second group of pathogenic Leptospira strains including the serogroups Grippotyphosa, Hebdomadis, Pomona, Tarassovi, Pyrogenes, Australia has been found to occupy an intermediate position between the first group of pathogenic Leptospira strains and the group of saprophytic ones. As the difference in the content of myristic acid in pathogenic and saprophytic Leptospira strains is a stable characteristic, it can be used for the differentiation of these strains. The present investigation has revealed that the distribution of the main fatty acids in Leptospira phospholipids is similar to their distribution in Leptospira neutral lipids with the exception of unsaturated fatty acid with 14 carbon atoms, occurring mainly in phospholipids.     

Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.     

A dried nutrient medium for the isolation of Vibrio cholerae

A dried differential nutrient medium for the isolation of V. cholerae has been developed. The medium is sufficiently sensitive, has pronounced differentiating properties and greatly inhibits the appearance of microbial associations. During the cultivation of V. cholerae with the use of this medium the cultural, morphological and agglutination properties of the initial strains are retained.     

Effect of cultivation conditions on the adhesive activity of Rhodococcus cells towards n-Hexadecane

The effect of cultivation conditions (the composition, acidity, and salinity of the cultivation medium; temperature; and the hydrodynamic conditions of cultivation) on the adhesion of actinobacteria of the genus Rhodococcus to n-hexadecane has been investigated. A study performed showed that the adhesive activity of rhodococci depends on the composition of the cultivation medium and on the cultivation temperature. The possible mechanisms underlying the effect of growth conditions on the adhesion of rhodococci to liquid hydrocarbons and involving changes in the cell lipid content or the zeta potential of cells are addressed. Rhodococcal strains displaying high adhesive activity (80–90%) at a low temperature (18°C), high salinity (5.0% NaCl), and acidity (pH 6.0) of the cultivation medium have been selected as a result of the present work; these strains have a considerable potential for use in bioremediation of soil and water contaminated by hydrocarbons.     

Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy

A low-foaming hydrophobin II deletant of the Trichoderma reesei strain Rut-C30 was used for production of cellulases by continuous cultivation on lactose medium in a laboratory fermenter. The control paradigm of the addition of new medium to the continuous process was based on the growth dynamics of the fungus. A decrease in the rate of base addition to the cultivation for pH-minimum control was used as an indicator of imminent exhaustion of carbon source for growth and enzyme induction. When the amount of base added per 5 min computation cycle decreased below a given value, new medium was added to the fermenter. When base addition for pH control thereafter increased above the criterion value, due to increased growth, the medium feed was discontinued or decreased. The medium feeding protocol employed was successful in locking the fungus in the stage of imminent, but not actual, exhaustion of carbon source. According to the results of a batch cultivation of the same strain on the same medium, this is the phase of maximal enzyme productivity. The medium addition protocol used in this work resulted in a very stable continuous process, in which cellulase productivity was maintained for several hundred hours at the maximum level observed in a batch cultivation for only about 10 h. Despite a major technical disturbance after about 420 h, the process was restored to stability. When the cultivation was terminated after 650 h, the level of enzyme production was still maximal, with no signs of instability of the process.     

Diethylpyrocarbonate—An effective agent for the sterilization of different types of nutrient media

A simple and convenient method has been tested for the steriltzation of nutrient media for long-term cultivation of plant cells. Diethylpyrocarbonate is suitable for this task in concentrations about 1000 mg l^-1 The cells cultivated for 15 subsequent passages on media treated by DPC had the same growth parameters, production pattern and ability to transform exogeneous organic compounds as did the controls. The method is suitable for the preparatian of both liquid and agar media, for stabilization of stock solutions and for sterilization of cultivation vessels and tubing.Abbreviations DPC diethylpyrocarbonate - medium MS nutrient medium according to Murashige and Skoog - NAA naphtaleneacetic acid     

Preparation and use of solid culture medium for Leptospira isolation

Recommendations on the preparation and use of a solid culture medium are given; age changes in the colonies of leptospires belonging to different serovars and serogroups have been studied in their dynamics; the absence of relationship between the form of the colonies, the serovar and serogroup of the cultures, their virulence, as well as the region, time and source of their isolation has been established, which makes it impossible to use these parameters for the differentiation of Leptospira strains belonging to different serovars on solid culture media.     

Nutrient medium for the isolation and cultivation of pneumococcus

A culture medium for the isolation and cultivation of pneumococci, produced in a solid or liquid form and based on raw material unsuitable for use as foodstuff (human placenta), has been developed. The amino acid composition of the medium has been studied. The medium has been found to contain 19 amino acids, to be free from ballast serum proteins and blood, and to ensure the good growth of pneumococci isolated from pathological material, the formation of the normal capsule, as well as active biological properties. The medium has proved to create elective and selective conditions enhancing the effectiveness of investigations and simplifying the isolation of pneumococci in the microbiological examination of patients.     

Conservation and Propagation of High Altitude Medicinal and Aromatic Plant: Hedychium spicatum

The micropropagation of H.spicatum, a medicinal and aromatic plant was investigated as an option for conservation and propagation, as wild populations are fast depleting. The source of raw material is rhizomes of plants that are collected from the wild. There is no planned cultivation of the plant. Multiple shoot cultures were established on MS medium supplemented with BAP and IAA from the pre-existing buds on the rhizome. Prolonged cultivation on the same medium or transfer to hormone free medium induced roots/rhizome formation; liquid medium proved more suitable. Greenhouse hardened plants were transferred to field. A successful protocol with 99% root formation and 80–85.5% field survival has been formulated.