A fluorescent-cytochemical method for selective detection of yeast mitochondrial DNA

Yeast mitochondrial DNA (mDNA) can selectively be detected using a specific dye (DAPI). Nuclear DNA (nDNA) was stained along with mDNA only in three out of the fifteen studied yeast strains. Visualisation with a luminescent microscope showed that mDNA content varied among different yeast species as well as the size and shape of fluorescent mitochondrial nuclei. Intensive nDNA fluorescence was detected when a fixed specimen was treated with DAPI. Under these conditions, mDNA was revealed only in yeast cells with its high content. The process of fixation was shown to interfere with the integrity of mitochondria. It is also possible that the structure of DNA was modified to affect its interaction with the dye and thus the level of fluorescence. The developed technique of selective mDNA staining and the experimental results make it possible to gain a more accurate quantitative information about DNA content at the cellular level using cyto- and spectrofluorimetric techniques. This study involves important aspects pertinent to the dye interaction with yeast nuclear and mitochondrial DNA, both native and subjected to different fixation procedures.     

Efficacy of brain heart infusion-egg albumen agar,yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

A method for the detection of Issatchenkia orientalis in a baker's yeast factory

Summary A method for the detection of Issatchenkia orientalis (Candida krusei), a contaminant of bakers' yeast, is described. Nine different liquid medium types were compared and maximum specific growth rates for the yeast contaminant were determined in each medium. Issatchenkia orientalis grew fastest in malt extract broth (0,37 h^-1) and potato dextrose broth (0,36 h^-1). Five antibiotics were tested for selective inhibition of seven different bakers' yeast strains in malt extract broth. Nystatin was the only antibiotic tested that inhibited the growth of all the bakers' yeast strains used, but did not affect the growth of I. orientalis.     

Light effects in yeast: relation between the respiratory deficiency and light sensitivity in yeast

Visible light of 5,000 lux intensity has been shown to photokill yeast cells at 12 degrees C. In the present report some of isogenic respiratory deficient mit- and nuclear mutants were compared for their sensitivity to light. No close correlation between the cytochromes spectra and light resistance was observed. Although, the nuclear and rho- mutants which lack cytochromes a + a3 and b are as a rule light resistant. Photokilling effect in yeast seems to be dependent both on the sufficiency of respiratory chain and on protein synthesis probably on cytoplasmic level.     

A new method of optical detection of yeast acidification power

We describe here a newly developed method for a contact-free optical pH measurement in yeast suspensions supplemented with glucose, and containing the pH sensitive triphenylmethane dye bromocresol green. It is suitable for performing the acidification power test (based on measuring the rate of pH drop of yeast suspension caused by active extrusion of acidity from cells after glucose addition) used for assessing yeast vitality in fermentation industries. Using this methodology we monitored the pH in yeast suspensions in the course of acidification in the pH range of 3.5–5.3. Optical pH measurement allows simultaneous testing of several samples, minimizes the sample volume, simplifies sample handling and reduces the hands-on time in sample processing.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Simple detection method for distinguishing dead and living yeast colonies

A rapid and simple assay was developed for detection of yeast colonies containing dying or dead cells. Methylene blue, phloxin B, rose bengal and trypan blue at concentrations of 5-10 micromol l(-1) were shown to stain non-viable cells in colonies of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Filobasidium capsuligenum without staining or affecting the viability of living cells of the colonies.     

Aminoacylation and conformational properties of yeast mitochondrial tRNA mutants with respiratory deficiency

We report the identification and characterization of eight yeast mitochondrial tRNA mutants, located in mitochondrial tRNA(Gln), tRNA(Arg2), tRNA(Ile), tRNA(His), and tRNA(Cys), the respiratory phenotypes of which exhibit various degrees of deficiency. The mutations consist in single-base substitutions, insertions, or deletions, and are distributed all over the tRNA sequence and structure. To identify the features responsible for the defective phenotypes, we analyzed the effect of the different mutations on the electrophoretic mobility and efficiency of acylation of the mutated tRNAs in comparison with the respective wild-type molecules. Five of the studied mutations determine both conformational changes and defective acylation, while two have neither or limited effect. However, variations in structure and acylation are not necessarily correlated; the remaining mutation affects the tRNA conformation, but not its acylation properties. Analysis of tRNA structures and of mitochondrial and cytoplasmic yeast tRNA sequences allowed us to propose explanations for the observed defects, which can be ascribed to either the loss of identity nucleotides or, more often, of specific secondary and/or tertiary interactions that are largely conserved in native mitochondrial and cytoplasmic tRNAs.     

Lecithin agar for detection of microbial phospholipases.

Lecithin agar was developed on which phospholipase C produced turbid zones and phospholipase A produced clear zones. Reactions on lecithin agar agreed 74% of the time with reactions in egg yolk broth. On lecithin agar, interpretation was easier, phospholipase A was detectable, and opaque zones were visible 1 or 2 days earlier than on egg yolk agar. All constituents of the medium can be autoclaved.     

A facile strategy for nonenzymatic glucose detection

Glassy carbon electrode modified with boron oxide nanoparticles supported on multiwall carbon nanotubes was obtained via a facile approach. The as-prepared modified electrode exhibits excellent electrocatalytic activity toward the redox of glucose in pH 7.0 phosphate buffer solution. The electrochemical response of the modified electrode to glucose shows a linear range of 1.5-260 μM with a correlation coefficient of 0.9986 and the calculated detection limit is 0.8 μM at a signal-to-noise ratio of 3, which makes it useful for developing the electrochemical determination of glucose concentrations without using glucose oxidase at physiological pH.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.