Clinico-experimental study of a dried selective medium for isolating fungi in the genus Candida

The trial of a newly developed dried selective medium for the isolation of fungi of the genus Candida, involving the inoculation of 103 museum yeast strains and 542 specimens of pathological material, has been carried out. The data obtained in this trial indicate that the proposed medium has advantages over wort agar and Sabouraud medium with antibiotics in the germination index, in the ability to ensure fungal growth after the inoculation of pathological material, and in selective properties. The medium ensures the intactness of the morpho-cultural, biochemical and serological properties of the fungi. The results of the trial recommend the newly developed preparation for laboratory practice for the isolation of yeast-like fungi from clinical material in the diagnosis of candidiasis, intestinal microflora disturbances, as well as for isolation of the fungi from various environmental objects.     

A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.     

Selective nutrient medium for the isolation of Bacillus cereus

A selective dried medium for the isolation of B. cereus from clinical material and foodstuffs has been developed. The medium has high selective properties which ensure the isolation of B. cereus from microbial association in pure culture, thus making it possible to accelerate further identification of the microorganisms.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

A New Isolation Method for Labyrinthulids Using a Bacterium, Psychrobacter phenylpyruvicus

A new isolation method for labyrinthulids, marine microbes with spindle-shaped vegetative cells and gliding movement, is presented. The method for isolating labyrinthulids has been found to be more difficult and less reproducible than that for thraustochytrids, classified in the same order. So far serum seawater agar fortified with antibiotics has been proposed to be the best for isolation of labyrinthulids. The method presented here involves placing plant samples on an agar medium on which a marine bacterium, Psychrobacter phenylpyruvicus, has been grown. The new method, which utilizes fallen mangrove leaves as source material, was more than twice as effective as isolation agar medium without the bacterium. The increased effectiveness appears to derive partly from the bacterial colonies' delaying extension of fungal mycelium. The bacterium was more effective for the isolation of labyrinthulids than either the bacterium Shewanella sp. or the yeast Rhodotorula rubra.     

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.     

A selective nutrient medium for isolating clinical strains of Escherichia coli O157:H7

A dried selective culture medium, electrolyte-deficient sorbitol agar (EDS agar), for the isolation and preliminary identification of E. coli O157:H7 from clinical material has been developed. The medium is not inferior in its quality to analogous foreign media and requires no scarce ingredients for its manufacture.     

Primary structure of bovine erythrocyte carbonic anhydrase CI. I. Tryptic peptides]

Bovine erythrocyte carbonic anhydrase CI consists of 259 amino acid residues including 18 lysines and 9 arginines. Its primary structure has been first investigated by isolation and sequence determination of the tryptic units. Acidification of the tryptic hydrolysate leads to the precipitation of 40% of the peptidic material. All the acid soluble peptides were isolated from the supernatant by chromatography on Dowex 50 W-X2 and Dowex 1-X2 followed by purification of heterogeneous fractions. Two of the three acid insoluble peptides were obtained in a pure form from the whole tryptic hydrolysate by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex in alkaline medium. The sequence of the so isolated tryptic units has been determined with the exception of two of them obtained in a very poor yield.     

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.     

Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.     

Pneumococcal Forssman antigen: enrichment in mesosomal membranes and specific binding to the autolytic enzyme of Streptococcus pneumoniae.

The choline-containing pneumococcal membrane teichoic acid (Forssman antigen) can be isolated with the membrane fractions of the bacteria. The small vesicle (mesosomal) fraction generated during the formation of protoplasts seems to be highly enriched in this material. Forssman antigen was identified in cell fractions on the basis of (i) radioactive choline label, (ii) autolysin-inhibitory activity, and (iii) the sedimentation profile in sucrose density gradients with and without detergent. A membrane teichoic acid could also be isolated from pneumococci grown in medium in which choline was replaced by ethanolamine as the nutritionally required amino alcohol. This material contained radioactive ethanolamine label and behaved similarly to the choline-containing membrane teichoic acid during centrifugation in detergent-containing and detergent-free density gradients. On the other hand, the material had only low autolysin-inhibitory activity. Binding of pure pneumococcal autolysin to micelles of purified Forssman antigen could be demonstrated by mixing these components in vitro and analyzing them by sucrose density gradients and by agarose chromatography. No binding could be observed between the pneumococcal enzyme and the micellar forms of either cardiolipin or polyglycerophosphate-type lipoteichoic acid isolated from Streptococcus lactis.     

An Effective and Low-Cost Culture Medium for Isolation and Growth of Xylella fastidiosa from Citrus and Coffee Plants

Buffered charcoal–yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-μm pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.     

Dried nutrient media for the cultivation of pneumococcus and meningococcus

Dried synthetic nutrient medium for the cultivation of meningococci and the accumulation of their biomass has been developed. The kinetics of the culture growth, changes in pH and in the content of dissolved oxygen in the medium during prolonged controlled processes of the cultivation of meningococci in a bioreactor with the use of this medium have been studied. The stable physico-chemical properties and composition of the polysaccharide-protein complex isolated from the biomass of meningococci grown in the above-mentioned medium have made it possible to use it for the preparation of the samples of group B meningococcal vaccine. In addition, dried semi-synthetic nutrient medium for the accumulation of pneumococci without the necessity of introducing blood or serum into the medium has been developed. As regards its biological properties, this newly developed medium make it not inferior to meat media, containing blood or serum, and ensures good yield of biomass.     

Glycine-containing selective medium for isolation of Legionellaceae from environmental specimens.

Glycine, at a final concentration of 0.3%, has been shown to be an excellent selective agent for the isolation of Legionellaceae. Stock cultures of Legionella pneumophila were not inhibited on buffered charcoal-yeast extract agar containing the amino acid. Among the other Legionellaceae tested, only one of two strains of L. dumoffii and two of six strains of L. micdadei were appreciably inhibited. This medium permitted the isolation of L. pneumophila from environmental specimens with marked inhibition of many non-Legionellaceae bacteria. The selectivity of the medium was subsequently improved by the incorporation of vancomycin (5 microgram/ml) and polymyxin B (100 U/ml). This selective medium, glycine-vancomycin-polymyxin B agar, should facilitate the recovery of Legionellaceae from environmental sources.     

Importance of carbon dioxide in the isolation of pneumococci

Austrian, Robert (The University of Pennsylvania School of Medicine, Philadelphia), and Patricia Collins. Importance of carbon dioxide in the isolation of pneumococci. J. Bacteriol. 92:1281-1284. 1966.-Of the strains of pneumococci isolated from man, 8% manifest a requirement for CO(2) if detectable growth is to occur on the surface of solid media. The pneumococci most frequently manifesting this requirement are types I, III, XVI, XXVIII, and XXXIII. These strains will grow in the presence or absence of oxygen provided the atmosphere in which they are incubated contains CO(2). Review of published data provides no unequivocal evidence for the existence of anaerobic pneumococci.     

Studies on cell-coat macromolecules in normal and virus-transformed BHK21/C13Cells

Cells have been grown in vitro in the presence of radioactive precursors; much of the glucosamine label is incorporated into the surface material, part of it is then spontaneously released into the medium, part is readily removed by an EDTA wash, and further amounts may be released by a mild trypsin treatment or by a period of chase.The material in the EDTA was from variously labelled cultures has been fractionated by Sephadex G-200 chromatography; the excluded peak has been analysed by Sepharose 6B and DEAE-cellulose chromatography and by electrophoresis on large pore polyacrylamide gels. Its sensitivity to pronase and hyaluronidase has been determined, as well as its sugar, lipids and amino acid composition.Cell-coat material from Polyoma and Rous sarcoma virus-transformed cell lines failed to show significant differences in chromatographic properties and in sugar and amino acid composition. Minor but reproducible differences were found in the polyacrylamide electrophoresis migration patterns, when comparing glucosamine-labelled materials.     

Type-specific pneumococcal agglutinating sera in precipitation tests

Diagnostic agglutinating sera to pneumococci of different serotypes have been studied with respect to their capacity for taking part in the reactions of precipitation with capsular pneumococcal polysaccharides. The sera have proved to be highly active and specific in the reactions of double immunodiffusion and immunoelectrophoresis. The sera under study have also been found to react with cattle serum, one of the components of the medium used for the cultivation of pneumococci.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.     

A selective and differential medium for Vibrio harveyi.

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.