scholarly journals A modified crystal violet assay is comparable to XTT reduction assay for quantification of biofilm formation by Candida albicans

2020 ◽  
Keyword(s):  
Candida Albicans ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Crystal Violet Assay ◽  
Reduction Assay

Abstract The authors have requested that this preprint be withdrawn due to a need to make corrections.

scholarly journals A modified crystal violet assay is comparable to XTT reduction assay for quantification of biofilm formation by Candida albicans

2020 ◽  
Author(s):  
Yue Qu ◽  
Shoufeng Yang ◽  
Zhangzhang Chen ◽  
Feifei Su
Keyword(s):  
Candida Albicans ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Quantitative Methods ◽  
Standard Procedure ◽  
Short Term ◽  
Biofilm Growth ◽  
Human Fungal Pathogen ◽  
Crystal Violet Assay ◽  
Reduction Assay

Abstract Background: The ability of the human fungal pathogen Candida albicans to form biofilms, for example on indwelling medical devices, is a major pathogenic mechanism and has been the focus of intense studies in the fungal pathogenesis field. A key research tool used is the quantitative methods for measuring biofilm formation of C. albicans. Objective: We sought to optimize the conventional crystal violet (CV) staining assay for quantification of biofilm formation by C. albicans and evaluate its performance. Methods: Individual modifications included (i) submerge-washing of microplates to remove non-adherent cells, (ii) heat-fixation, (iii) short-term staining for 3 min, (iv) submerge-washing to remove unbound CV dye, and (v) short-term destaining for 15 min were compared with the standard procedure, and those were superior were incorporated. Performance analysis was carried out for the modified CV assay, in comparison to the conventional CV assay and the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide] reduction assay. Results: The modified CV assay demonstrated several advantages in quantitative assessment of biofilm formation of C. albicans over the conventional CV assay, including greater accuracy and reproducibility, shorter experimental time and reduced labor intensity, and was at least comparable to the XTT reduction assay.Conclusion: The modified CV method can be used as an alternative to the XTT reduction assay in quantification of biofilm growth by C. albicans.

scholarly journals Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans

2021 ◽  
Author(s):  
Harikrishnan Pandurangan ◽  
Balamani Arayambath ◽  
Vijay Karthik Jayaraman ◽  
Kanimozhi Ekambaram ◽  
Emad A Ahmed ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Reduction Assay ◽  
Ergosterol Synthesis ◽  
Inhibitory Potential ◽  
Anti Fungal ◽  
Time Kill

Abstract Candida albicans is a commensal human fungal pathogen that colonizes and develops dental biofilm which cause Oral candidiosis. This study investigates the effects of a new molecule Thidiazuron against the growth and biofilm formation properties of C. albicans. This study applied computational and in vitro approaches such as broth microdilution, SEM, time-kill dynamics, crystal violet assay, XTT reduction assay, ergosterol quantification and quantitative RT PCR analysis of gene expression to validate the growth and biofilm inhibitory potential of thidiazuron against C. albicans. Preliminary molecular docking study revealed potential interaction between thidiazuron and amino acids residues of CYP51. Further in vitro anti-fungal susceptibility test, SEM and time kill analysis revealed anti-fungal potency of thidiazuron in dose and time dependent passion. Crystal violet staining, XTT reduction assay and Acridine Orange staining visually confirmed biofilm inhibitory potential of thidiazuron. Gene expression study shows that thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis, cell adhesion and hyphae development in C. albicans. This study identified thidiazuron as CYP51 inhibitor and a new antibiofilm agent against C. albicans.

scholarly journals Green Tea Polyphenols and Padma Hepaten Inhibit Candida albicans Biofilm Formation

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Yosi Farkash ◽  
Mark Feldman ◽  
Isaac Ginsburg ◽  
Doron Steinberg ◽  
Miriam Shalish
Keyword(s):  
Candida Albicans ◽  
Green Tea ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Host Cells ◽  
Green Tea Polyphenols ◽  
Crystal Violet Staining ◽  
Biofilm Inhibition ◽  
Therapeutic Concept ◽  
Biofilm Development

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

scholarly journals Improvement of XTT assay performance for studies involving Candida albicans biofilms

2008 ◽  
Vol 19 (4) ◽  
pp. 364-369 ◽  
Author(s):  
Wander José da Silva ◽  
Jayampath Seneviratne ◽  
Nipuna Parahitiyawa ◽  
Edvaldo Antonio Ribeiro Rosa ◽  
Lakshman Perera Samaranayake ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Biofilm Formation ◽  
Cell Activity ◽  
Growth Yield ◽  
Xtt Assay ◽  
Biofilm Growth ◽  
Assay Performance ◽  
Reduction Assay ◽  
Cell Layers ◽  
Biofilm Development

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†

2020 ◽  
Vol 58 (7) ◽  
pp. 887-895 ◽  
Author(s):  
Judith Díaz-García ◽  
Maiken C Arendrup ◽  
Rafael Cantón ◽  
Julio García-Rodríguez ◽  
Ana Gómez ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Candida Spp ◽  
Biofilm Production ◽  
Hospital Environments ◽  
Marked Variability ◽  
Inert Surfaces ◽  
Biofilm Development

Abstract The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries—and the same number of singleton genotypes used as controls—were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters—particularly in the case of C. albicans—show significantly more biomass production and metabolic activity than singleton genotypes.

scholarly journals The Effect of Oral Probiotic Streptococcus Salivarius K12 on Candida Albicans Biofilm Formation

2020 ◽  
Vol 18 (2) ◽  
Author(s):  
Munirah Mokhtar ◽  
Alia Risma Rismayuddin ◽  
Ridhwan Abdul Wahab ◽  
Muhamad Ashraf Rostam ◽  
Mohd Hamzah Mohd Nasir ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Oral Cancer ◽  
Biofilm Formation ◽  
Streptococcus Salivarius ◽  
Yeast Form ◽  
Cell Numbers ◽  
Crystal Violet Assay ◽  
Common Cancer ◽  
The Media ◽  
Candida Albicans Infection

Introduction:  Oral cancer is the sixth most common cancer worldwide with  Candida albicans  infection being one of the aetiological factors for the disease. Meanwhile,  Streptococcus salivarius  K12 is an oral probiotic that is beneficial to the oral cavity. The objective of the present study is to determine the effect of  S. salivarius  K12 on  C. albicans  biofilm-forming ability with the hypothesis that  S. salivarius  K12 inhibits biofilm formation of  C. albicans  Materials and method: To assess the effect of  S. salivarius  K12 on  C. albicans  biofilm formation,  S. salivarius  K12, lab strain  C. albicans  MYA-4901 and clinical isolates from oral cancer, ALC2 and ALC3 were grown in both nutrient broth (NB) and RPMI. In a mono-species biofilm, 105 of  C. albicans  cells and 106 of  S. salivarius  K12 cells were grown separately in a 96-well plate. In contrast, both microorganisms were combined for polymicrobial biofilms with similar cell numbers as in mono-species. The biofilms were incubated for 72 hours at 37°C and the media were replenished every 24 hours. Finally, the crystal violet assay was conducted, and the optical density was measured at OD620nm.  Results: Polymicrobial biofilms of  C. albicans  (MYA-4901 and ALC3) with  S. salivarius K12 when grown in NB, exhibited a decrease by 64.5 ± 25.8% and 83.7 ± 5.4%, respectively when compared to the expected biofilms which were predominated by yeast form.  Furthermore, polymicrobial biofilms of  C. albicans  (ALC2 and ALC3) with  S. salivarius  K12 showed a decrease by 62.5 ± 25.6% and 55.9 ± 17.1 %, respectively when compared to the expected biofilms when grown in RPMI that were predominated by hyphal form.  Conclusion:  S. salivarius  K12 inhibited polymicrobial biofilms formation of  C. albicans  yeast and hyphal forms, thus supported the hypothesis that  S. salivarius  K12 inhibits biofilm formation of  C. albicans.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation

2015 ◽  
Vol 61 (4) ◽  
pp. 247-255 ◽  
Author(s):  
Tzu-Ling Tseng ◽  
Wei-Chung Lai ◽  
Tai-Lin Lee ◽  
Wan Hua Hsu ◽  
Yu Wen Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Negative Regulation ◽  
Null Mutant ◽  
Auxotrophic Strain ◽  
Reduction Assay ◽  
Mutant Strains

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1–S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

scholarly journals Inhibitory effects of antifungal drugs on biofilm producing Aspergillus spp. recovered from drinking water system

2020 ◽  
Author(s):  
Noorulain Nazir ◽  
Abubakar Siddique ◽  
Nisar A Khan
Keyword(s):  
Drinking Water ◽  
Amphotericin B ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Respiratory Diseases ◽  
Antifungal Susceptibility ◽  
Antifungal Drugs ◽  
Microtitre Plate ◽  
Plate Method ◽  
Crystal Violet Assay

Abstract Aims: Biofilms formed in drinking water distribution systems serve as a continuous source of fungal infections. Biofilms are thick aggregates of adherent microorganisms including pathogenic species of fungi. Respiratory diseases and skin allergy reactions are caused by drinking water containing biofilm forming fungus and bacteria. One of the main causes of nosocomial infections and respiratory diseases in hospitals is due to the fungal biofilm formation in machines, catheters and other surgical instruments. There are some antifungal drugs which are used to control biofilm formation to minimize the infection rate. Methodology and results: The present study was conducted to isolate and identify Aspergillus species which are the main fungal spp. responsible for the biofilm formation in drinking water and to check their antifungal susceptibility against antifungal drugs. The isolated fungal samples from drinking water were cultivated on Potato dextrose agar for the isolation of Aspergillus species. Isolated Aspergillus species were identified on the basis of cultural, morphological and microscopic examination. Then in-vitro ability of biofilm produced by isolated Aspergillus species was estimated using microtitre plate method and quantification by crystal violet assay. Antifungal susceptibility testing against isolated fungal spp. was done by antifungal drug Amphotericin B.Results: From results, it is concluded that drinking water of labs, hospitals and common water chillers were more prevelant by Aspergillus species whereas water from reverse osmosis plants showed negative results. From microtitre plate method and crystal violet assay, it was concluded that Aspergillus spp. are Susceptible against Amphotericin B drug as compared to miconazole.

scholarly journals The Inhibitory Effects of Ficin on Streptococcus mutans Biofilm Formation

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Yan Sun ◽  
Wentao Jiang ◽  
Mingzheng Zhang ◽  
Lingjun Zhang ◽  
Yan Shen ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Streptococcus Mutans ◽  
Metabolic Activity ◽  
Crystal Violet ◽  
Mtt Assay ◽  
Total Protein ◽  
Biofilm Formation ◽  
Acid Production ◽  
Extracellular Proteins ◽  
Crystal Violet Assay

To investigate the effects of ficin on biofilm formation of conditionally cariogenic Streptococcus mutans (S. mutans). Biomass and metabolic activity of biofilm were assessed using crystal violet assay, colony-forming unit (CFU) counting, and MTT assay. Extracellular polysaccharide (EPS) synthesis was displayed by SEM imaging, bacteria/EPS staining, and anthrone method while acid production was revealed by lactic acid assay. Growth curve and live/dead bacterial staining were conducted to monitor bacterial growth state in both planktonic and biofilm form. Total protein and extracellular proteins of S. mutans biofilm were analyzed by protein/bacterial staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), severally. qRT-PCR was conducted to detect acid production, acid tolerance, and biofilm formation associated genes. Crystal violet assay, CFU counting, and MTT assay showed that the suppression effect of ficin on S. mutans biofilm formation was concentration dependent. 4 mg/mL ficin had significant inhibitory effect on S. mutans biofilm formation including biomass, metabolic activity, EPS synthesis, and lactic acid production ( p < 0.05 ). The growth curves from 0 mg/mL to 4 mg/mL ficin were aligned with each other. There was no significant difference among different ficin groups in terms of live/dead bacterial staining result ( p > 0.05 ). Protein/bacterial staining outcome indicated that ficin inhibit both total protein and biofilm formation during the biofilm development. There were more relatively small molecular weight protein bands in extracellular proteins of 4 mg/mL ficin group when compared with the control. Generally, ficin could inhibit biofilm formation and reduce cariogenic virulence of S. mutans effectively in vitro; thus, it could be a potential anticaries agent.

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