Biomass and metabolic activity staining biofilm techniques are not reliable enough to be used in microbiology laboratories.

Routine staining procedures often pose a problem in differentiating a mitotic cell from an apoptotic cell, deteriorating the reliability of histology grading. Although various new methods have been recommended for identifying mitotic figures (MFs) in tissues, the time factor and cost makes them less feasible. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen reaction in identifying MFs and also to see for any variation in the number of MFs in various grades of Epithelial dysplasia. 1. Using crystal violet and Feulgen stain in the identification and counting of MFs on diagnosed cases of epithelial dysplasia and thereby to evaluate their efficacy. 2. To evaluate the variation in the number of MFs in various grades of epithelial dysplasia. The study sample includes retrieval of 30 formalin fixed paraffin embedded tissue sections diagnosed for different grades of epithelial dysplasia (WHO grading system, 2005) from the archives, Department of Oral Pathology, MSRDC, Bengaluru. Ten tissue sections each of mild, moderate and severe epithelial dysplasia were stained with H&E, Feulgen and 1% crystal violet stains and the number of MFs were counted. Five cases of cervical carcinoma were taken as control. Stained sections were compared, and data obtained was statistically analyzed using the Kruskal-Wallis test. A significant increase in the number of MFs (p = 0.02) was observed in Feulgen stained sections as compared to H&E stain. Feulgen stain can be considered as a simple, reliable, cost-effective and reproducible method of staining MFs.

The objectives of this study were to quantitatively estimate the number of mitotic figures (MFs) and evaluate the cellular and nuclear features of various histological grades of oral squamous cell carcinoma (OSCC) using Feulgen and 1% crystal violet stains. This case-control study took place at the Dr D. Y. Patil Dental College & Hospital in Mumbai, Maharashtra, India, between June and December 2016. A total of 51 samples were retrieved from the hospital archives. Of these, 15 well-differentiated, 15 moderately-differentiated and six poorly-differentiated OSCC samples formed the case group while 15 samples of normal gingival mucosa constituted the control group. Each sample was dyed using Feulgen and 1% crystal violet stains and the mitotic count, nuclear area (NA), cellular area (CA), nuclear perimeter (NP), cellular perimeter (CP) and nuclear-to-cytoplasmic (N/C) ratio was calculated using computer-aided morphometry techniques. The number of MFs visible per field was significantly higher in Feulgen-stained sections as compared to those stained with crystal violet (P = 0.050). In addition, the NA, NP, CA and CP values and N/C ratios of samples in the experimental group increased significantly in accordance with an increase in OSCC grade (P <0.001). The Feulgen stain is more reliable than 1% crystal violet in terms of the selective staining of MFs. Moreover, the findings of this study indicate that computer-based morphometric analysis is an effective tool for differentiating between various grades of OSCC.

Context: Mitosis is the process of nuclear cell division. Increased mitosis causes excessive proliferation of cells which is observed in oral squamous cell carcinoma (OSCC). However, there is always problem in differentiating a mitotic cell from an apoptotic cell, during routine staining procedures, which affect the reliability of histological grading of OSCC. Recently, several methods have been implemented in identifying these mitotic figures (MFs), but they seem to be time-consuming and expensive which makes them less feasible. Thus, an effort was done to evaluate the efficacy of crystal violet and Feulgen stains in identifying MFs along with routine hematoxylin and eosin (H and E) in OSCC. Aim: In the identification of MFs in diagnosed cases of OSCC using crystal violet and Feulgen stains and H and E stain. Settings and Design: This was a retrospective study. Materials and Methods: The study sample includes five tissue sections of moderately-differentiated and well-differentiated OSCC each and four sections of poorly differentiated which were stained with H and E, Feulgen, and 1% crystal violet stains, and the number of MFs was enumerated. Statistical Analysis Used: One-way ANOVA was used for statistical analysis. Results: The results from our study showed that 1% crystal violet-stained MFs are better than H and E and Feulgen stains. Conclusion: Crystal violet stain can be considered as a simple, reliable, cost-effective, and reproducible method of staining MFs.

Background: Oral cancer is one of the most prevalent cancers worldwide. The oral squamous cell carcinoma (OSCC) is graded for the proper treatment planning, and it has been a subjective phenomenon. This grading depends on several features, of which the presence of mitotic figures and apoptotic bodies is one of the important criteria. The routine hematoxylin and eosin (H and E) staining may help in identifying the mitotic figures and apoptotic bodies, but it is difficult to identify accurately. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen stain in identifying the mitotic figures and apoptotic bodies and to observe any variation in different grades of carcinoma. Aims: This study was aimed at using crystal violet and Feulgen stain in identifying mitotic figures and apoptotic bodies in different grades of OSCC. Materials and Methods: Fifteen diagnosed cases of OSCC were retrieved and stained with routine H and E, crystal violet stain, and Feulgen stain. All the sections were scanned for mitotic figures and apoptotic bodies. Apoptotic index (AI) and mitotic index (MI) were calculated. AI and MI were expressed as the average of a total number of apoptotic and mitotic cells counted in ten high-power fields. Results: A significant increase in MI and AI was found in Feulgen stain than crystal violet and H and E stain. Conclusion: Feulgen stain can be considered as the best, cost-effective, relatively cheap stain to visualize mitotic figures and apoptotic bodies.

Magnified endoscopic observation of early colorectal cancer by linked color imaging with crystal violet staining (with video).

Bacterial Vaginosis (BV) involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, but its in-depth study has been limited due to the complexity of the bacterial community, which makes the design of in vitro models challenging. Perhaps the most common experimental technique to quantify biofilms is the crystal violet (CV) staining method. Despite its widespread utilization, the CV method is not without flaws. While biofilm CV quantification within the same strain in different conditions is normally accepted, assessing multi-species biofilms formation by CV staining might provide significant bias. For BV research, determining possible synergism or antagonism between species is a fundamental step for assessing the roles of individual species in BV development. Herein, we provide our perspective on how CV fails to properly quantify an in vitro triple-species biofilm composed of Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, and Prevotella bivia, three common BV-associated bacteria thought to play key roles in incident BV pathogenesis. We compared the CV method with total colony forming units (CFU) and fluorescence microscopy cell count methods. Not surprisingly, when comparing single-species biofilms, the relationship between biofilm biomass, total number of cells, and total cultivable cells was very different between each tested method, and also varied with the time of incubation. Thus, despite its wide utilization for single-species biofilm quantification, the CV method should not be considered for accurate quantification of multi-species biofilms in BV pathogenesis research.

Several studies have validated the effectiveness of narrow-band imaging (NBI) in estimating invasion depth of early colorectal cancers. However, comparative diagnostic accuracy between NBI and chromoendoscopy remains unclear. Other than crystal violet, use of acetic acid as a new staining method to diagnose deep submucosal invasive (SM-d) carcinomas has not been extensively evaluated. We aimed to assess the diagnostic accuracy and interobserver agreement of NBI, acetic acid enhancement, and crystal violet staining in predicting invasion depth of early colorectal cancers.A total of 112 early colorectal cancers were prospectively observed by NBI, acetic acid, and crystal violet staining in sequence by 1 expert colonoscopist. All endoscopic images of each technique were stored and reassessed. Finally, 294 images of 98 lesions were selected for evaluation by 3 less experienced endoscopists.The accuracy of NBI, acetic acid, and crystal violet for real-time diagnosis was 85.7%, 86.6%, and 92.9%, respectively. For image evaluation by novices, NBI achieved the highest accuracy of 80.6%, compared with that of 72.4% by acetic acid, and 75.8% by crystal violet. The kappa values of NBI, acetic acid, and crystal violet among the 3 trainees were 0.74 (95% CI 0.65–0.83), 0.68 (95% CI 0.59–0.77), and 0.70 (95% CI 0.61–0.79), respectively.For diagnosis of SM-d carcinoma, NBI was slightly inferior to crystal violet staining, when performed by the expert endoscopist. However, NBI yielded higher accuracy than crystal violet staining, in terms of less experienced endoscopists. Acetic acid enhancement with pit pattern analysis was capable of predicting SM-d carcinoma, comparable to the traditional crystal violet staining.

To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections. A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp. were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods. Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm. Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.

INTRODUCTION: Histopathological grading depends on several features, of which appearance of mitotic figures and apoptotic bodies in the tissue sections plays an important role. The mitotic figures and apoptotic bodies help assess the cellular proliferation and the turnover of the tumor. As mitotic figures (MF) and apoptotic bodies are difficult to assess under routine H and E sections, other methods such as immunohistochemistry (IHC) and flow cytometry have been implemented but, these methods are expensive and more time-consuming and are highly technique sensitive. Hence, stains, which should be cheap, and can be used routinely, and can be tried in identifying the mitotic figures and apoptotic bodies, which has come out with a better result. Routine histological procedures cannot differentiate between pyknotic nuclei, apoptotic cells and MFs which makes the diagnosis difficult.&#x0D; MATERIAL AND METHODS: Study samples were divided in to 3 groups, group 1- case group in which 40 samples of paraffin blocks diagnosed as oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) and group 2- control group in which 40 samples from the healthy individuals. 5 micron thickness sections were made from each tissue specimen. One section was stained with H &amp; E stain nd another section was stained with 1% crystal violet stain.&#x0D; RESULTS: In case group there were 24(60%) male and 16(40%) female while in control group there were 22 (55%) male and 18(45%) female. Mean age in case group was 36.2±15.4 and in control group was25.7±12.7. There is a significant increase in mitotic figures of H &amp; E (P&lt; 0.01) and crystal violet (P &lt; 0.01). In case, there was a significant (P&lt;0.05) increase in mitotic figures when stained with crystal violet. As compared to H&amp;E stain there was a significant (P &lt; 0.01) increase in metaphase observed in crystal violet stain. Mean mitotic figure by H&amp;E in case group was 4.15±1.07 while in crystal violet it was 5.97±1.87.&#x0D; CONCLUSION: % crystal violet stain is the cheaper stain and can be performed in small laboratories and resource constraint settings. Crystal violet can be used for the localization of mitotic figures and assessing proliferation.

The role and relationship with efflux pump of biofilm formation in Klebsiella pneumoniae

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48h of incubation in Dulbecco's modified Eagle's medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24h and 48h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

Background: Mitotic figure counting is simplest and oldest method for determining proliferative activity of cell. It is considered as one of the important diagnostic aid in cancer pathology. Though advanced methods to evaluate dysplastic features are more precise and definite but expense and time makes them less practicable for routine use. Therefore an effort was made to use economical as well as simple approach involving crystal violet stain (1%) to study the mitotic figures in oral squamous cell carcinoma. Materials and Methods: This descriptive research included samples, consisting of thirty three cases of the oral squamous cell carcinoma (OSCC). Representative sections were stained with H&amp;E stain and 1% crystal violet stain respectively. The stained sections were viewed under optical microscope to count mitotic figures for evaluating the effectiveness of 1% crystal violet stain. Data obtained was statistically analyzed by using sample t-test. Results: There was noteworthy increase in the mean mitotic count among the crystal violet stained sections of OSCC in contrast to the OSCC sections stained with H&amp;E (P = 0.00). Conclusion:1% Crystal violet stain can be considered as one of the optimum stains to observe the mitotic figure. Practice of staining with 1% crystal violet during routine histopathological procedures will be cost effective and may be used as a selective stain.

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

In many natural and technological applications, microbial biofilms grow under fluid flow. In this project, we investigated the influence of flow on the formation and growth of biofilms produced by gram-positive Lactobacillus plantarum strains WCFS1 and CIP104448. We used an in-house designed device based on a 48-well plate with culture volumes of 0.8 ml, and quantified total biofilm formation under static and flow conditions with flow rates 0.8, 1.6, 3.2 and 4.8 ml/h (with 1, 2, 4 and 6 volume changes per hour) using crystal violet (CV) staining, and determined the number of viable biofilm cells based on plate counts. The amount of total biofilm under flow conditions increased in the CIP 104448 strain, with significantly increased staining at the wall of the wells. However, in the WCFS1 strain, no significant difference in the amount of biofilm formed under flow and static conditions was observed. Plate counts showed that flow caused an increase in the number of viable biofilm cells for both strains. In addition, using enzyme treatment experiments, we found that for WCFS1 in the static condition, the amount of mature biofilm was declined after DNase I and Proteinase K treatment, while for flow conditions, the decline was only observed for DNase I treatment. The CIP104448 biofilms formed under both static and flow conditions only showed a decline in the CV staining after adding Proteinase K, indicating different contributions of extracellular DNA (eDNA) and proteinaceous matrix components to biofilm formation in the tested strains.