Gentian Violet Stain Research Articles

Identification and characterization of Acinetobacter nosocomialis BfmRS, two-component regulatory system, essential for biofilm development.

Biofilm development by bacteria is considered to be an essential stage in the bacterial infection. Acinetobacter nosocomialis is an important nosocomial pathogen causing a variety of human infections. However, characteristics and specific determinants of biofilm development have been poorly characterized in A. nosocomialis. The aim of this study was to investigate the factors involved in the biofilm development by A. nosocomialis. Library of random transposon mutants was constructed using the Tn5 mutagenesis. The mutant strains, in which the ability of biofilm formation was significantly impaired, were screened by gentian violet staining. The roles of BfmR and BfmS were determined by constructing a bfmR and bfmS deletion mutant and analyzing the effects of bfmR and bfmS mutation on the biofilm development and motility of A. nosocomialis. We identified a biofilm-defective mutant in which a transposon insertion inactivated an open reading frame encoding the BfmR in a two-component regulatory system consisting of BfmR and BfmS. The bfmR mutant revealed a significant reduction in biofilm formation and motility compared to wild-type strain. Deficiency in the biofilm formation and motility of the bfmR mutant was restored by single copy bfmR complementation. In contrast, the bfmS mutant had no effect on biofilm formation. A. nosocomialis has a two-component regulatory system, BfmRS. BfmR is a response regulator required for the initial attachment and maturation of biofilm during the biofilm development as well as the bacterial growth. BfmR could be a potential drug target for A. nosocomialis infection.

Mekanisme Antibakteri Ekstrak Etanol Daun Kemangi (Ocimum basilicum L.) terhadap Salmonella typhi

Infectious diseases are conditions that can be brought on by a variety of different organisms, including bacteria, viruses, fungi, and parasites. Typhoid fever is an example of an infectious disease that frequently causes the body's defenses to come under attack. The fever associated with typhoid is a leading cause of death around the world. Infection with the bacterium Salmonella typhi is considered to be one of the potential causes of typhoid fever. When antibiotics are used to treat bacterial infections, there is a risk that the bacteria will become resistant to the medication. In day-to-day life, basil leaves are utilized both as a cooked vegetable and as a fresh vegetable. The antibacterial properties of kemangi leaves have yet to be fully uncovered. The purpose of this study was to investigate the various mechanisms that are involved in antibacterial activity. These mechanisms include cell leakage, minimum inhibitory concentration, minimum killing concentration, and antibiofilm activity. The minimum inhibitory concentration (MIC) was determined with the agar diffusion method, the minimum biofilm forming concentration (MBC) was determined with the staining method, and cell leakage was determined with UV-VIS spectrophotometry at wavelengths of 260 nm (DNA) and 280 nm (protein). Additionally, the formation of anti-biofilms was determined with microtiter plates and gentian violet staining. In the moderate category, the MIC value tests showed a concentration of 0.3125% with an inhibition zone value of 6.80 0.05 mm. These results were obtained from the MIC value. Because there was no detectable bacterial growth on the streaked medium, KBM was obtained at a concentration of 10%. At wavelengths of 260 and 280 nanometers, leakage cells show absorbance absorption. The percentage of biofilm activity that is measured when the concentration is increased to 25% is 44.82%. In conclusion, the ethanol extract of basil leaves possesses a possible antibacterial mechanism against Salmonella typhi. This was discovered through testing.

Norepinephrine Effects on Uropathogenic Strains Virulence.

The degree of virulence correlates with adhesion, biofilm formation, motility and the capacity to quickly colonize biological surfaces. The virulence of the bacteria that have colonized the urinary tract may be modified by substances dissolved in urine. One such substance is the norepinephrine (NE) hormone, which may be present in human urine, especially in times of stress and under changes in the activity of the renin-angiotensin-aldesterone system. In this work, we study the influence of NE on the biomass, biofilm formation, matrix production, adhesion, motility and metabolism of uropathogenic strains of E. coli and S. aureus. We used Congo red and gentian violet staining for detection of matrix and biomass formation, respectively. The optical density was measured by a multichannel spectrophotometer. The motility of bacterial cells was measured on semi-solid agar at 24 h and 48 h. The metabolic activity was analyzed by MTT assay. It was shown that the metabolic activity of E. coli was stimulated by NE, which led to the increasing synthesis of virulence factors such as biofilm production, adhesion, and motility. At the same time, NE did not activate the S. aureus strain's metabolism and did not change its adhesion and motility. Thus, the virulence activity of uropathogenic E. coli may be modified by NE in urine.

Dissociated forms of moraxella isolated from the affected eyes of cattle

The dissociation phenomenon of epizootic cultures of Moraxella was studied. The study was conducted in economic entities of Almaty region of the Republic of Kazakhstan for 233 heads of cattle with clinical signs of keratoconjunctivitis. Isolation of the causative agent of Moraxella was performed by bacteriological washes from the conjunctival sacs of the eyes of animals. The laboratory study was carried out according to the approved methodological guidelines. It was found that bacteria of the genus Moraxella dissociate when grown on a solid nutrient medium for more than 6 hours in a thermostat at 37 °C. The bacteria were studied by the following methods: staining according to White-Wilson, thermoagglutination and acriflavine assay. When evaluating the grown colonies according to White-Wilson, the optimal dilution for crystal violet was found to be 1 : 2000, and for gentian violet stain 1 : 1000. In this case, the colonies in the S-form have a dark purple color with a metallic tint, and the dissociated colonies in the R-form do not stain. In the presence of dissociated cells, precipitation (thermoagglutination), sediment formation and clearing of the supernatant fluid at 90 °C for 30 minutes were noted. The suspension of undissociated colonies remained cloudy. When weighing microbial cells isolated by a bacterial loop from individual grown colonies in a solution of acriflavine, dissociated bacteria stick together to form conglomerates. When studying the antigenic activity of the S-, R- forms of Moraxella, it was revealed that the activity of the S-antigen significantly exceeded that of the R-forms. Data on the dissociation of Moraxella cultures can be used for the development of diagnostic and prophylactic drugs against moraxellosis in cattle.

Formation of biofilms by representatives of the oral microflora on the surfaces of basic materials

The aim of the work is to conduct a comparative analysis of the biofilm formation by representatives of the oral microflora on the surfaces of basic materials. Materials and methods. The process of biofilm formation was examined on 7 types of basic plastic samples: Polyan, Breflex, Nylon, Protakryl, Vinakryl, Biocryl, which were used for the manufacture of removable prosthetic basis constructions, and SYNMA, which was used for comparison. Biofilm formation was analyzed by the method Y. Zhang (2017) with minor modifications. The test sample was placed in a test tube with 2.0 ml of nutrient broth Brain Heart Infusion to model the biofilm growth of microorganisms (HiMedia Laboratories Pvt. Ltd., India) supplemented with 1 % glucose, pre-inoculated with test strains at a final concentration of 1 × 104 CFU/ml. The strains were cultivated for 24 hours at a temperature of 37 °C under continuous stirring in a shaker MR-1 (SIA BIOS AN, Latvia) at 20 rpm. Evaluation of the biofilm massiveness was performed after gentian violet staining followed by elution of the stain with ethanol and registration of the eluent optical density (OD). The OD was measured with a Synergy™ HTX S1LFTA microplate multimode photometer (BioTek Instruments, Inc., USA) at 595 nm wavelength using Gen5™ Data Analysis Software. The number of viable bacterial cells in the formed biofilms was determined by the method of ten-fold serial dilutions. The obtained results were converted per unit area of the sample tested. Processing of the results was performed using a two-sample t-test with the software package Statistica 13.0 and Microsoft Office Excel, the differences were considered statistically significant at a P value of < 0.05. Statistical analysis of the obtained data was presented as mean values of measurements ± standard deviation for three independent experiments. Results. According to the microbiological analysis results it was found that α-hemolytic streptococci S. oralis and S. sanguinis showed the ability to form biofilms on the surfaces of basic materials, namely Protacryl and Vinacryl, the total biomass of S. sanguinis biofilms was 47.7 % (P < 0.01) and 14.7 % (P > 0.05) greater, respectively, in comparison to a glass slide. Inhibition of biofilm formation processes was observed on the surfaces of Nylon and Biocryl basic materials. S. oralis and S. gordonii showed the highest ability to survive in biofilms. The intensity of C. albicans biofilms formation on Biocryl basic materials, comparative plastics SINMA and Breflex basic materials was greater than on glass slides by 48.3 %, 43.0 % and 34.9 % (P < 0.01), respectively. The least massive C. albicans biofilms were formed on Breflex surfaces and SINMA comparative plastics in comparison to glass slides by 33.6 % and 24.8 % (P < 0.01), respectively. Both Candida strains had the highest level of fungal viability in biofilms on Breflex, Polyan and Protacryl basic materials (P < 0.01), and C. tropicalis biofilms on Biocryl and Vinacryl basic materials (P < 0.05). Integral coefficients indicated the inhibition of the oral microflora ability to form biofilms on the surfaces of basic materials. Conclusions. Oral α-hemolytic and β-hemolytic streptococci have the ability to intensive biofilm growth on the surfaces of the basic materials Protacryl and Vinacryl. Oral Candida albicans form massive biofilms on the surfaces of Biocryl and Vinacryl basic materials and comparative SYNMA plastics. The basic materials Breflex, Nylon and comparative plastics SYNMA are the most inert to biofilm formation by the oral microflora representatives.

Lactobacillus rhamnosus and Lactobacillus casei Affect Various Stages of Gardnerella Species Biofilm Formation.

Bacterial vaginosis (BV) and its recurrence are most commonly associated with the formation of Gardnerella species biofilm. Probiotics are typically used to treat BV; however, the optimal period of Lactobacillus probiotic application in BV treatment remains uncertain. The present study aimed to explore the effects of Lactobacillus rhamnosus and Lactobacillus casei on various stages of biofilm formation in Gardnerella species. The biofilm-forming ability of seven strains, including one Gardnerella vaginalis ATCC 14018 and six clinically isolated Gardnerella species, was determined via gentian violet staining assay. Moreover, the sensitivity of the planktonic and biofilm forms toward metronidazole and clindamycin was assessed via microdilution broth method. L. rhamnosus Xbb-LR-1 and L. casei Xbb-LC-1 were added during various stages of biofilm formation in Gardnerella species and were cocultured for 24 h. The biofilm thickness of each sample was determined via confocal laser scanning microscopy (CLSM). The absolute quantities of Gardnerella species in each sample was obtained via real time polymerase chain reaction method, and the pH value was obtained using a pH indicator paper. Biofilm formation by Gardnerella species in a medium with distinct pH values was observed via gentian violet staining, CLSM, and scanning electron microscopy (SEM). The biofilm increased the resistance of Gardnerella species toward metronidazole and clindamycin. L. rhamnosus added at the initial biofilm formation stage in Gardnerella species exhibited highest inhibitory effect, with a percentage inhibition of 38.17% ± 1.35%. When the pH value of the culture medium was <4.5 or >6.5, ATCC 14018 could hardly form a biofilm; however, at pH ≥4.5 and ≤6.5, it was able to form a stronger biofilm. The amount of biofilm attained maximum value at optical density of 3.29 ± 0.28 (595 nm), pH 5.5, and at 36 h. Biofilm formation increases the resistance of Gardnerella species toward antibiotics. Maintaining an acidic vaginal environment with pH <4.5 and a vaginal microbiota dominated by Lactobacillus remarkably prevents the formation of Gardnerella species biofilm at the initial stage, which further has a significant impact on the treatment and prevention of biofilm-related infections.

A crossover clinical study to evaluate pain intensity from microneedle insertion in different parts of the oral cavity

The objective of the present study was to evaluate discomfort and safety of microneedle (MN) insertion in several intraoral regions. A device was developed to standardize MN insertions. MNs were inserted in the following regions of the oral cavity: gingiva, palatine alveolar process, buccal mucosa, dorsum of the tongue and inner portion of the lower lip. Perforations from MNs post insertion were confirmed with topical gentian violet stain. Pain was evaluated in a randomized, double-blinded, crossover study in 30 volunteers. Each volunteer received a MN patch, a 30G hypodermic needle (positive control) and an identical MN patch with its needles laying flat in the plane of the patch (negative control). Adverse events were visually evaluated immediately after (0 h) and 24 h post MN application. The application device developed a consistent application force (10 N) and promoted perforation of all individual MNs on a patch. At all sites, insertion of the hypodermic needle promoted more pain when compared to the negative control (p < 0.001). Application of the MNs promoted less pain than the hypodermic needle (p < 0.05), but slightly more pain as compared to the negative control (p < 0.05) at all sites except the tongue, where the MN did not differ from the negative control (p > 0.05). Hypodermic needle caused bleeding at all insertion sites. In contrast, MNs did not cause bleeding at most sites except in some cases of insertion into the hard gingiva and the palatine alveolar process where tiny blood spots appeared immediately after MN application for few of the MNs on the patch. There were no cases of bleeding at 24 h post MN application. In conclusion, MNs can perforate different sites of the oral cavity in a safe and significantly less painful manner as compared to the 30G hypodermic needle. Thus, analogous to the skin, MN-based approaches could be an attractive approach for drug delivery in the oral cavity.

Proud Flesh: a Complicated Wound Healing—Case Report and Review of Literature

Granulation tissue fills dead space, decreases the size of the wound and provides a barrier to external contaminants in wound healing. Sometimes it is impaired by the formation of extreme granulation tissue in conditions like casted or pressure-bandaged wound, accumulated inflammatory cells which result in the production of lactic acid generating low oxygen tension and low-pH environment. Exuberant granulation tissue, commonly known as proud flesh, is a frustrating complication of wound healing. It leads to the development of significant elevation above the wound edges, causing inhibition of epithelial migration. This is mostly seen in the equine species (horses). A 37-year-old man met with a high-velocity road traffic accident was brought to the Subharti Hospital, Meerut. Examination revealed significant head injury with clinical evidence of pan-facial fracture. Ten days post-trauma, maxillofacial fixation was performed. Postoperatively, a small exuberant growth of soft tissue was noted over the upper lip region which gradually extended to all over the face. Considering the antiangiogenic property of gentian violet stain, it was applied over the lesion subsequently along with intravenous injections of hydrocortisone. The operator later observed regression in the nature of lesion, which is resolved subsequently at the end of 4 weeks. Definitive management of proud flesh is variable and depends upon the phase of the wound, the extent of granulation tissue and the cosmetic outcome desired. The clinicians should have a thorough understanding of wound healing so that they can apply the specific treatment at a specific time in a specific manner.

Durable visible light self-cleaning surfaces imparted by TiO2/SiO2/GO photocatalyst

This research intends to enhance the photocatalytic efficiency and durability of substrates coated with TiO2-based nanocomposites under visible light illumination. More specifically, TiO2/SiO2/graphene oxide nanocomposites are successfully sono-synthesized and sono-fabricated onto a polymeric fabric through a facile one-step method. The morphologies, phase structures and optical properties of the samples are studied. The photocatalytic activity of the functionalized samples is assessed through a gentian violet stain removal test and then photodecomposition of methylene blue under visible light, and tested again after 10 washes to check on its durability. It is corroborated that TiO2/SiO2/graphene oxide nanocomposites have shown significantly better visible light self-cleaning performance than just using the pure TiO2 alone. In addition, even after 10 washes, the samples virtually maintain the functionality. Herein, sono-synthesis of TiO2/SiO2/graphene oxide represents a remarkable step forward in visible light-driven self-cleaning treatment for flexible materials and, hence, holds a great potential for commercialization.

Abstract 15649: Open-Chest Intramyocardial Delivery of Exosomes From Cardiosphere-Derived Cells (CDCs) Mimics Cellular Postconditioning: Decreased Infarct Size and Improved Perfusion in a Pig Model of Acute Myocardial Infarction

Background: Various lines of evidence indicate that exosomes mediate the therapeutic effects of cardiosphere-derived cells (CDCs). However, retention and efficacy of exosomes may be limited after intracoronary (IC) delivery due to their very small size (30-150 nm diameter). Objectives: We sought to assess the optimal dosing and delivery techniques for exosomes, and their ability to reduce microvascular obstruction (MVO) and infarct size (IS) in a pig model of ischemia/reperfusion. Methods: Acute myocardial infarction (AMI) was induced in 23 mini-pigs by balloon occlusion of the mid-LAD for 1.5h. Thirty minutes after reperfusion, pigs received, by prior random assignment, IC injection of CDC-derived exosomes (1.5mg [n=3] or 15mg [n=5] of exosome protein), vehicle [n=6], or transepicardial intramyocardial injection (IM) of exosomes (15mg [n=4] or 7.5mg [n=5]) via median sternotomy. Pigs were sacrificed 48 hr post-MI to measure area at risk (AAR), IS and MVO utilizing gentian violet, TTC (2,3,5-triphenyl tetrazolium chloride) and thioflavin staining respectively. Left ventricular ejection fraction (LVEF) was measured by contrast ventriculography before injection and at endpoint. Results: AAR was identical in all groups (averaging 22±4% of LV, P=0.65 among groups). Consistently, LVEF was similar in all groups before treatment. IC delivery of high-dose exosomes (15mg) decreased MVO (43±6% vs. 56±7%, P=0.02) whereas IC low-dose (1.5mg) did not (54±6%, P=0.66). However, IC exosomes failed to decrease IS regardless of dose. In contrast, both IM doses decreased MVO (43±6% for 15mg and 25±14% for 7.5mg, P=0.04 and P&lt;0.0001 respectively) and IS (67±6% for 15mg, 57±15% for 7.5mg vs. 80±6 for placebo, P=0.04 and P=0.003 respectively). At both IM doses, LVEF at endpoint was higher in exosome-injected pigs compared to vehicle (P=0.03). Conclusion: Intracoronary injection of CDC-secreted exosomes decreases MVO at high doses, but fails to reduce IS. In contrast, both low- and high-dose exosomes reduce IS and MVO after intramyocardial delivery, mimicking the cardioprotection seen with CDCs themselves (cellular postconditioning). These results indicate that IM delivery may be more suitable for exosome therapy of myocardial disease.

The Effect of Gentian Violet on Human Anterior Lens Epithelial Cells

Purpose: To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application.Materials and methods: Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca2+]i) in response to pharmacological stimulation by acetylcholine. The [Ca2+]i homeostasis is the indicator of cellular function. The changes in [Ca2+]i were compared between the two groups.Results: Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (n = 17) and non-stained ones (n = 33). No significant differences of the elicited responses were found in rise time (p = 0.89), decay time (p = 0.61) or amplitude of [Ca2+]i (p = 0.96 for 63× and p = 0.26 for 40× objectives) between the two groups of capsules (Student t test).Conclusions: The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca2+]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

Progressive Disseminated Superficial Actinic Porokeratosis: a Case Report with a Family History in Three Generations

Abstract Disseminated superficial actinic porokeratosis is the most common form of porokeratosis in adults, which develops in the third or fourth decade of life, but may also occur later or earlier, more frequently in females, particularly in countries with high sun exposure. Lesions are numerous, uniform, superficial with central atrophy, demarcated by a distinct peripheral ridge, and usually found on sun exposed areas or elsewhere. The disease is inherited as an autosomal dominant condition. We present a 57-year-old retired woman. Her initial skin changes, affecting the face, developed in her thirties, and they have not changed their features since. In the following years, changes developed on the extensor surface of her arms and legs, with more prominent erythema, and then also on other parts of the body, including palms and soles, presenting as dark brown pigmented patches. Her mother had similar changes, and her daughter, who lives abroad, also has them. On examination, the patient presented with facial lesions, patches 2-3 mm wide, with peripheral hyperpigmentation and a pale center. There were multiple, 2-3 mm wide, dark brown lesions on the extremities and trunk. The lesions were either flat or with atrophic center with darker filiform corneal rim. Pathohistolgical examination revealed a "cornoid lamella", which is pathognomonic for the diagnosis of porokeratosis. Auxiliary diagnostic methods were also used - dermoscopy and Gentian violet staining. The patient was advised to avoid sun exposure and to apply photoprotective sunscreens. In conclusion, this is a case report of a disseminated superficial actinic porokeratosis that affected three generations of a family. Our patient developed lesions on palms and soles as well. A review of available world literature shows that this is the second case report of disseminated superficial actinic porokeratosis with palmoplantar involvement.

Measurement of calprotectin in ascitic fluid to identify elevated polymorphonuclear cell count

To evaluate the diagnostic capability of calprotectin in ascitic fluid for detecting a polymorphonuclear (PMN) cell count > 250/μL ascites. In this prospective observational study, a total of 130 ascites samples were analysed from 71 consecutive patients referred for paracentesis. Total and differential leukocyte cell counts were determined manually with a Neubauer chamber and gentian-violet stain. Calprotectin was measured in 1 mL ascetic fluid by enzyme-linked immunosorbent assay (ELISA) and a point-of-care (POC) lateral flow assay with the Quantum Blue(®) Reader (Bühlmann Laboratories). All measurements were carried out in a central laboratory by senior personnel blinded to patient history. A PMN count > 250/μL was the primary endpoint of the study. The diagnostic value of ascitic calprotectin measurement was assessed by comparing to the final diagnosis of each patient that had been adjudicated by investigators blinded to calprotectin values. The PMN count was > 250/μL in 19 samples (14.6%) from 15 patients (21.1%) and varied widely among the study population (range 10-19 800/mL and 1-17 820/mL, respectively). Spontaneous bacterial peritonitis (SBP) was the final diagnosis in four patients (5.6%). All patients with PMN ≤ 250/μL had negative bacterial culture. PMN count was elevated in five patients with peritoneal carcinomatosis, three with lymphoma, one with neuroendocrine carcinoma, and two with secondary peritonitis due to abdominal perforation. PMN cell counts correlated with ascitic calprotectin values (Spearman's rho; r = 0.457 for ELISA, r = 0.473 for POC). A considerable range of ascitic calprotectin concentrations was detected by ELISA [median 0.43 μg/mL, interquartile range (IQR) 0.23-1.23 (range 0.10-14.93)] and POC [median 0.38 μg/mL, IQR 0.38-0.56 (range 0.38-13.31)]. Ascitic calprotectin levels were higher in samples with PMN > 250/μL, by both ELISA [median (IQR) 2.48 μg/mL (1.61-3.65) vs 0.10 μg/mL (0.10-0.36), P < 0.001] and POC [2.78 μg/mL (2.05-5.37) vs 0.38 μg/mL (0.38-0.41), P < 0.001]. The area under the receiver operating characteristics curve for identifying an elevated PMN count was 0.977 (95%CI: 0.933 to 0.995) for ELISA and 0.982 (95%CI: 0.942 to 0.997) for POC (P = 0.246 vs ELISA). Using the optimal cut-off value for ELISA (0.63 μg/mL), ascitic calprotectin had 94.8% sensitivity, 89.2% specificity, positive and negative likelihood ratios of 8.76 and 0.06 respectively, positive and negative predictive values of 60.0% and 99.0% respectively, and 90.0% overall accuracy. Using the optimal cut-off value for POC (0.51 μg/mL), the respective values were 100.0%, 84.7%, 6.53, 0.00, 52.8%, 100% and 87.7%. Correlation between ELISA and POC was excellent (r = 0.873, P < 0.001). The mean ± SD of the difference was -0.11 ± 0.48 μg/mL with limits of agreement of + 0.8 μg/mL (95%CI: 0.69 to 0.98) and -1.1 μg/mL (95%CI: -1.19 to -0.91). Ascitic calprotectin reliably predicts PMN count > 250/μL, which may prove useful in the diagnosis of SBP, especially with a readily available bedside testing device.

Corneal Edema After Descemet Membrane Stripping Automated Endothelial Keratoplasty With the Use of Gentian Violet Staining

Corneal Edema After Descemet Membrane Stripping Automated Endothelial Keratoplasty With the Use of Gentian Violet Staining

A Novel Cell-Associated Protection Assay Demonstrates the Ability of Certain Antibiotics To Protect Ocular Surface Cell Lines from Subsequent Clinical Staphylococcus aureus Challenge

In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Delineating the extent of anterior capsulorhexis with gentian violet using capsulorhexis marker: a preliminary study of efficacy and toxicity in an animal model

Background:The purpose of this study was to evaluate the efficacy and safety of a new instrument to optimize the size, shape, and centration of anterior capsulorhexis.Methods:The study was divided into two parts. The first part was done in 10 goats’ eyes. A specially designed capsulorhexis marker was used. The lower edge of the instrument was stained by gentian violet and then applied to the anterior capsule to produce a circular mark of 5.5 mm. In five eyes, the gentian violet was applied to the marker using a corneal marking pen; in the remaining five eyes, the gentian violet was applied using a corneal marking pad. In the second part, the toxicity of gentian violet was tested as follows: ten albino rabbits received a gentian violet injection into the anterior chamber in one eye (experimental group) and an equal volume of balanced salt solution in the fellow eye (control group). Five rabbits were sacrificed one day after surgery and the remaining five rabbits after one week.Results:In the first part, there was no difficulty in the introduction or removal of the instrument from all eyes. In the first two eyes, the circular mark was diffuse due to sliding of the marker on the surface of the anterior capsule. In the remaining eight eyes, there was a well-centered, rounded mark which was adequately stained. In the second part, there was no histopathological evidence of corneal toxicity in either group. There was loss of ganglion cells from the neurosensory retina one day after surgery in one eye from the experimental group. At one week, there was no evidence of retinal toxicity in any of the rabbits.Conclusion:This capsulorhexis marker can guide the surgeon to a better centration and proper sizing of anterior capsulorhexis using gentian violet staining. More refinement of the instrument is needed to be able to use it in human eyes.

A New Look at the Pathogenesis of the Meconium Aspiration Syndrome: A Role for Fetal Pancreatic Proteolytic Enzymes in Epithelial Cell Detachment

We hypothesized that fetal pancreatic digestive enzymes play a role in the lung damage after meconium aspiration. We studied the effect of meconium on the A549 alveolar epithelial cell line. The exposure of the cells to 0.5 to 5% meconium resulted in significant disruption of connections between A549 cells and caused dose-dependent cell detachment, without signs of cell death. A protease inhibitor cocktail prevented the A549 cell detachment induced by meconium. After the exposure to 2.5% meconium, a protective effect was quantified by measuring light absorbance by gentian violet stain of still attached cells. The protease inhibitor cocktail and chymostatin showed significant protective effects, increasing the number of attached cells by 135 and 123%, respectively (p < 0.05). Other individual protease inhibitors tested in the detachment assay (AEBSF, leupeptin, E-64, aprotinin, benzamidine, phosphamidon, and aminohexanoic acid) did not offer statistically significant protection. These results afford a new perspective on the pathophysiology of meconium aspiration syndrome (MAS). We speculate that disruption of intercellular connections and cell detachment from the basement membrane are key events in the pathology associated with MAS. The observed protective effects of protease inhibitors suggest that they may be useful in the treatment and/or prophylaxis of MAS.

The Change of Rectus Muscle Length Caused by Traction With Muscle Hooks in Strabismus Surgery-the First Report

Purpose: To evaluate changes in muscle length when retracting the extraocular rectus muscle with muscle hooks during strabismus surgery. Methods: Forty-four rectus muscles of 42 patients consecutively resected in two hospitals (A, B) were included in this study. After isolation of the rectus muscle, the length of the muscle was recorded and the portion to be resected was marked using gentian violet stain on the tip of calipers. After the rectus muscle was retracted with two muscle hooks in either direction, its length was measured again with the calipers. Results: The length of the rectus muscle was not changed by retraction in 25 of 44 muscles (56.8%). The length of the muscle was changed by 0.5 mm in 13 muscles (29.5%) and by 1mm in six muscles (13.6%). Changes of rectus muscle length over 0.5 mm were observed in 15 of 27 muscles of patients treated at hospital A (55.5%) and four of 17 muscles of patients treated at hospital B (23.5%). The results for the two hospitals were significantly different (p=0.037). Conclusions: Small errors in measurement can occur when muscle resections are performed during strabismus surgery. Institutional or clinician related factors may explain variations in error rates between hospitals.

An epidemiologic analysis of staphylococcus aureus‐associated keratitis in Boston

Abstract Purpose S. aureus is a normal commensal of the human skin and nasopharynx. It is therefore of interest to determine whether S. aureus keratitis is caused by a subset of these organisms. In this study, the phenotypic and genotypic characteristics of S.aureus keratitis isolates were analyzed. Methods All S. aureus clinical isolates were prospectively collected over a 24 month period at the MEEI (2006‐2008). The diagnosis of clinical keratitis and associated risk factors was by medical record review. Keratitis‐associated S. aureus strains were assessed for: 1) antibiotic susceptibility, 2) biofilm robustness by gentian violet staining using an in vitro microtiter plate assay, and 3) genetic lineage by multi‐locus sequence typing (MLST). Results 26 cases of keratitis were identified from the 600 S. aureus clinical isolates. Risk factors associated with S.aureus keratitis included trauma, prior surgery, soft contact lens wear, and the presence of a foreign body. Ocular surface disease does not appear to be an independent risk factor. All 26 isolates were tetracycline‐ and trimethoprim‐sulfamethoxazole‐ sensitive. All the MRSA strains were found to be ciprofloxacin‐resistant (10/26). Nearly one‐half of all the S.aureus keratitis‐associated isolates were caused by a single clone, ST5. Both methicillin sensitive and resistant S. aureus strains were represented within ST5. Conclusion These results suggest that there may be specific S.aureus lineages which possess phenotypic and genotypic characteristics that enable S. aureus to more effectively cause sight‐threatening keratitis. Future work will examine their virulence traits and a comparison to commensal S.aureus strains.

Antiviral activity of nano carbon fullerene lipidosome against influenza virus in vitro

The activity of nano carbon fullerene lipidosome (NCFL) against influenza virus H1N1 in vitro was studied by observing the cytotoxicities and its activity rendered by different intensities of lighting with various periods of time. Rimantadine hydrochloride was used as the positive control drug. By using microcultural technique, the morphological changes of cells were observed and by using the gentian violet staining, antiviral activity of the NCFL against influenza virus was assayed. The results showed that: (1) The maximal concentration of the NCFL was 7 microg/mL and the 50% toxic concentration (TC(50)) was 13.54 microg/mL respectively; (2) NCFL had a significant activity of directly killing the influenza virus, while the activities in antiadsorption and antireplication were not obvious; (3) There was a dose-activity relationship between the dosages of NCFL and the direct killing effect against the influenza virus, and the periods of lighting-time could influence the activity partly. It was concluded that NCFL had a significant activity of directly killing the influenza virus.