Production, purification and characterization of phytase from Pichia kudriavevii FSMP-Y17and its application in layers feed.

INTRODUCTION: Phytase, recognized for its ability to enhance the nutritional value of phytate-rich foods, has has gained significant prominence. The production of this enzyme has been significantly boosted while preserving economic efficiency by utilizing natural substrates and optimizing essential factors. This study focuses on optimizing phytase production through solid-state fermentation and evaluating its effectiveness in enhancing nutrient utilization in chicken diets. OBJECTIVE: The objective is to optimize phytase production via solid-state fermentation, characterize purified phytase properties, and assess its impact on nutrient utilization in chicken diets. Through these objectives, we aim to deepen understanding of phytase's role in poultry nutrition and contribute to more efficient feed formulations for improved agricultural outcomes. METHODOLOGY: We utilized solid-state fermentation with Pichia kudriavzevii FSMP-Y17 yeast on orange peel substrate, optimizing variables like temperature, pH, incubation time, and supplementing with glucose and ammonium sulfate. Following fermentation, we purified the phytase enzyme using standard techniques, characterizing its properties, including molecular weight, optimal temperature and pH, substrate affinity, and kinetic parameters. RESULTS: The optimized conditions yielded a remarkable phytase yield of 7.0 U/gds. Following purification, the enzyme exhibited a molecular weight of 64 kDa and displayed optimal activity at 55 °C and pH 5.5, with kinetic parameters (Km = 3.39 × 10-3 M and a Vmax of 7.092 mM/min) indicating efficient substrate affinity. CONCLUSION: The addition of purified phytase to chicken diets resulted in significant improvements in nutrient utilization and overall performance, including increased feed intake, improved feed conversion ratio, enhanced bird growth, better phosphorus retention, and improved egg production and quality. By addressing challenges associated with phytate-rich diets, such as reduced nutrient availability and environmental pollution, phytase utilization promotes animal welfare and sustainability in poultry production.

A novel cost-effective methodology for the screening of nanocellulose producing micro-organisms.

In this paper, the work has been done to develop a cost-effective methodology, for the isolation of the potential producer of bacterial nanocellulose. No report is available in the literature, on the use of gram flour and table sugar for the screening of nanocellulose-producing isolates. Since commercially used, Hestrin-Schramm medium is expensive for the isolation of nanocellulose-producing micro-organisms, the possibility of using gram flour-table sugar medium was investigated in this work. Qualitative screening of micro-organisms was done using cost-effective medium, i.e., gram flour-table sugar medium. Qualitative analysis of various nanocellulose-producing bacteria depicted that cellulose layer production occurred on both HS medium and gram flour-table sugar medium. The yield of nanocellulose was also better on air-liquid surface in case of gram flour-table sugar medium as compared to HS medium. 16S rRNA was used for molecular characterization of bacterial strain and the best nanocellulose producer was identified as Novacetimonas hansenii BMK-3_NC240423 (isolated from rotten banana). FTIR and FE-SEM studies of nanocellulose pellicle produced on HS medium and gram flour-table sugar medium demonstrated equivalent structural, morphological, and chemical properties. The cost of newly designed medium (0.01967 $/L) is nearly 90 times lower than the Hestrin-Schramm medium (1.748 $/L), which makes the screening of nanocellulose producers very cost-effective. A strategy of using gram flour extract-table sugar medium for the screening of nanocellulose-producing micro-organisms is a novel approach, which will drastically reduce the screening associated cost of cellulose-producing micro-organisms and also motivate the researchers/industries for comprehensive screening programme for getting high cellulose-producing microbes.

Chylothorax in Infants and Children After Congenital Heart Surgery: Approach and Review.

Chylothorax in infants after surgery for congenital heart disease is associated with significant morbidity and mortality. Numerous management modalities applied alone or in various combinations have been utilized but definitive guidelines have not yet been established. We present two infants who developed refractory chylothorax after congenital heart surgery. We also reviewed evidence for the use of available treatment modalities. In our experience, the use of lymphangiography followed by pleurodesis by povidone-iodine was safe and our impression was that it may have played a decisive role in controlling the lymph leak.

Comparison of Levosimendan Versus Milrinone After the Arterial Switch Operation for Infants ≤3†kg.

Background: Various inotropes and inodilators have been utilized to treat low cardiac output syndrome after the arterial switch operation. The use of levosimendan, a calcium sensitizer has been limited in this setting. This study compares the effects of levosimendan with milrinone in managing low cardiac output after the arterial switch operation. Methods: A retrospective, comparative study was conducted in a tertiary care hospital on patients weighing up to 3 kg undergoing the arterial switch operation between January 2017 and January 2022. Patients received a loading dose followed by continuous infusion of either levosimendan or milrinone. Echocardiographic, hemodynamic and biochemical parameters were compared. Results: Forty-three patients received levosimendan and 42 patients received milrinone as the primary test drug. Cardiac index of less than 2.2 L/min/m2 on postoperative day 1 and 2 was found in 9.3% and 2.3% of patients receiving levosimendan versus 26.2% and 11.9% in those receiving milrinone, respectively (P = .04 and .08, respectively). Early lactate-clearance and better central venous oxygen saturations were noted in the levosimendan group. Prevalence of acute kidney injury was higher in the milrinone group (50% vs 28%; P = .03). Use of peritoneal dialysis in the milrinone group versus levosimendan was 31% and 16.3%, respectively (P = .11). There was no difference in hospital mortality between the groups (milrinone, 3; levosimendan, 2, P = .62). Conclusions: Levosimendan is safe and as effective as milrinone to treat low cardiac output syndrome occurring in neonates after the arterial switch operation. In addition we found that levosimendan was renal protective when compared with milrinone.

Pan-India influenza-like illness (ILI) and Severe acute respiratory infection (SARI) surveillance: epidemiological, clinical and genomic analysis.

Background: Over time, COVID-19 testing has significantly declined across the world. However, it is critical to monitor the virus through surveillance. In late 2020, WHO released interim guidance advising the use of the existing Global Influenza Surveillance and Response System (GISRS) for the integrated surveillance of influenza and SARS-CoV-2. Methods: In July 2021, we initiated a pan-India integrated surveillance for influenza and SARS-CoV-2 through the geographically representative network of Virus Research and Diagnostic Laboratories (VRDLs) across 26 hospital and laboratory sites and 70 community sites. A total of 34,260 cases of influenza-like illness (ILI) and Severe acute respiratory infection (SARI) were enrolled from 4 July 2021 to 31 October 2022. Findings: Influenza A(H3) and B/Victoria dominated during 2021 monsoon season while A(H1N1)pdm09 dominated during 2022 monsoon season. The SARS-CoV-2 "variants of concern" (VoC) Delta and Omicron predominated in 2021 and 2022, respectively. Increased proportion of SARI was seen in extremes of age: 90% cases in < 1 year; 68% in 1 to 5 years and 61% in ≥ 8 years age group. Approximately 40.7% of enrolled cases only partially fulfilled WHO ILI and SARI case definitions. Influenza- and SARS-CoV-2-infected comorbid patients had higher risks of hospitalization, ICU admission, and oxygen requirement. Interpretation: The results depicted the varying strains and transmission dynamics of influenza and SARS-CoV-2 viruses over time, thus emphasizing the need to continue and expand surveillance across countries for improved decision making. The study also describes important information related to clinical outcomes of ILI and SARI patients and highlights the need to review existing WHO ILI and SARI case definitions.

Enhancement of bacterial cellulose production by ethanol and lactic acid by using Gluconacetobacter kombuchae.

The current study intended to analyze the impact of ethanol and lactic acid on the bacterial cellulose yield as well as physicochemical and mechanical properties, by using Gluconacetobacter kombuchae. The optimization of ethanol and lactic acid concentration has been done by using one-way ANOVA. Both the supplements significantly enhance the yield of bacterial cellulose (BC) as compared to the standard Hestrin-Schramm medium (control). Optimization leads to significant increase in BC yield as compared to the control, i.e., the addition, of optimized concentration of lactic acid (0.6%) increases the yield from (0.78 ± 0.026) g to (4.89 ± 0.020) g dry weight, and optimized concentration of ethanol (1%) increases the yield from (0.73 ± 0.057) g to (3.7 ± 0.01) g dry weight. Various physicochemical and mechanical properties of BC films produced in different media (i.e., HS, HS + Ethanol, and HS + Lactic acid), such as the crystallinity, structure, tensile strength, strain at break, Young's modulus, and water holding capacity, were also examined, by employing various techniques such as SEM, FTIR, XRD, etc. BC produced in medium supplemented with the optimum concentration of both the additives were found to possesses higher porosity. Though, slight decline in crystallinity was observed. But the tensile strength and strain at break, were upgraded 1.5-2.5 times, 2-2.5 times, respectively. This article attempted to present a method for enhancing BC yields and characteristics that may lead to more widespread and cost-effective use of this biopolymer.

Enhanced Xylanolytic enzyme production from Parthenium hysterophorus through assessment of the RSM tool and their application in saccharification of lignocellulosic biomass.

Parthenium hysterophorous, a widespread weed in India, contributes a substantial amount of lignocellulosic biomass. The key objective of this study is to evaluate the feasibility of producing xylanase enzyme from P. hysterophorus weed biomass using the fungus Aspergillus niger. The impact of various physiological factors was confirmed through a two-step approach: first, a one-factor-at-a-time (OFAT) investigation, and subsequently, employing the RSM-based CCD method in statistical design. This research revealed that the RSM-based model led to the optimization of enzyme activity, resulting in a value of 2098.08 IU/gds for xylanase. This was achieved with an incubation time of 4.5 days, a medium pH of 6, and a cultivation temperature of 32.5 °C. Additionally, a pretreatment involving 1% NaOH and a 30-min autoclave treatment was found to alter the chemical composition of lignocellulose substrates (cellulose 43.87% and xylan 28.7%), thereby enhancing the efficiency of enzymatic hydrolysis. Moreover, fermentable sugars were produced by autoclave-assisted alkali pretreatment (NaOH-1.0% w/v) at rates of 219.6 ± 2.05 mg/gds-1 by utilizing the crude xylanase from A. niger and 291.3 ± 1.2 mg/gds-1 from commercial xylanase enzyme. Our study revealed that P. hysterophorus served as a viable and affordable substrate for fermentable sugar liberation, and xylanase is a rate-limiting enzyme in enzymatic saccharification.

External quality assessment for laboratories in pan-India ILI/SARI surveillance for simultaneous detection of influenza virus and SARS-CoV-2.

Introduction: The Indian Council of Medical Research has set up a nationwide network of 28 laboratories for simultaneous surveillance of influenza virus and SARS-CoV-2 in ILI/SARI patients, using an in-house developed and validated multiplex real-time RTPCR assay. The aim of this study was to ensure the quality of testing by these laboratories by implementing an external quality assessment program (EQAP). Methods: For this EQAP, a proficiency test (PT) panel comprising tissue-culture or egg-grown influenza virus and SARS-CoV-2 was developed. The PT panel was distributed to all the participant laboratories, which tested the panel and submitted the qualitative results online to the EQAP provider. The performance of the laboratories was evaluated on qualitative criteria but cycle threshold (Ct) values were also gathered for each sample. Results: On a qualitative basis, all the laboratories achieved the criteria of 90% concordance with the results of the PT panel provider. Ct values of different samples across the laboratories were within ≤ ±3 cycles of the corresponding mean values of the respective sample. The results of this EQAP affirmed the quality and reliability of testing being done for simultaneous surveillance of influenza virus and SARS-CoV-2 in India.

"Prolonged" venoarterial extracorporeal membrane oxygenation support for respiratory failure: Outcome in an infant.

Extracorporeal membrane oxygenation (ECMO) is a form of extracorporeal life support which provides cardiorespiratory support to patients with potentially reversible pathophysiological processes. ECMO has evolved over the past few decades as a standard technology for neonatal severe respiratory support. However, its use in the pediatric population has increased only since 2009. We report a case of a 9-month infant who required a prolonged (789 h) venoarterial ECMO for severe acute respiratory distress consequent to pneumonia probably secondary to aspiration. He was discharged after this prolonged ECMO run without any obvious unfavorable outcome and is neurodevelopmentally sound at a 26-month follow-up.

Protocol for establishing a model for integrated influenza surveillance in Tamil Nadu, India.

The potential for influenza viruses to cause public health emergencies is great. The World Health Organisation (WHO) in 2005 concluded that the world was unprepared to respond to an influenza pandemic. Available surveillance guidelines for pandemic influenza lack the specificity that would enable many countries to establish operational surveillance plans. A well-designed epidemiological and virological surveillance is required to strengthen a country's capacity for seasonal, novel, and pandemic influenza detection and prevention. Here, we describe the protocol to establish a novel mechanism for influenza and SARS-CoV-2 surveillance in the four identified districts of Tamil Nadu, India. This project will be carried out as an implementation research. Each district will identify one medical college and two primary health centres (PHCs) as sentinel sites for collecting severe acute respiratory infections (SARI) and influenza like illness (ILI) related information, respectively. For virological testing, 15 ILI and 10 SARI cases will be sampled and tested for influenza A, influenza B, and SARS-CoV-2 every week. Situation analysis using the WHO situation analysis tool will be done to identify the gaps and needs in the existing surveillance systems. Training for staff involved in disease surveillance will be given periodically. To enhance the reporting of ILI/SARI for sentinel surveillance, trained project staff will collect information from all ILI/SARI patients attending the sentinel sites using pre-tested tools. Using time, place, and person analysis, alerts for abnormal increases in cases will be generated and communicated to health authorities to initiate response activities. Advanced epidemiological analysis will be used to model influenza trends over time. Integrating virological and epidemiological surveillance data with advanced analysis and timely communication can enhance local preparedness for public health emergencies. Good quality surveillance data will facilitate an understanding outbreak severity and disease seasonality. Real-time data will help provide early warning signals for prevention and control of influenza and COVID-19 outbreaks. The implementation strategies found to be effective in this project can be scaled up to other parts of the country for replication and integration.

Determination of a cut-off value for the serological diagnosis of scrub typhus by detecting anti-Orientia tsutsugamushi immunoglobulin M.

Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.

Multifunctional chromone-incorporated poly(hydroxybutyrate) luminescent film for active and intelligent food packaging.

The major objective of present work was to fabricate poly(hydroxybutyrate) based luminescent films for genuine food packaging applications. These films were synthesized by incorporating varying Chromone (CH) concentrations (5, 10, 15, 20, and 25 wt%) into poly(hydroxybutyrate) (PHB) matrix through solvent-casting. Different characteristics of prepared films were examined using Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Thermogravimetric analysis (TGA), Mechanical testing, and Time-resolved photoluminescence (TRPL). UV-blocking properties and water vapor permeation were also examined. FTIR spectra indicated the occurrence of hydrogen bonding between PHB and CH. Among all prepared film samples, PHB/CH15 showed maximum tensile strength (22.5 MPa) with enhanced barrier ability against water vapor and UV rays, thermal stability, and luminescent performance. After overall analysis, PHB/CH15 film was selected to investigate its X-ray diffraction, release behavior, DPPH scavenging, and antimicrobial potential. Release kinetics revealed that the cumulative release percentage of CH was higher in fatty acid stimulant. Moreover, results suggested that this film demonstrated antioxidant activity (>55 %) and superior antimicrobial potential against Aspergillus niger, Staphylococcus aureus, and Escherichia coli. Furthermore, packaging of bread samples using PHB/CH15 film demonstrated the complete inhibition of microbial growth in bread up to 10 days of storage and ensure the safety of genuine food products.

Perfusion Strategy to Prevent Right Ventricular Decompression on Cardiopulmonary Bypass During Extracardiac Fontan for Right Ventricle-Dependent Coronary Circulation.

Early and long-term outcomes in patients with pulmonary atresia-intact ventricular septum undergoing staged univentricular palliations have been known to be adversely affected by the presence of right ventricle-dependent coronary circulation. We describe a surgical technique to circumvent the coronary insufficiency caused by acute decompression of the right heart.

Association of glutathione-S-transferase polymorphism with genetic damage in paint workers.

BACKGROUND: Occupational exposure to toluene causes serious health problems ranging from drowsiness to lethal diseases such as cancer. Paint workers are exposed to toluene through inhalation or the dermal route, which can induce genetcic damage. The increased DNA damage could be linked to genetic polymorphism. Therefore, we evaluated the association of glutathione-S-transferase polymorphism with DNA damage in paint workers. METHODS: First, we included skilled paint workers (n = 30) as exposed and healthy individuals (n = 30) as control belonging to the same socio-economic strata. The genotoxicity biomarkers, Cytokinesis-block micronucleus (CBMN), and single-cell gel electrophoresis (SCGE)/Comet assay were used to assess genotoxicity while Multiplex-PCR and PCR-RFLP were used to assess polymorphism in glutathione-s-transferase (GST) genes. Using linear curve regression analysis, we assessed the association between genetic damage and polymorphism in the glutathione-s-transferase (GST) gene in the exposed and control subjects. RESULTS: A significantly higher frequency of CBMN (4.43 ± 1.50) and tail moment (TM) (11.23 ± 1.0) respectively in paint workers as compared to the control(1.50 ± 0.86 and (0.54 ± 0.37) underlined significantly high genetic damage in paint workers.Regression curve analysis reveals that polymorphism in the GST gene is significantly associated with higher MN and TM in paint workers. CONCLUSION: Overall, our study provides a strong rationale for identifying a clear association between glutathione-S-transferase polymorphism and genetic damage in paint workers.

Comparative study of ethanol production from sodium hydroxide pretreated rice straw residue using Saccharomyces cerevisiae and Zymomonas mobilis.

Rice straw is a suitable alternative to a cheaper carbohydrate source for the production of ethanol. For pretreatment efficiency, different sodium hydroxide concentrations (0.5-2.5% w/v) were tested. When compared to other concentrations, rice straw processed with 2% NaOH (w/v) yielded more sugar (8.17 ± 0.01 mg/ml). An alkali treatment induces effective delignification and swelling of biomass. The pretreatment of rice straw with 2% sodium hydroxide (w/v) is able to achieve 55.34% delignification with 53.30% cellulose enrichment. The current study shows the effectiveness of crude cellulolytic preparation from Aspergillus niger resulting in 80.51 ± 0.4% cellulose hydrolysis. Rice straw hydrolysate was fermented using ethanologenic Saccharomyces cerevisiae (yeast) and Zymomonas mobilis (bacteria). Overall, superior efficiency of sugar conversion to ethanol 70.34 ± 0.3% was obtained with the yeast compared to bacterial strain 39.18 ± 0.5%. The current study showed that pretreatment with sodium hydroxide is an effective method for producing ethanol from rice straw and yeast strain S. cerevisiae having greater fermentative potential for bioethanol production than bacterial strain Z. mobilis.

Bacterial cellulose: Nature's greener tool for industries.

Bacteria are considered mini chemical factories that help us in providing a wide range of products for various purposes. These days, bacterial cellulose (BC) is getting attention by researchers due to its quality, eco-friendly nature, and excellent physical-mechanical qualities. It is being used in the fabrication of nanocomposites. Its nanocomposites can be used in various industries, including medicine, food, leather, textiles, environment, electronics, and cosmetics. This area of research is emerging and still in its infancy stage, as new applications are still coming up. Most of the work on BC has been done during the last two decades and serious inputs are required in this direction in order to make the production process commercially viable and ultimately the application part. Biowastes, such as fruits and vegetables wastes, can be used as a cost-effective medium to minimize the cost for large-scale production of BC-based nanocomposites thus will valorize the biowaste material into a valuable product. Using biowaste as media will also aid in better waste management along with reduction in detrimental environmental effects. This review will help the readers to understand the potential applications of BC and its nanocomposites as well as their vital role in our daily lives.

Development of poly(hydroxybutyrate) film incorporated with nano silica and clove essential oil intended for active packaging of brown bread.

The objective of current study was to develop Poly(hydroxybutyrate) (PHB) based active packaging film with long lasting antimicrobial potential in food-packaging applications. For developing such films, PHB was incorporated with poly(ethylene glycol) (PEG) as a plasticizer, nano-silica (n-Si) as strengthening material and clove essential oil (CEO) as an antimicrobial agent. These solvent-casted films with varying concentration of n-Si (0.5, 1, 1.5, 2 %) and 30 % CEO of total polymer matrix weight i.e., PHB/PEG (90/10) were prepared and studied on the basis of morphological, mechanical, thermal, degradation and antimicrobial behaviours. The presence of CEO and n-Si was confirmed by Fourier transform infrared spectroscopy (FTIR). Scanning Electron Microscopy (SEM) and X-ray diffraction (XRD) were used to investigate homogeneous dispersal of n-Si in polymer matrix. PHB/PEG/CEO/Si 1.0 film was selected as optimized one after mechanical testing and therefore further carried for antimicrobial testing. This selected film extended the shelf-life of brown bread up to 10 days comparable to bread wrapped in polyethylene. This revealed that PHB/PEG/CEO/Si 1.0 exhibited superior antibacterial activity against the food borne microbes i.e., Escherichia coli, Staphylococcus aureus and Aspergillus niger. Our findings indicate that this film improved the shelf-life of packaged bread and has promising features for active food packaging.

Bacterial cellulose: Nano-biomaterial for biodegradable face masks - A greener approach towards environment.

The use of face masks aids to stop the transmission of various deadly communicable ailments, and therefore widespread mask wearing habit is advocated by nearly all health organisations including the WHO to curb the COVID-19 pandemic. Recent studies predicted a shocking requirement of masks globally, approximately billions of masks per week in a single country, and maximum of them are disposable masks, which are made up of nonbiodegradable material such as polypropylene. With expanding review on improper masks disposal, it is imperative to perceive this inherent environmental hazard and avert it from resulting in the subsequent problematic situation due to plastic. The shift towards biodegradable biopolymers alternatives such as bacterial cellulose and newly evolving sustainable scientific knowledge would be significant to dealt with upcoming environmental problem. Bacterial cellulose possesses various desirable properties to replace the conventional mask material. This review gives an overview of data about accumulation of waste masks and its potential harm on environment. It also focuses on diverse characteristics of bacterial cellulose which make it suitable material for making mask and the challenges in the way of bacterial cellulose production and their possible solution. The current review also discussed the report on global bacterial cellulose market growth.

Evaluation of the Role of Toluidine Blue Paint as an Adjunctive Method to Biopsy in Suspicious Oral Lesion: A Hospital Based Study.

Early detection is a major step in the success of cancer therapy. Histopathology report is considered as the gold standard in the formulation of management protocol of any malignancy worldwide. But unfortunately, there is a delay in the detection of oral cancer very often due to inconclusive histopathology reports. The main reason behind it is obtaining a biopsy specimen from the non-representative area of the lesion. A hospital-based evaluation of the role of Toluidine Blue dye, used as an adjunctive method prior to biopsy was conducted in a tertiary care hospital on 200 patients presenting with oral lesions persistent for more than 3 weeks. The participants were divided into two equal groups by alternate sampling. In one group biopsy was taken by clinical judgment and in others, Toluidine Blue was used prior to obtaining a biopsy to decide the area to be biopsied. Data was collected using a predesigned proforma and was analyzed with the help of SPSS version 20. Results in two groups were compared with respect to sensitivity, specificity, positive and negative predictive values, false positive and false negative percentages. The Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of wedge biopsy without staining were 73.68, 58.14, 70.00, and 62.50% respectively. These values were 95.08, 82.05, 89.23, and 91.43% respectively when Toluidine Blue staining was done as an adjunctive before the biopsy procedure. These results indicate the promising role of Toluidine blue staining before the biopsy to diagnose oral malignancy more efficiently than obtaining biopsy specimens on clinical assessment only and in avoiding the delay in initiating the treatment in case of oral malignant lesions.