The use of triphenyltetrazolium chloride in the study of dehydrogenase activity of Brucellae

Experiments for the investigation of dehydrogenase activity of washed cells of a strains of Br. abortus and another of Br. suis in presence of different single added substrates are reported. The activity was measured as the amount of formazan produced by the reduction of 2, 3, 5-triphenyltetrazolum chloride acting as a hydrogen ions acceptor, at pH 7.0. In a general manner the dehydrogenase activity of Br. suis was much more intense than that of Br. abortus (fig. 5). In the conditions of the experiments Br. abortus oxidized L-arabinose, D-galactose, D-glucose, glycerol, D-xylose, DL-alanine, D-fructose, and D-sorbitol. Brucella suis oxidized D-xylose, L-arabinose, D-glucose, D-galactose, DL-alanine, sodium acetate, maltose, glycine, D-fructose, and D-sorbitol. Glycerol was oxidized by Br. abortus but its oxidation by Br. suir was very slight. Sodium acetate and maltose were intensely oxidized by Br. suir but not by Br. abortus. The sites of more intense enzymatic acitivity were seen as small red colored round granules located in one pole of the cells.

Performance of Petrifilm Aerobic Count plates on enumeration of lactic acid bacteria in fermented milks

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using the de Man-Rogosa-Sharpe (MRS) agar for the enumeration of lactic acid bacteria (LAB) in fermented milks (FMs), with different starter cultures added. FM samples (n = 66) were collected and plated on both methodologies, with incubation under anaerobic conditions at 35C for 48 h. The count results were compared by analysis of variance (P <= 0.05) and regression analysis. No differences between the mean counts obtained by both methodologies were observed, even when distinct FMs were compared. Considering all samples, a high correlation level was obtained between Petrifilm AC and MRS agar (r = 0.92), but these indexes were lower in FMs with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (r = 0.90) and Lactobacillus fortis (r = 0.81). Despite some slight interferences, Petrifilm AC has proven to be a convenient methodology on enumerating LAB in FM.

In vitro pollen germination and pollen viability in passion fruit (Passiflora spp.)

The use of Passiflora species for ornamental purposes has been recently developed, but little is known about pollen viability and the potential for crossing different species. The objective of this study was to evaluate the pollen viability of six Passiflora species collected from different physiological stages of development through in vitro germination and histochemical analysis using dyes. The pollen was collected in three stages (pre-anthesis, anthesis and post-anthesis). Three compositions of culture medium were used to evaluate the in vitro germination, and two dyes (2,3,5-triphenyltetrazolium chloride, or TTC, and Lugol's solution) were used for the histochemical analysis. The culture medium containing 0.03% Ca(NO3) 4H2O, 0.02% of Mg(SO4 ).7H2O, 0.01% of KNO3, 0,01% of H3BO3, 15% sucrose, and 0.8% agar, pH 7.0, showed a higher percentage of pollen grains germinated. Anthesis is the best time to collect pollen because it promotes high viability and germination. The Lugol's solution and TTC dye overestimated the viability of pollen, as all accessions showed high viability indices when compared with the results obtained in vitro.

Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain

A gravimetric method was evaluated as a simple, sensitive, reproducible, low-cost alternative to quantify the extent of brain infarct after occlusion of the medial cerebral artery in rats. In ether-anesthetized rats, the left medial cerebral artery was occluded for 1, 1.5 or 2 h by inserting a 4-0 nylon monofilament suture into the internal carotid artery. Twenty-four hours later, the brains were processed for histochemical triphenyltetrazolium chloride (TTC) staining and quantitation of the schemic infarct. In each TTC-stained brain section, the ischemic tissue was dissected with a scalpel and fixed in 10% formalin at 0ºC until its total mass could be estimated. The mass (mg) of the ischemic tissue was weighed on an analytical balance and compared to its volume (mm³), estimated either by plethysmometry using platinum electrodes or by computer-assisted image analysis. Infarct size as measured by the weighing method (mg), and reported as a percent (%) of the affected (left) hemisphere, correlated closely with volume (mm³, also reported as %) estimated by computerized image analysis (r = 0.88; P < 0.001; N = 10) or by plethysmography (r = 0.97-0.98; P < 0.0001; N = 41). This degree of correlation was maintained between different experimenters. The method was also sensitive for detecting the effect of different ischemia durations on infarct size (P < 0.005; N = 23), and the effect of drug treatments in reducing the extent of brain damage (P < 0.005; N = 24). The data suggest that, in addition to being simple and low cost, the weighing method is a reliable alternative for quantifying brain infarct in animal models of stroke.

Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications

Triphenyltetrazolium chloride (TTC) staining and echocardiography (ECHO) are methods used to determine experimental myocardial infarction (MI) size, whose practical applicability should be expanded. Our objectives were to analyze the accuracy of ECHO in determining infarction size in rats during the first days following coronary occlusion and to test whether a simplified single measurement by TTC correctly indicates MI size, as determined by the average value for multiple slices. Infarction was induced in female Wistar rats by coronary artery occlusion and MI size analysis was performed after the acute (7th day) and chronic periods (after 4 weeks) by ECHO matched with TTC. ECHO and TTC showed similar values of MI size (% of left ventricle perimeter) in acute (ECHO: 33 ± 11, TTC: 35 ± 14) and chronic (ECHO: 38 ± 14, TTC: 39 ± 13 periods), and also presented an excellent correlation (r = 0.92, P < 0.001). Although measurements from different heart planes showed discrepancies, a single measurement acquired from the mid-ventricular level by TTC was a good estimate of MI size calculated by the average of multiple planes, with minimal disagreement (Bland-Altman test with mean ratio bias of 0.99 ± 0.07) and close to an ideal correlation (r = 0.99, P < 0.001). In the present study, ECHO was confirmed as a useful method for the determination of MI size even in the acute phase. Also, the single measure of a mid-ventricular section proposed as a simplification of the TTC method is a satisfactory prediction of average MI extension.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

A forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model

We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P<0.05) and similar to the treadmill group. The experimental data demonstrated that the proposed platform can be applied to test the benefit of exercise-preconditioning-induced neuroprotection using the animal stroke model. Additional advantages of the FNESRW system include stand-alone capability, independence of subjective human adjustment, and ease of use.

In vitro evaluation of the antimicrobial activity of a castor oil-based irrigant

The antimicrobial activity of irrigating solutions - Endoquil (castor oil detergent), 2% chlorhexidine gluconate solution, and 0.5% NaOCI solution - was evaluated against Gram-positive cocci (Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus sobrinus), Gramnegative rods (Escherichia coli and Pseudomonas aeruginosa), and the yeast Candida albicans. Activity was evaluated using the two-layer agar diffusion technique. The base layer was obtained by pouring 10.0 ml of Muller Hinton Medium or 10.0 ml of Brain Heart Infusion agar in a Petri dish. After solidification a 5.0 ml seed layer of Muller Hinton Medium or Brain Heart Infusion agar with inoculum (106/ml) was added. Absorbent paper disks (6.0 mm in diameter) immersed in the solutions were placed at equidistant points. Plates were maintained at room temperature for 2 h for prediffusion of the solutions and incubated at 37°C for 24 h. The candle jar system was used for the Brain Heart Infusion agar plates. All tests were performed in duplicate. After incubation the medium was optimized with 0.05 g% triphenyltetrazolium chlorate gel and inhibition halos were measured. All bacterial strains were inhibited by 2.0% chlorhexidine gluconate. Endoquil was effective against Grampositive microorganisms, and 0.5% NaOCI was effective only against S. aureus. Copyright © 2001 by The American Association of Endodontists.

In vitro antimicrobial activity of different gutta-percha points and calcium hydroxide pastes

The aim of this study was to evaluate the antimicrobial activity of different trademarks and compositions of gutta-percha points and calcium hydroxide pastes used in endodontic therapy. The evaluated material consisted of gutta-percha points containing calcium hydroxide (Roeko™), gutta-percha points containing chlorhexidine (Roeko™), two convencional gutta-percha points (Endo Points™ and Roeko™) and two calcium hydroxide pastes (Calen™ and Calen/PMCC™). Antimicrobial tests included five species of microorganisms: Escherichia coli (ATCC10538), Staphylococcus epidermidis (ATCC12228), Staphylococcus aureus (ATCC6538), Pseudomonas aeruginosa (ATCC27853), and Micrococcus luteus (ATCC9341). The Agar difusion method was employed. The plates were kept at room temperature for 2 h for prediffusion and then incubated at 37°C for 24 h. The triphenyltetrazolium chloride gel was added for optimization and the zones of inhibition were measured. Statistical evaluation was carried out using analysis of variance and Tukey Test. The obtained results showed that all microbial species used in the study were inhibited by the gutta-percha points containing chlorhexidine and by the calcium hydroxide pastes (Calen™ and Calen/ PMCC™), with similar results (p > 0.05). No antimicrobial activity was observed for the other groups. It was concluded that the gutta-percha points containing chlorhexidine presented antimicrobial activity, whereas the gutta-percha points containing calcium hydroxide did not.

In vitro antimicrobial activity of endodontic sealers, MTA-based cements and Portland cement.

The aim of this study was to evaluate the antimicrobial activity of different root-end filling materials - Sealer 26, Sealapex with zinc oxide, zinc oxide and eugenol, white and gray Portland cement, white and gray MTA-Angelus, and gray Pro Root MTA - against six different microorganism strains. The agar diffusion method was used. A base layer was made using Müller-Hinton agar (MH) and wells were formed by removing the agar. The materials were placed in the wells immediately after manipulation. The microorganisms used were: Micrococcus luteus (ATCC9341), Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC10538), Pseudomonas aeruginosa (ATCC27853), Candida albicans (ATCC 10231), and Enterococcus faecalis (ATCC 10541). The plates were kept at room temperature for 2 h for prediffusion and then incubated at 37 degrees C for 24 h. Triphenyltetrazolium chloride 0.05% gel was added for optimization, and the zones of inhibition were measured. Data were subjected to the Kruskal-Wallis and Dunn tests at a 5% significance level. The results showed that all materials had antimicrobial activity against all the tested strains. Analysis of the efficacy of the materials against the microbial strains showed that Sealapex with zinc oxide, zinc oxide and eugenol and Sealer 26 created larger inhibition halos than the MTA-based and Portland cements (P < 0.05). On the basis of the methodology used, it may be concluded that all endodontic sealers, MTA-based and Portland cements evaluated in this study possess antimicrobial activity, particularly the endodontic sealers.

Aplicabilidade do TTC para a detecção de bacteriúria

Laboratory tests to detect urinary infection at a lawer cost than uroculture are necessary. Triphenyl tetrazolium chloride was evaluated in parallel with uroculture in 342 urine samples, for the detection of significant bacteriuria. The results showed that the test has high sensitivity (91.3%) but low specificity (64.3%), and negative predictive value 99.0%. Although the test cannot replace uroculture as a diagnostic method, it can be recommended for preliminary screening, and could eliminate the need for uroculture of negative samples.

Controle rápido da eficiência e segurança do processo de pasteurização do leite (HTST - High Temperature Short Time)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Atividade antimicrobiana in vitro de extratos hidroetanólicos de Astronium sp incorporados ou não em sistemas nanoestruturados

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tetrazolium test for assessment of seed viability of atemoya (Annona cherimola Mill. x A. squamosa L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do potencial biológico de extratos hidroetanólicos de Astronium sp incorporados ou não em sistemas nanoestruturados: in vitro e in vivo: in vitro e in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)