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@#AIM:To investigate the application value of fluorescent staining technique in the detection of amoebic pathogens in corneal tissue biopsy, and to apply fluorescent staining technique in the histopathological diagnosis of Acanthamoeba keratitis(AK), comparing the results with those of hemotoxyiln-eosin staining(HE staining)and periodic acid-schiff staining(PAS staining), and analyzing the sensitivity and specificity of these three staining methods.<p>METHODS:Specimens of infected corneal tissue were collected from 74 cases(75 eyes), and then they were divided into an AK group and a non-Acanthamoeba keratitis(NAK)group based on the results of corneal scraping, culture and histopathological diagnosis. The tissues of consecutive sections were stained with HE staining, PAS staining and fluorescence respectively, and the sensitivity and specificity of the three staining methods for the diagnosis of AK were analyzed. Area under the curve(AUC)was calculated using the receiver operating characteristic(ROC)curve. Further analysis was performed to count the number of Acanthamoeba pathogens found by the three staining methods under the same magnification field of view at the same site, and to clarify the diagnostic value of fluorescent staining technique for AK.<p>RESULTS: The sensitivity of HE staining was 69%(27/39)with a specificity of 92%; the sensitivity of PAS staining was 62%(24/39)with a specificity of 97%, and the sensitivity of fluorescent staining was 95%(37/39)with a specificity of 97%. There were differences in the sensitivity of the three staining methods for the diagnosis of AK(χ2=19.857, <i>P</i><0.001), and pairwise comparison revealed that the differences between HE staining and fluorescent staining, PAS staining and fluorescent staining for the diagnosis of AK were statistically significant(<i>P</i>=0.003,<0.001), while the difference in sensitivity between HE staining and PAS staining for the diagnosis of AK was not statistically significant(<i>P</i>=0.978). The maximum AUC was 0.960 for fluorescence staining, followed by 0.804 for HE staining and 0.794 for PAS staining, respectively. The median number of amoeba cysts detected by HE staining, PAS staining and fluorescent staining at the same site under the same magnification field of view was 4(0, 11), 2(0, 9)and 12(3, 33), respectively(χ2=56.561, <i>P</i><0.001). Pairwise comparison revealed that the differences in the number of amoeba cysts found by HE staining and fluorescence staining, PAS staining and fluorescence staining were statistically significant(<i>P</i><0.001), while the difference in the number of amoeba cysts found by HE staining and PAS staining was not statistically significant(<i>P</i>=0.210). Fluorescently stained histopathological sections make it easier to identify amoebic pathogens.<p>CONCLUSION:Fluorescent staining technique is more sensitive to histopathological diagnosis of AK than HE staining and PAS staining, which can significantly improve the positive rate of detection of amoebic pathogens.
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Objective@#To observe the permeability of four kinds of self-etching adhesives in aged glass-fiber-reinforced composite (GFRC).@*Methods@#After light polymerization following the manufacturers’ instructions, a total of 80 pieces of bisphenol-A-glycodal-methacrylate (Bis-GMA)+ polymethyl methacrylate (PMMA) based GFRC were randomly divided into two main groups: test group and control group, each group was then divided into four subgroups with 10 samples for each subgroup. While the test group was conducted to be aged through thermocycling at 5 ℃/55 ℃, the control group remained fresh. After the addition of a fluorescent dye (rhodamine-B-isothiocyanate), four self-etching adhesives AdperEasy One (AEO), S 3 BOND (S 3B), Tetric N-Bond Self-Etch (TNB), G-Bond (GB) were correspondently applied to the test and control groups and were light polymerized. Specimens were sectioned using hard tissue cutting and grinding system. Slices from each subgroup were observed under a confocal laser scanning microscope, the depth of dye permeation (DDP) under the surface of GFRC in each group was measured and the Results were statistically analyzed.@*Results@#The DDP of AEO was the deepest (32.58 ± 6.06) μm, and that of TNB was the shallowest (6.19 ± 1.38)μm among the four self-etching adhesive subgroups in the control group. The order of each group was AEO > GB > S 3B > TNB. The DDP of the four subgroups in the test group was significantly shallower than that in the control group (P < 0.05). The change in GB was the greatest (9.05 ± 2.35)μm/(28.93 ± 5.32)μm. In the test group, the DDP in AEO was the deepest (28.42 ± 5.32)μm, and the DDP in TNB was shallowest (1.93 ± 0.22)μm again. The order of each group was AEO > S 3B > GB > TNB. In the test group, while the layer of fluorescent dye of AEO and S 3 B could still be seen distinctly, that of TNB and GB was hard to recognize. @*The DDP of AEO was the deepest (32.58 ± 6.06) μm, and that of TNB was the shallowest (6.19 ± 1.38)μm among the four self-etching adhesive subgroups in the control group. The order of each group was AEO > GB > S 3B > TNB. The DDP of the four subgroups in the test group was significantly shallower than that in the control group (P < 0.05). The change in GB was the greatest (9.05 ± 2.35)μm/(28.93 ± 5.32)μm. In the test group, the DDP in AEO was the deepest (28.42 ± 5.32)μm, and the DDP in TNB was shallowest (1.93 ± 0.22)μm again. The order of each group was AEO > S 3B > GB > TNB. In the test group, while the layer of fluorescent dye of AEO and S 3 B could still be seen distinctly, that of TNB and GB was hard to recognize. @#The self-etching adhesives of AEO and S 3 B still have good permeation effect in this kind of aged GFRC, which can help to establish a good bond between these aged GFRC and the subsequent repair of composite resin.
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Objective To observe the effect of different fixative solutions on cancer cell morphology and membrane permeability. Methods Human pancreatic acinar epithelial carcinona(HPAC) cells of human pancreatic cancer and HeLa cells of human cervical cancer were fixed with 4 fixation solutions: Freshly prepared 0.25% paraformaldehyde solution; Freshly prepared 4% paraformaldehyde solution; 75% ethanol solution; 90% ethanol solution. The fixation lime is 30 minutes. PBS solution and complete medium were used as the controls. Cell morphology of each group was observed under optical microscope. Changes in cell jncmbrane permeability were observed by fluorescence staining with 7-aminoactinomycin (7-AAD) , which is not cell membrane permeable in intact cells but permeable in damaged cells. Hoechst33342 was used for staining both intact and damaged cells. Results The cells in the complete medium group were similar to unfixed cells in morphology, and the fluorescence staining of 7-AAD was the weakest. The cells in the complete medium group have typical eel! morphology and low 7-AAD permeability. The 0.25% paraformaldehyde solution group had similar cell morphology to the complete medium group, and the 7-AAD fluorescence staining was weak. The morphology of cells in the 4% paraformaldehyde solution group was typical, but the fluorescence staining of 7-AAD was strong. The cells in the 90% ethanol solution group showed swelling, with a larger volume than the unfixed cells and a stronger fluorescence staining of 7-AAD. The cell swelling in 75% ethanol solution group was not as obvious as that in 90% ethanol solution group, and the fluorescence staining of 7-AAD was strong. The cells in PBS group were round, and the fluorescence staining of 7-AAD was strong. Conclusion 0. 25% paraformaldehyde solution can not only fix tumor cells, but also maintain the integrity of cell membrane.
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Objective To compare the diagnostic value of fluorescent staining versus KOH wet-mount microscopy in detecting superficial fungal infection.Methods Totally,600 specimens from cases of clinically diagnosed superficial fungal infections and 102 from cases of clinically diagnosed Malassezia infection (including 54 cases of pityriasis versicolor and 48 cases of Malassezia folliculitis) were collected from the dermatology clinic of Tenth People's Hospital of Tongji University between July 2017 and February 2018.These specimens were subjected to fluorescent staining and KOH wet mount separately followed by direct microscopy,and the positive rate and average time for slide reading were compared between the two methods.Culture served as the gold standard method,and the missed diagnosis rate was compared between the two methods.Statistical analysis was carried out using chi-square test or Fisher's exact test for comparing enumeration data,and paired t test for comparing emeasurement data.Results Of the 600 specimens from clinically diagnosed superficial fungal infection cases,fungi were detected in 546 (91.00％) and 489 (81.50％) by fluorescent staining and KOH wet-mount microscopy respectively (x2 =22.83,P ＜ 0.05).Fluorescent staining showed significantly shorter average reading time (73.67 ± 13.56 s)compared with KOH wet-mount microscopy (87.12 ± 15.83 s,t =14.60,P ＜ 0.05).Among the 54 specimens from pityriasis versicolor cases,fluorescent staining and KOH wet-mount microscopy positive results in 51 (94.44％) and 50 (92.59％) specimens respectively (adjusted x2 =0,P ＞ 0.05),with the average reading time being 38.36 ± 8.79 s and 41.25 ± 15.67 s respectively (t =1.14,P ＞ 0.05).Of the 48 specimens from Malassezia infection cases,43 (89.58％) and 11 (22.92％) specimens were detected to be positive for fungi by fluorescent staining and KOH wet-mount microscopy respectively (x2 =43.34,P ＜ 0.05),and fluorescent staining showed shorter average reading time (42.14 ± 12.61 s) compared with KOH wet-mount microscopy (103.56 ± 9.48 s,t =17.83,P ＜ 0.05).Among the 600 specimens from superficial fungal infection cases,culture yielded fungi in 479.Moreover,476 specimens were found positive by fluorescent staining,and 3 were found negative (0.63％),while KOH wet-mount microscopy showed 465 positive results and 14 negative results (2.92％).There was a significant difference in the missed diagnosis rate between the two methods (x2 =7.25,P ＜ 0.05).Conclusion Compared with KOH wet-mount microscopy,fluorescent staining can increase the detection rate,reduce missed diagnosis rate and shorten reading time.
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Objective To evaluate the value of modified calcofluor white fluorescent staining in the histopathological diagnosis of subcutaneous mycosis,in order to provide a new method for histopathological diagnosis of subcutaneous mycosis.Methods A total of 21 lesional skin tissues were collected from patients with subcutaneous mycosis in the Affiliated Hospital of Chengde Medical University between 1987 and 2017,and embedded in paraffin.Then,each paraffin-embedded tissue section was cut into 4 4-μm-thick serial sections,and subjected to modified calcofluor white fluorescent staining,hematoxylin and eosin (HE) staining,periodic acid Schiff (PAS) staining and Gomori methenamine silver nitrate (GMS) staining respectively.Positive rates and staining outcomes were compared among the above staining methods.Statistical analysis was carried out with SPSS 19.0 software by using chi-square test for comparing the positive rates among the above 4 staining methods.Results Of 21 patients with fungal infections,14 (66.67％) were positive for modified calcofluor white fluorescent staining,5 (23.80％) for HE staining,6 (28.57％) for PAS staining,and 11 (52.38％) for GMS staining.The positive rate by modified calcofluor white fluorescent staining was significantly higher than that by HE staining and PAS staining (x2 =6.718,5.200,respectively,both P ＜ 0.05),while no significant difference was observed between the modified calcofluor white fluorescent staining and GMS staining (x2 =0.693,P =0.530).Conclusion The modified calcofluor white fluorescent staining is an accurate method for detecting fungi,and has a certain application value in the histopathological diagnosis of subcutaneous mycosis.
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Objective To evaluate the performance of an enhanced fluorescent staining for the rapid diagnosis of invasive mycosis, especially rare cases, considering the traditional culture method always leads to delays in clinical diagnosis for its time consuming. Methods Cases of invasive mycosis identified by fluorescent staining in our hospital from September, 2017 to September, 2018 were retrospectively analyzed. Three rare in-vasive infections were reported in this study. Clinical specimens were pretreated using standard procedures and then smeared on slides along with the enhanced fluorescent dye. Species of the pathogens were identified accord-ing to their morphology under fluorescent microscope. The traditional culture method was used as a standard method to identify the pathogenic species based on their colony morphology, followed by PCR and sequencing analysis for further confirmation. Results Three cases of invasive mycosis caused by rare pathogens of Talaro-myces marneffei, Mucorales and Prototheca were rapidly diagnosed with the fluorescent staining method. Sequen-cing results indicated the species were Talaromyces marneffei, Rhizopus arrhizus and Prototheca wickerhamii. Conclusions Fluorescent staining is a rapid, economic and direct method for the diagnosis of invasive mycosis. The morphology of fungi is clear and easy to identify after fluorescence staining, which could be used for indica-tive diagnosis of highly suspected invasive mycosis and serve as an important complement to the traditional cul-ture method, especially for the diagnosis of rare or uncultured fungal pathogens.
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Tuberculosis remains world's leading cause of death from a single infectious agent. Pulmonary tuberculosis (PTB) is the most common presentation of tuberculosis. Here we compare Ziehl-Neelsen (ZN), Gabbet's and Fluorescent staining (FS) methods to determine efficiency and cost effectiveness of diagnosing pulmonary tuberculosis from sputum samples. Three smears from 148 sputum samples of PTB cases were prepared and stained by the above methods. Of the samples examined, 71.6% and 53.4% and 85.8% cases were detected by Ziehl-Neelsen, Gabbet's and Fluorescence staining respectively. Fluorescent staining was found to be more reliable and cost effective method particularly when dealing with large sample loads than Ziehl-Neelsen and Gabbet's staining technique.
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· AIM:To assess the effect of bandage contact lens on the corneal epithelium healing condition,degree of pain and corneal surface after recurrent pterygium excision.· METHODS:Retrospective case-series study.A total of 64 patients (64 eyes) with recurrent pterygium who received treatment in the First Affiliated Hospital of Huzhou University from September 2015 to September 2017 were divided into Group A (34 cases with bandage contact lens group) and Group B (30 cases without bandage contact lens group).The healing status of corneal epithelium was evaluated by fluorescent staining between the two groups at 1d and 1wk after surgery.The degree of pain was assessed between the two groups at 2h,1d and 1wk after recurrent pterygium excision by visual analogue score.Computerized corneal topography was performed on all cases with recurrent pterygium before and 1 mo after successful excision surgery.Statistical analysis of surface regularity index (SRI),surface asymmetry index (SAI) and corneal astigmatism (CA),was done before and 1mo after surgery.· RESULTS:Average scores of corneal epithelium healing condition at 1d and 1wk were better in Group A than that in Group B (P＜0.01).The mean scores of pain values at 2h,1d and 1wk after surgery in Group A were significantly lower than that in Group B respectively (P＜ 0.01).The indicators reflecting corneal surface at 1mo after surgery,including SRI,SAI,CA,were significantly lower in Group A than that in Group B (P＜0.01),while they were not significantly different before surgery between the two groups (P＞0.05).· CONCLUSION:Bandage contact lenses could significantly promote the healing status of corneal epithelium,release pain response and improve corneal refractive status after recurrent pterygium excision.
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Objective To evaluate the application of novel fluorescent staining method with hypersensitivity and enhancement for microscopic examination of superficial fungal.Methods A total of 200 cases with clinically suspected superficial fungal infection were screened by the hypersensitive,enhanced fluorescent staining,calcofluor white staining and microscopic examination with 1.78 mol/L potassium hydroxide (KOH) based smear assay,respectively.The positive percentages of the different methods were calculated and compared.Results In the developed fluorescent staining examination,clean background was shown in the view field.The hyphae and spores displayed in bright blue,thus fungal morphology was so distinct that they were easy to be distinguished.Compared with calcofluor white staining and KOH based smear,the fluorescent staining showed high positive rate with significant difference respectively (x2 =17.984,P ＜ 0.05;x2 =32.063,P ＜ 0.05).Conclusion The novel fluorescent staining should be a rapid,accurate method for microscopic examination of fungi,which is worthy to be widely used in clinical laboratories for early diagnosis of fungal infection.
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OBJECTIVE: To investigate the protection of hepatocyte growth factor (HGF) on 5 kinds of tumor cells (B cell lymphoma cell line Raji, human acute myeloid leukemia cell line HL-60, cervical cancer cell line HeLa, prostate cancer cell line PC-3, and non-small cell lung cancer cell line A549) from apoptosis induced by etoposide (VP-16). METHODS: Normal control group, drug group, and HGF protection group were set. CCK-8 assay was used to measure proliferation inhibition on 5 kinds of tumor cells by VP-16. Quantitative and qualitative analysis on 5 kinds of tumor cells was performed through acridine orange (AO) fluorescent staining, flow cytometry, HE staining, and transmission electron microscopy. RESULTS: CCK-8 assay revealed that the concentrations of VP-16, which can significantly inhibit the proliferation of Raji, HL-60, PC-3, HeLa, and A549 cell lines, were 100, 1,400, 200, and 200 μg·mL-1, respectively. The typical morphologic changes of cell apoptosis were observed under transmission electronic microscope. Flow cytometry showed that the apoptotic rates of tumor cells in the drug groups were significantly higher than those in normal control group (P>0.01, P>0.05) and in HGF protection groups (P>0.01, P>0.05). AO and HE staining revealed that cells in normal control group appeared to have regular cell morphology, but the cells apoptotic rates in the drug groups were significantly higher than those in normal control groups (P>0.01, P>0.05) and in HGF protection groups (P>0.01, P>0.05). CONCLUSION: HGF can significantly protect 5 kinds of tumor cells from apoptosis induced by VP-16. The mechanism need further investigation.
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Se evaluó la coloración diferencial de fluorescencia modificada en Pseudomonas spp. aisladas de suelos de cultivos agrícolas del estado Sucre, a fin de observar eventos microscópicos relacionados con el ciclo celular. Cada especie de Pseudomonas identificada bioquímicamente se sembró en caldos incubados a temperatura ambiente, aerobiosis, durante 15, 20, 30 y 45 minutos, y 1, 24, 48 y 72 horas; luego, se elaboraron y colorearon los extendidos. En las 24 cepas de Pseudomonas identificadas, P. mendocina (41,67 por ciento), P. aeruginosa (37,50 por ciento) y P. putida (20,83 por ciento), se observaron variaciones de tinción en los diferentes tiempos de incubación como verde, amarilla y anaranjada, fluorescentes y de baja fluorescencia. La coloración emplea naranja de acridina que se intercala al ADN, provocando fluorescencia verde, e interactúa con el ARN provocando fluorescencia anaranjada; el decolorante remueve el naranja de acridina no unido al material genético y la fluoresceína de sodio produce color amarillo en bacterias que retienen suficiente cantidad de naranja de acridina. Las variaciones de tinción citoplasmática en Pseudomonas spp., están asociadas a la cantidad de ARN y ADN presente en la célula de acuerdo a la fase de su ciclo celular.
The modified fluorescence staining differential was evaluated using Pseudomonas spp. isolated from cultivated agricultural soils in the State of Sucre, in order to observe microscopic events related to the cellular cycle. Each species of biochemically identified Pseudomonas was inoculated into a broth and incubated at room temperature, aerobiosis, for 15, 20, 30 and 45 minutes and 1, 24, 48 and 72 hours; then, slides were made and stained. For the 24 identified strains of Pseudomonas, P. mendocina (41.67 percent), P. aeruginosa (37.50 percent) and P. putida (20.83 percent), staining variations such as green, yellow and orange, fluorescent and low fluorescence were observed for the different incubation times. The stain uses acridine orange that interacts with DNA by intercalation, causing green fluorescence; it interacts with RNA by electrostatic attraction causing orange fluorescence; the alcohol-acetone decolorant removes the acridine orange not united with the genetic material and sodium fluorescein produces a yellow color in bacteria that retain a sufficient amount of acridine orange. Cytoplasmatic staining variations in Pseudomonas spp., are associated with the amount of RNA and DNA present in the cells according to the phase of their cellular cycle.
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Orange acridine/analyse , Orange acridine/composition chimique , Fluorescence , Pseudomonas/classification , Pseudomonas/composition chimique , Analyse du Sol , Microbiologie , Biologie moléculaire , Biologie du SolRÉSUMÉ
Fluorescent staining method is a sensitive cytochemical staining method, it can reflect minimal changes of biochemical components of cells. The 7690-Xu fluorescent staining solution prepared with thioflavine can show the heterogeneo- us fluorescence of cells in different stages of differentiation. The younger cells with low degree of differentiation show blue fluorescence, the lower the degree the deeper the color. Along with the differentiation of cells, the blue color of fluorescence deflects to the red color of spectrum gradually, the blue color of fluorescence of cytoplasm changes gradually to bluish, bluish-green, and finally yellow color. The blue color of fluorescence of nucleus changes through greyish- blue to orangeyellow or orange-red.The cytoplasm of cells in various organs with high degree of differentiation appears yellow, and the nucleus orange-yellow or orange-red in color.