Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.     

Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.     

A colour reaction for the differentiation of ascomycetous and hemibasidiomycetous yeasts

Seventy yeast strains, representative of twenty-six ascogenous genera, four saprobic hemibasidiomycetous genera and thirteen genera of the Cryptococcales were tested for their reaction with the stabilized aromatic diazonium compound, Diazonium Blue B salt. An aqueous, buffered solution of this compound gave a characteristic red colouration with the colonies of the hemibasidiomycetous species and those Cryptococcales characterized by the hemibasidiomycetous cell-wall type. The characteristic colour reaction was not observed with colonies of either the ascomycetous yeasts or those Cryptococcales characterized by the ascomycetous cell-wall type.The possible taxonomic use of the colour reaction with Diazonium Blue B salt as an affinitive characteristic is discussed.     

A procedure for the rapid screening of Maillard reaction inhibitors

A procedure for the rapid screening of inhibitors of glycation reaction, based on their ability to protect RNase against sugar induced inactivation of the enzyme is described. Glycation is implicated in variety of disorders including diabetes, atherosclerosis various micropathies yet is a slow process both in vivo and in vitro. In order to speed up glycation, the reaction was carried out at 60 °C using a thermostable protein RNase and ribose, a sugar that is known to react rapidly than glucose in the glycation reaction. It was observed that incubation of RNase with ribose at 60 °C in rapid inactivation of the enzyme with a parallel decrease in tyrosine fluorescence, enhancement in new fluorescence and hyperchromicity in the UV-region. No such alterations in the enzyme activity were observed when the incubation was carried out in absence of the sugar. Compounds and drugs that are known to act as inhibitors of glycation reaction restricted the ribose-induced inactivation of RNase. RNase immobilized on CNBr-activated Sepharose was also sensitive to exposure to ribose and appeared a better system to screen inhibitors of glycation from natural sources that contain substances that interfere with the assay of enzyme as well as in the study of post Amadori inhibitors of glycation.     

Beta-amylase-resistant amylose. Effect of urea on the limited hydrolysis of amylose by beta-amylase.

Amylose prepared from starch dispersed in 10M-urea, pH6.2, was found to be resistant to the action of beta-amylase and phosphorylase, though it was degraded by alpha-amylase. Amylose isolated by conventional methods was similarly refractory after urea treatment, and was hydrolysed by beta-amylase to the extent of 32-35%; it had no inhibitory effect towards beta-amylase. The physical and chemical properties of the modified amylose were in general comparable with those of normal amylose with a beta-amylolysis limit of 94-98%. Starch and amylopectin were unaffected by urea treatment, i.e. the presence of amylopectin protected amylose against changes induced in it by urea. It is speculated that urea treatment "freezes" amylose molecules in a conformation that renders non-reducing termini inaccessible to the active site of the exo-enzymes. Such changes may limit the degradative action of beta-amylase and phosphorylase.     

A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction

We have developed an efficient process for rapidly isolating campylobacter DNA using mechanical disruption combined with the guanidine-based reagent DNAzol. Template DNA was isolated by this method from cultures of Campylobacter jejuni resistant to lysis by boiling or enzymes and identified following polymerase chain reaction (PCR) amplification using primers specific for the hippuricase gene. Direct detection of campylobacters in poultry-processing samples by PCR is demonstrated in chicken carcass rinses spiked with lysis-resistant C. jejuni. Our results indicate that this method of DNA isolation may be ideal for direct PCR detection of pathogenic bacteria in complex samples of widely varied origin, especially when the target organisms are difficult to lyse by other means.     

Observations on paraffin baiting as a laboratory diagnostic procedure in nocardiosis

The efficacy of paraffin bait technique in the isolation ofNocardia asteroides from clinical specimens has been investigated. In a comparative study 1091 clinical specimens, mostly sputa and bronchial aspirates collected from 639 patients of bronchopulmonary diseases and 11 of meningitis, were examined by paraffin baiting and the conventional technique. Thirty-six clinical specimens originating from 12 of the patients yieldedN. asteroides by the paraffin bait technique but only 4 by the conventional technique. Approximately 95 % of 125 sputum samples inoculated withN. asteroides yielded the pathogen by paraffin baiting as against 49 % by the conventional technique. Paraffin baiting was more productive than the conventional technique in the isolation ofN. asteroides from mixed suspensions with a number of fungi and bacteria. It is concluded that paraffin baiting can be profitably adopted as a suitable technique for the isolation ofN. asteroides from clinical specimens, such as, sputum, gastric lavage, etc., which are often contaminated. The technique has no particular advantage with non-contaminated specimens.This work forms a part of the Ph. D. thesis of S.K.M. submitted in 1971 to the University of Delhi, and was presented at the Vth meeting of ISHAM held in Paris from 5th–10th July, 1971.     

Rate of hydrolysis of urea as influenced by thiourea and pellet size

Summary Two incubation experiments and a number of field experiments were conducted to determine the effect of soil moisture tension, pellet size and addition of thiourea to urea on the rate of urea hydrolysis. In the incubation experiments at 20°C, the rate of hydrolysis of urea increased from 15 bar to 1/3 bar soil moisture tension, with the largest change (doubling) occurring from 15 bar to 7 bar moisture tension. Increasing pellet size reduced the rate of urea hydrolysis by about 12% with urea pellets weighing 0.21 g as compared to 0.01 g urea pellets after 114h. When thiourea (a metabolic inhibitor) was pelleted with urea in a ratio of two parts urea and one part thiourea, the rate of hydrolysis was halved.In a field experiment, the addition of thiourea to urea and increasing pellet size suppressed the rate of urea hydrolysis considerably for 8 days. The amount of urea hydrolyzed with urea+thiourea (21) pellets weighing 2.51 g was one-fourth of the amount of urea hydrolyzed with 0.01 g pellets of urea alone. In the other six field experiments which were set out in October, only 22% to 39% of urea +thiourea (21) was hydrolyzed at two weeks after application, while almost all of the urea was hydrolyzed when it was mixed into the soil without an inhibitor.Unter our field conditions, we would estimate that the hydrolysis of urea can be inhibited for at least one week. The inhibition of urea hydrolysis appears to be great enough that the problems encountered from the rapid hydrolysis of urea, wherever these occur, may be reduced by combined use of thiourea and either increased pellet size or band placement.     

A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino-n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages.

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

Microbiological transformations of lipids: acyl-specific hydrolysis of lard by yeasts.

The fatty acid and positional specificities of Saccharomyces cerevisiae (UI-SACCH) and Schizosaccharomyces octosporus (NRRL Y-854) in the hydrolysis of lard were studied by using gas-liquid chromatography. Synthetic triglycerides were used to determine the positional specificities of the lipases of both organisms. Palmitic acid is specifically cleaved from all three triglyceride ester positions by S. cerevisiae, while S. octosporus was able to cleave stearic acid at either position 1 or position 3 of the glycerol moiety. Preparative scale fermentation with 200 g of lard per liter yielded 48.4 g of palmitic acid per liter with S. cerevisiae and 42 g of stearic acid per liter with S. octosporus. The free fatty acids produced by microbial transformation of lard were characterized spectrally (1H and C nuclear magnetic resonance and mass spectrometry) and chromatographically (thin-layer and gas chromatographies).