Effect of hydroalcoholic extract of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on the viability and activity of microcosm biofilm and on enamel demineralization

Abstract Objectives: The aim of this study was to assess the effect of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves hydroalcoholic extracts on viability and metabolism of a microcosm biofilm and on enamel demineralization prevention. Methodology: Microcosm biofilm was produced on bovine enamel using inoculum from pooled human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. The biofilm was daily-treated with the extracts for 1 min. At the end, it was analyzed with respect to viability by fluorescence, CFU counting and extracellular polysaccharides (phenol-sulphuric acid colorimetric assay) and lactic acid (enzymatic assay) production. The demineralization was measured by TMR. The data were compared using ANOVA or Kruskal-Wallis (p<0.05). Results: M. urundeuva All. at 100, 10 and 0.1 μg/mL and Q. grandiflora Mart. at 100 and 0.1 μg/mL reduced biofilm viability similarly to positive control (chlorhexidine) and significantly more than the negative-vehicle control (35% ethanol). M. urundeuva at 1000, 100 and 0.1 μg/mL were able to reduce both lactobacilli and mutans streptococci CFU counting, while Q. grandiflora (1000 and 1.0 μg/mL) significantly reduced mutans streptococci CFU counting. On the other hand, the natural extracts were unable to significantly reduce extracellular polysaccharides and lactic acid productions neither the development of enamel carious lesions. Conclusions: The extracts showed antimicrobial properties on microcosm biofilm, however, they had no effect on biofilm metabolism and caries protection.

Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite

Abstract The aim of this study was evaluated the biofilm formation by Staphylococcus aureus 4E and Salmonella spp. under mono and dual-species biofilms, onto stainless steel 316 (SS) and polypropylene B (PP), and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. The biofilms were developed by immersion of the surfaces in TSB by 10 d at 37 °C. The results showed that in monospecies biofilms the type of surface not affected the cellular density (p > 0.05). However, in dual-species biofilms on PP the adhesion of Salmonella spp. was favored, 7.61 ± 0.13 Log10 CFU/cm2, compared with monospecies biofilms onto the same surface, 5.91 ± 0.44 Log10 CFU/cm2 (p < 0.05). The mono and dual-species biofilms were subjected to disinfection treatments; and the most effective disinfectant was peracetic acid (3500 ppm), reducing by more than 5 Log10 CFU/cm2, while the least effective was cetrimonium bromide. In addition, S. aureus 4E and Salmonella spp. were more resistant to the disinfectants in mono than in dual-species biofilms (p < 0.05). Therefore, the interspecies interactions between S. aureus 4E and Salmonella spp. had a negative effect on the antimicrobial resistance of each microorganism, compared with the monospecies biofilms.

Development and characterization of an innovative synbiotic fermented beverage based on vegetable soybean

Abstract Soymilk was produced from vegetable soybean and fermented by probiotics (Lactobacillus acidophilus La-5, Bifidobacterium animalis Bb-12) in co-culture with Streptococcus thermophilus. The composition of the fermented beverage and oligosaccharides content were determined. The effect of fructooligosaccharides and inulin on the fermentation time and viability of probiotic microorganisms throughout 28 days of storage at 5 °C were evaluated. The soymilk from vegetable soybeans was fermented in just 3.2 h, when pH reached 4.8. Fermentation reduced the contents of stachyose and raffinose in soymilk. Prebiotics had no effect on acidification rate and on viability of B. animalis and S. thermophilus in the fermented beverage. The viable counts of B. animalis Bb-12 remained above 108 CFU mL-1 in the fermented soymilk during 28 days of storage at 5 °C while L. acidophilus La-5 was decreased by 1 log CFU mL-1. The fermented soymilk from vegetable soybeans showed to be a good food matrix to deliver probiotic bacteria, as well as a soy product with a lower content of non-digestible oligosaccharides.

Removal and killing of multispecies endodontic biofilms by N-acetylcysteine

ABSTRACT Removal of bacterial biofilm from the root canal system is essential for the management of endodontic disease. Here we evaluated the antibacterial effect of N-acetylcysteine (NAC), a potent antioxidant and mucolytic agent, against mature multispecies endodontic biofilms consisting of Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans and Enterococcus faecalis on sterile human dentin blocks. The biofilms were exposed to NAC (25, 50 and 100 mg/mL), saturated calcium hydroxide or 2% chlorhexidine solution for 7 days, then examined by scanning electron microscopy. The biofilm viability was measured by viable cell counts and ATP-bioluminescence assay. NAC showed greater efficacy in biofilm cell removal and killing than the other root canal medicaments. Furthermore, 100 mg/mL NAC disrupted the mature multispecies endodontic biofilms completely. These results demonstrate the potential use of NAC in root canal treatment.

Effect of γ-lactones and γ-lactams compounds on Streptococcus mutans biofilms

Abstract Considering oral diseases, antibiofilm compounds can decrease the accumulation of pathogenic species such as Streptococcus mutans at micro-areas of teeth, dental restorations or implant-supported prostheses. Objective To assess the effect of thirteen different novel lactam-based compounds on the inhibition of S. mutans biofilm formation. Material and methods We synthesized compounds based on γ-lactones analogues from rubrolides by a mucochloric acid process and converted them into their corresponding γ-hydroxy-γ-lactams by a reaction with isobutylamine and propylamine. Compounds concentrations ranging from 0.17 up to 87.5 μg mL-1 were tested against S. mutans. We diluted the exponential cultures in TSB and incubated them (37°C) in the presence of different γ-lactones or γ-lactams dilutions. Afterwards, we measured the planktonic growth by optical density at 630 nm and therefore assessed the biofilm density by the crystal violet staining method. Results Twelve compounds were active against biofilm formation, showing no effect on bacterial viability. Only one compound was inactive against both planktonic and biofilm growth. The highest biofilm inhibition (inhibition rate above 60%) was obtained for two compounds while three other compounds revealed an inhibition rate above 40%. Conclusions Twelve of the thirteen compounds revealed effective inhibition of S. mutans biofilm formation, with eight of them showing a specific antibiofilm effect.

Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel

Abstract Titanium tetrafluoride (TiF4) is known for interacting with enamel reducing demineralization. However, no information is available about its potential antimicrobial effect. Objectives This study evaluated the antimicrobial and anti-caries potential of TiF4 varnish compared to NaF varnish, chlorhexidine gel (positive control), placebo varnish and untreated (negative controls) using a dental microcosm biofilm model. Material and Methods A microcosm biofilm was produced on bovine enamel previously treated with the varnishes, using inoculum from human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. All experiments were performed in biological triplicate (n=4/group in each experiment). Factors evaluated were: bacterial viability (% dead and live bacteria); CFU counting (log10 CFU/mL); and enamel demineralization (transverse microradiography - TMR). Data were analysed using ANOVA/Tukey's test or Kruskal-Wallis/Dunn's test (p<0.05). Results Only chlorhexidine significantly increased the number of dead bacteria (68.8±13.1% dead bacteria) compared to untreated control (48.9±16.1% dead bacteria). No treatment reduced the CFU counting (total microorganism and total streptococci) compared to the negative controls. Only TiF4 was able to reduce enamel demineralization (ΔZ 1110.7±803.2 vol% μm) compared to both negative controls (untreated: ΔZ 4455.3±1176.4 vol% μm). Conclusions TiF4 varnish has no relevant antimicrobial effect. Nevertheless, TiF4 varnish was effective in reducing enamel demineralization under this model.

In vitro analysis of a local polymeric device as an alternative for systemic antibiotics in Dentistry

Abstract The development of a biodegradable material with antimicrobial properties for local applications is required in the prevention and treatment of infectious diseases. The objective of this study was to produce blends of poly-L-lactide acid (PLLA) synthetic polymer associated with several antimicrobials, as an alternative in the prevention and treatment of infections, as well as to evaluate its cytotoxicity, release of antimicrobials and inhibit bacteria growth. Blends of PLLA added with 20% Amoxicillin, Metronidazole, Clindamycin or Azithromicyn were used to produce Films (F) or Meshs (M) by casting and electrospinning methods, respectively. Standardized discs of the films and meshs were stored in buffer solutions (pH 5 or 7.4) and aliquots were analyzed by high performance chromatography (HPLC) during 168 hours. Cytotoxicity on human gingival fibroblasts was tested after 24, 48 and 72h by MTT reaction. The antimicrobial capacity was determined against P. gingivalis and S. pyogenes. The specimens were weighed after 3 and 6 months of storage for degradation analysis. SEM was performed to control interfaces and degradation. Antimicrobials presented a continuous and exponential drug release. Analysis showed that both M and F were able to inhibit S. pyogenes and P. gingivalis growth, indicating the release of active antimicrobial agents. The products were not toxic to the fibroblasts. Amoxicillin-film showed more degradation than PLLA at both pHs (p < 0.05), whereas Azithromycin-meshes were more degraded than PLLA at pH 7.4 (p < 0.05). PLLA association with antimicrobials is biocompatible and may represent a potential tool for the local delivery of antimicrobials.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment

ABSTRACT Objective The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. Material and Methods Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. Results The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). Conclusion The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Exopolysaccharide dispelled by calcium hydroxide with volatile vehicles related to bactericidal effect for root canal medication

ABSTRACT Objective: Enterococcus faecalis is the dominant microbial species responsible for persistent apical periodontitis with ability to deeply penetrate into the dentin. Exopolysaccharides (EPS) contribute to the pathogenicity and antibiotic resistance of E. faecalis. Our aim was to investigate the antimicrobial activity of calcium hydroxide (CH), camphorated parachlorophenol (CMCP), and chlorhexidine (CHX) against E. faecalis in dentinal tubules. Material and Methods: Decoronated single-canal human teeth and semicylindrical dentin blocks were incubated with E. faecalis for 3 weeks. Samples were randomly assigned to six medication groups for 1 week (n=10 per group): CH + 40% glycerin-water solution (1:1, wt/vol); CMCP; 2% CHX; CH + CMCP (1:1, wt/vol); CH + CMCP (2:3, wt/vol); and saline. Bacterial samples were collected and assayed for colony-forming units. After dentin blocks were split longitudinally, confocal laser scanning microscopy was used to assess the proportion of viable bacteria and EPS production in dentin. Results: CMCP exhibited the best antimicrobial activity, while CH was the least sensitive against E. faecalis (p<0.05). CHX showed similar antimicrobial properties to CH + CMCP (1:1, wt/vol) (p>0.05). CH combined with CMCP inhibited EPS synthesis by E. faecalis, which sensitized biofilms to antibacterial substances. Moreover, increasing concentrations of CMCP decreased EPS matrix formation, which effectively sensitized biofilms to disinfection agents. Conclusion: The EPS matrix dispelled by CH paste with CMCP may be related to its bactericidal effect; the visualization and analysis of EPS formation and microbial colonization in dentin may be a useful approach to verify medicaments for antimicrobial therapy.

Inactivation of Listeria monocytogenes ATCC 7644 on fresh-cut tomato using nisin in combinations with organic salts

ABSTRACT The inhibition of Listeria monocytogenes ATCC 7644 on fresh-cut tomato was investigated using nisin alone, and in combinations with organic salts. Nisin at a concentration of 5000 UI/mL was introduced alone or in combination with an organic salt (sodium citrate or sodium acetate each at 3 and 5 g/100 mL each) on fresh-cut tomato previously inoculated with 108 CFU/mL of L. monocytogenes ATCC 7644. Chlorine at 200 ppm was used as a control. The inoculated samples were incubated at different temperatures (4, 10 and 25 °C) and examined at 0, 24, 48 and 72 h. The effects of the antimicrobial treatments on quality parameters of tomato (pH, soluble solids, titratable acidity and vitamin C) were also evaluated, and colour parameters were observed at the lowest storage temperature for 10 days. Both nisin and the organic salts inhibited growth of L. monocytogenes, but the combinations of two compounds were more effective. The nisin-sodium citrate (5%) combination was significantly (p ≤ 0.05) effective, while chlorine was least effective against L. monocytogenes. The quality parameters were substantially retained, especially at 4 °C, suggesting good shelf stability at a low temperature. These results substantiate the use of the cheap and eco-friendly approach to reducing this pathogen of health concern in common fresh produce.

NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying

Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.

Analysis of the reaction of subcutaneous tissues in rats and the antimicrobial activity of calcium hydroxide paste used in association with different substances

The aim of this study was to evaluate the subcutaneous tissue response in rats and the antimicrobial activity of intracanal calcium hydroxide dressings mixed with different substances against E. faecalis. Fifty four rats were divided into three experimental groups according to the vehicle in the calcium hydroxide treatment: 0.4% chlorohexidine in propylene glycol (PG),Casearia sylvestris Sw in PG and calcium hydroxide+PG (control group). The pastes were placed into polyethylene tubes and implanted into the subcutaneous tissue. After 7, 14 and 30 days, the samples were processed and histologically evaluated (hematoxylin and eosin). The tissue surface in contact with the material was analyzed, and the quantitative analysis determined the volume density occupied by the inflammatory infiltrate (giant cells, polymorphonuclear cells and mononuclear cells), fibroblasts, collagen fibers and blood vessels. For the antimicrobial analysis, 20 dentin blocks infected with E. faecalis were treated with calcium hydroxide pastes in different vehicles; 0.4% chlorhexidine in PG, PG, extract fromCasearia sylvestris Sw in PG and a positive control (infection and without medication) for 7 days. The efficiency of the pastes was evaluated by the live/dead technique and confocal microscopy. The results showed that 0.4% chlorhexidine induced a higher inflammatory response than the other groups. The Casearia sylvestris Sw extract showed satisfactory results in relation to the intensity of the inflammatory response. In the microbiological test, there were no statistical differences between the evaluated intracanal dressings and the percentage of bacterial viability was between 33 and 42%. The control group showed an 86% viability. Antimicrobial components such as chlorhexidine or Casearia sylvestris Sw did not improve the antimicrobial activity against E. faecalis in comparison to the calcium hydroxide+PG treatment. In addition, the incorporation of chlorhexidine in the calcium hydroxide paste promoted the highest inflammatory response.

Differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis

The aim of this study was to determine the differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis. Thirty-two strains of Moraxella spp. isolated from cattle and sheep with infectious keratoconjunctivitis were tested via broth microdilution method to determine their susceptibility to ampicillin, cefoperazone, ceftiofur, cloxacillin, enrofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline and penicillin. The results demonstrated that Moraxella spp. strains could be considered sensitive for most of the antimicrobials tested in this study, but differences between the antimicrobial susceptibility profiles of these three Moraxella species were found. M. bovis might differ from other species due to the higher MIC and MBC values it presented.

Characteristics of dr1790 disruptant and its functional analysis in Deinococcus radiodurans

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.

Evaluation of some pharmacological activities of Budmunchiamine - A isolated from Albizia amara

The present investigations were aimed to evaluate the antimicrobial and antioxidant efficacies of budmunchiamine-A (BUA) of Albizia amara. The activity-guided isolation leaded to isolate the bioactive compound budmunchiamine-A from alkaloid extract of A. amara. The budmunchiamine-A showed significant broad-spectrum antimicrobial activity with zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values varied from 7.3 to 24.5 mm, 0.95 to 62.5 μg/mL, and 1.9 to 250 μg/mL, respectively. The budmunchiamine-A exhibited moderate antioxidant activity with inhibitory concentration 50% (IC50) value of 400 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and percent inhibition of β-carotene/linoleic acid was 67.8%. The results suggest the possible use of budmunchiamine-A as a molecular entity for drug development in pharmaceutical industry.

Different methods to quantify Listeria monocytogenes biofilms cells showed different profile in their viability

Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR). Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI), and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05). When, viability dyes (CTC/DAPI) combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05). Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Antiplasmodial Potential of Traditional Medicinal Plant Thlaspi arvense.

Aim: Antiplasmodial potential of traditional medicinal plant Thlaspi arvense against Plasmodium falciparum in vitro has been evaluated. Cytotoxicity of plant extract against HeLa cell lines and normal fibroblasts has also been observed. Place and Duration of the Study: Department of Zoology, Panjab University, Chandigarh, India, between May 2013 to April 2014. Materials and Methods: Ethanolic whole plant extract of Thlaspi arvense (EWETA) was analyzed for its phytochemical constituents. In vitro cytotoxicity was determined colorimetrically by MTT assay. WHO protocol, based on assessment of schizont maturation inhibition, was employed for the evaluation of in vitro antiplasmodial activity of plant extract. Results: Phytochemical screening of EWETA revealed the presence of diterpenes, triterpenes, steroids, anthraquinones and phytosterols. EWETA was observed to inhibit schizont maturation of both chloroquine-sensitive (MRC-2) and resistant (RKL-9) strains of P. falciparum with IC50<5μg/ml and =5μg/ml respectively. The extract was revealed to be safe against both HeLa cells and normal fibroblasts with CC50>1000μg/ml. Selectivity index for Thlaspi arvense was calculated to be >200 and =200 both for chloroquine sensitive and chloroquine-resistant strains of P. falciparum with both HeLa and normal fibroblasts. Conclusion: Plant extract possesses considerable in vitro antimalarial activity with high selectivity index (SI>10) pointing field pennycress to be an active antimalarial. Hence, present study provides scientific evidence for traditional usage of the plant as an antipyretic agent.

Antibacterial activity of α-terpineol may induce morphostructural alterations in Escherichia coli

The antibacterial effect of α-terpineol from Cinnamomum longepaniculatum (Gamble) N. Chao leaf essential oils were studied with special reference to the mechanism of inhibiting the standard strain of Escherichia coli (CMCC (B) 44102) growth at ultrastructural level. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) and time-kill curves of α-terpineol were determined; Escherichia coli was treated with α-terpineol and observed under a transmission electron microscope. The MIC and MBC values of α-terpineol were all 0.78 µL/mL, and time-kill curves showed the concentration-dependent. Under the transmission electron microscopy (TEM), Escherichia coli exposed to MIC levels of α-terpineol exhibited decreased cell size and irregular cell shape, cell wall and cell membrane were ruptured, nucleus cytoplasm was reduced and nuclear area gathered aside. Results suggest that α-terpineol has excellent antibacterial activity and could induce morphological changes of Escherichia coli.

Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability. .