Fast Staining Method Research Articles

Comparative evaluation of ziehl neelsen staining and knowledge, attitudes and practices of laboratory personnel in relation to ziehl nielsen.

To describe laboratory personnel's attitude and practices toward phenol exposure during Ziehl Neelsen (ZN) acid fast staining method and to evaluate the feasibility of an alternate modified Kinyoun cold (MKC) stain. A total of 187 sputum samples were collected from suspected tuberculosis cases and stained by the MKC method and ZN stain and were read by an experienced microscopist and a researcher. A cross-sectional questionnaire survey of 35 laboratory personnel was also conducted. Modified Kinyoun cold stain gave sensitivity, specificity, positive and negative predictive values of 100%, 99.4%, 94.1% and 100%, respectively. Both stains corresponded with an agreement rate of 99.5%. Almost 94.7% of respondents reported that they worked in a closed area when staining and 57.1% did the staining method without ventilation. Material safety data sheet (MSDS) of phenol was not known to 77.1% of laboratory personnel. All of the participants (100%) in this study welcomed a similar, non heating method for acid-fast bacillus (AFB). There was significant association between those not comfortable with phenol exposure (77.1%) and complaints of irritation (48.6%) and headache (2.9%) [c2 = 10.98, r = 0.55, p = 0.001]. The MKC is suitable for use as a substitute for the ZN method for the demonstration of AFB in the primary diagnosis and treatment assessment of pulmonary tuberculosis. Focus should be given on educating laboratory staff on the hazards, risks and precautions associated with the phenol/ZN method.

Molecular Characterization of Cryptosporidium spp. Isolated From Immunocompromised Patients and Children

Background:Cryptosporidium is known to be one of the most important causes of diarrhea in children and immunocompromised patients. Genotype characterization of Cryptosporidium species in each region would help in the treatment of this disease, as well as to locate the source of infection and to prevent the disease.Objectives:This current research was conducted in order to analyze the molecular characterization of isolated Cryptosporidium spp. in the Southwest of Iran.Materials and Methods:In this survey, 390 fecal samples were collected from immunocompromised individuals and children under five-years-of-age. Parasitic infection was evaluated using wet mount preparation, formalin ether, a modified acid fast staining method and microscopic examination. Finally, a PCR-RFLP assay was performed on the extracted DNA collected from fecal samples that were positive for Cryptosporidium by the acid fast method.Results:Among the 390 fecal samples, 16 cases (4.1%) were infected with Cryptosporidium. Molecular and genotype characterization found the following protozoan species; 11 Cryptosporidium parvum (68.8%), 4 C. hominis (25%), and one case of C. meleagridis (6.2%).Conclusions:The present study emphasized the public health importance of Cryptosporidium spp. in the study area. In addition, it seems that zoonotic species are the most important causes of infection in the region. As far as we are aware this the first report of a C. meleagridis infection in Iran.

A Method for Sensitive Staining of DNA in Polyacrylamide Gels Using Basic Fuchsin

PAGE is a widely used analytical method to resolve components of a DNA mixture based on their size. Various DNA visualization methods including fluorescence, visible dye and silver have been used for the detection of gel-separated DNA, with each having different advantages and disadvantages in terms of sensitivity, safety and simplicity. A fast and sensitive visible dye-based staining method for DNA in polyacrylamide gels using basic fuchsin (BF) is described. As low as 10-20 pg of DNA can be visualized within 10 min; the sensitivity is fourfold more sensitive than that of SYBR® Gold stain, the most sensitive commercial fluorescent probe, but similar to silver staining kit from GE Healthcare. In addition, the mechanism studies suggest that the interaction of BF with DNA is mainly contributed by non-intercalative binding mode. By comprehensive studies of this visible dye-based protocol, we concluded that BF stain is a fast and sensitive method currently available for detecting DNA in polyacrylamide gels.

Teratogenic Effect of Crude Ethanolic Root Bark and Leaf Extracts of Rauwolfia vomitoria (Apocynaceae) on Nissl Substances of Albino Wistar Rat Fetuses.

Rauwolfia vomitoria is a plant used for the treatment of insanity. The possible adverse effects of crude ethanolic root bark and leaf extract of the plant on Nissl substances of albino Wistar rat fetuses were studied using 25 mature female Wistar rats. The animals were divided equally into 5 groups, labeled A, B, C, D, and E. Group A was the control, while groups B, C, D, and E were the experimental. The female rats were mated with the males overnight, and the sperm positive day was designated as day zero of pregnancy. Oral doses of 150 mg/kg and 250 mg/kg body weight of the root bark extract were administered to groups B and C animals, respectively, while groups D and E animals received 150 mg/kg and 250 mg/kg body weight of the leaf extract, respectively, from day 7 to 11 of gestation. On day 20 of gestation, the rats were sacrificed, the fetuses brains extracted, and the cerebral cortices excised and routinely processed for Nissl substances using Cresyl fast violet staining method. Results showed reduced staining intensity of Nissl substances in the treated groups, especially those that received the root extract. Thus, the herbs may have adverse effects on protein synthesis within the cerebral cortex.

Crypto-Giardia antigen rapid test versus conventional modified Ziehl-Neelsen acid fast staining method for diagnosis of cryptosporidiosis

ObjectiveTo evaluate the validity of Crypto-Giardia antigen rapid test (CA-RT) in comparison with the conventional modified Ziehl-Neelsen acid fast (MZN-AF) staining method for the diagnosis of cryptosporidiosis. MethodsFifteen preserved stool samples from previously confirmed infections were used as positive controls and 40 stool samples from healthy people were used as negative control. A total of 85 stool samples were collected from suspected patients with cryptosporidiosis over 6 months during the period from January till June, 2011. The study was conducted in the department of parasitology, central laboratory, Alnoor Specialist Hospital, Makkah, Saudi Arabia. All samples were subjected to CA-RT and conventional MZN-AF staining method. Validation parameters including sensitivity (SN), specificity (SP), accuracy index (AI), positive predictive value (PPV), and negative predictive value (NPV) were evaluated for both tests. ResultsOut of 15 positive controls, CA-RT detected 13 (86.7%) while MZN-AF detected 11(73.3%) positive cases. However, CA-RT detected no positive case in 40 normal controls but MZN-AF detected 2(5%) as positive cases. Based on the results, the SN, SP, AI, PPV and NPV were high in CA-RT than MZN-AF staining method, ie., 86.7%vs. 73.3%, 100%vs. 95%, 96.4%vs. 89.1%, 100%vs. 84.6% and 95.2%vs. 90.5%, respectively. Out of a total of 85 suspected specimens, CA-RT detected 7(8.2%) but MZN-AF detected 6(7.1%) cases as positive. ConclusionsCA-RT immunoassay is more valid and reliable than MZN-AF staining method.

Can Stem Cells Home to the Injured Intervertebral Disk Via Intravenous Infusion? A Murine Model

IntroductionPatients who suffer traumatic injury of the spine can also present with intervertebral disk injury, both of which are associated with low back pain (LBP). While conventional treatments mainly serve to alleviate pain, these treatments are not biological. Various biological therapies such as growth hormones/factors and cells have been studied for treatment of different conditions including that of IVD degeneration, which is also associated with LBP. In various animal models, such biological therapies have been demonstrated to slow or even reverse degeneration.1,2 The use of stem cells to treat injury is very promising due to their ability to regenerate and differentiate into other cell types—thus lies their potential to repair tissue damage. Of great interest is a population of cord-blood stem cells with expanded multipotency and mesenchymal stem cell (MSC)-like properties.3 These cells are reported to home to injured sites after intravenous (IV) injection and are demonstrated in a rat spinal cord injury model to induce beneficial effects to injured rats.4 The use of such homing stem cells is advantageous as this could potentially bypass invasive surgery. We investigated the potential of these stem cells to aid IVD repair by determining (1) if these cells could home to injured IVD after intravenous infusion and (2) induce any beneficial effects on the morphology of the injured IVD using a murine model of IVD disk injury. Materials and MethodsStem cells were isolated from human umbilical cord blood as previously reported.3 Animal ethics was approved by the CULATR at the University of HK, and NOD/SCID mice aged 6–8 weeks (5/group) were used. Mice were anaesthetized and X-rayed to identify the caudal disk levels to be punctured and unpunctured controls. A 31G needle was used to perpendicularly puncture disks, the wounds were sutured and mice underwent standard postoperative procedures. On the day of puncture or 1 week postpuncture, cells were thawed and resuspended in DMEM and injected via the tail vein (10E6). IVDs were harvested at 7 days or 1 month postcell injection and processed for histology. IVD morphology was visualized with the FAST staining method.5 Stem cells were tracked by staining for antihuman mitochondrial antibodies. ResultsFrom the FAST staining, the IVD morphology was disrupted after puncture of the disk with a large loss of cells from the NP and the loss of the AF/NP border (Figs. 1 and 2). Little difference in morphology of the disks were observed between groups with cells injected versus no cells injected after 1 month (Fig. 2), however, both yielded similar staining intensity of the NP matrix. Immunostaining for the stem cells indicated that few cells migrated to the punctured disk. In the sections that did yield cells, they were usually single cells and found in the growth plate area or adjacent to the EP blood vessels, but not in the NP or AF. Cells were also found in adjacent unpunctured disks, and also in other organs including the spleen and lungs. ConclusionThe intravenous introduction of the stem cells did not arrest degeneration nor regenerate the IVD after 1 month. Few cells were found to be located at the sites of puncture, and of the cells that were found, these were limited to the growth plate area or vessels around the end plate, but not in the NP or AF. It is possible that the little change in morphology of the IVD, after 1 month of injury and intravenous introduction of the stem cells, was due to the fact that few cells were able to migrate to the injured disk. This may have been caused by a number of factors including: (1) the homing signal that was not sufficient to attract the stem cells to the injured site, (2) the avascular nature of the IVD which further limited cell penetration to the injured site, and (3) that the cells lodged in other organs such as the lungs and spleen. Ongoing studies are being carried out to quantitatively determine any localized changes to the IVD including proteoglycan and gene expression levels.This work is supported by the Seed Funding Programme for Basic Research, The University of Hong Kong.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredHo G, et al. Connective Tissue Research 2008;49(1):15–21Yang F, et al. Molecular Therapy 2009;17(11):1959–1966Rogers I, et al. Experimental Cell Research 2007;313(9):1839–1852Chua SJ, et al. Spine (Phila Pa 1976), 2010;35(16):1520–1526Leung VY, et al. Journal of Histochemical Cytochemistry 2009;57(3):249–256

Can Stem Cells Home to the Injured Intervertebral Disk Via Intravenous Infusion? A Murine Model

Introduction Patients who suffer traumatic injury of the spine can also present with intervertebral disk injury, both of which are associated with low back pain (LBP). While conventional treatments mainly serve to alleviate pain, these treatments are not biological. Various biological therapies such as growth hormones/factors and cells have been studied for treatment of different conditions including that of IVD degeneration, which is also associated with LBP. In various animal models, such biological therapies have been demonstrated to slow or even reverse degeneration.1,2 The use of stem cells to treat injury is very promising due to their ability to regenerate and differentiate into other cell types—thus lies their potential to repair tissue damage. Of great interest is a population of cord-blood stem cells with expanded multipotency and mesenchymal stem cell (MSC)-like properties.3 These cells are reported to home to injured sites after intravenous (IV) injection and are demonstrated in a rat spinal cord injury model to induce beneficial effects to injured rats.4 The use of such homing stem cells is advantageous as this could potentially bypass invasive surgery. We investigated the potential of these stem cells to aid IVD repair by determining (1) if these cells could home to injured IVD after intravenous infusion and (2) induce any beneficial effects on the morphology of the injured IVD using a murine model of IVD disk injury. Materials and Methods Stem cells were isolated from human umbilical cord blood as previously reported.3 Animal ethics was approved by the CULATR at the University of HK, and NOD/SCID mice aged 6–8 weeks (5/group) were used. Mice were anaesthetized and X-rayed to identify the caudal disk levels to be punctured and unpunctured controls. A 31G needle was used to perpendicularly puncture disks, the wounds were sutured and mice underwent standard postoperative procedures. On the day of puncture or 1 week postpuncture, cells were thawed and resuspended in DMEM and injected via the tail vein (10E6). IVDs were harvested at 7 days or 1 month postcell injection and processed for histology. IVD morphology was visualized with the FAST staining method.5 Stem cells were tracked by staining for antihuman mitochondrial antibodies. Results From the FAST staining, the IVD morphology was disrupted after puncture of the disk with a large loss of cells from the NP and the loss of the AF/NP border (Figs. 1 and 2). Little difference in morphology of the disks were observed between groups with cells injected versus no cells injected after 1 month (Fig. 2), however, both yielded similar staining intensity of the NP matrix. Immunostaining for the stem cells indicated that few cells migrated to the punctured disk. In the sections that did yield cells, they were usually single cells and found in the growth plate area or adjacent to the EP blood vessels, but not in the NP or AF. Cells were also found in adjacent unpunctured disks, and also in other organs including the spleen and lungs. Conclusion The intravenous introduction of the stem cells did not arrest degeneration nor regenerate the IVD after 1 month. Few cells were found to be located at the sites of puncture, and of the cells that were found, these were limited to the growth plate area or vessels around the end plate, but not in the NP or AF. It is possible that the little change in morphology of the IVD, after 1 month of injury and intravenous introduction of the stem cells, was due to the fact that few cells were able to migrate to the injured disk. This may have been caused by a number of factors including: (1) the homing signal that was not sufficient to attract the stem cells to the injured site, (2) the avascular nature of the IVD which further limited cell penetration to the injured site, and (3) that the cells lodged in other organs such as the lungs and spleen. Ongoing studies are being carried out to quantitatively determine any localized changes to the IVD including proteoglycan and gene expression levels. This work is supported by the Seed Funding Programme for Basic Research, The University of Hong Kong. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Ho G, et al. Connective Tissue Research 2008;49(1):15–21 Yang F, et al. Molecular Therapy 2009;17(11):1959–1966 Rogers I, et al. Experimental Cell Research 2007;313(9):1839–1852 Chua SJ, et al. Spine (Phila Pa 1976), 2010;35(16):1520–1526 Leung VY, et al. Journal of Histochemical Cytochemistry 2009;57(3):249–256

Inquisition of the Human Intervertebral Disk Histopathology with the Use of Two Histological Staining Methods

IntroductionChronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown.Materials and MethodsHuman disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination.ResultsThe FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&E only.ConclusionThe combined use of H&E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredLeung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249

Inquisition of the Human Intervertebral Disk Histopathology with the Use of Two Histological Staining Methods

Introduction Chronic low back pain (LBP) affects over 10% of the adult population resulting in profound decreases in the quality of life. The intervertebral disk (IVD) is prone to degeneration which may contribute toLBP. To prevent disk degeneration-related LBP, better understanding of disk structure and the degenerative process is needed to understand the underlying mechanisms. Standard Hematoxylin and Eosin (H&E) staining, alone or in combination with other dyes, have been used to assess the normal biology and degeneration of IVDs. Recently, the multichromatic FAST (Alcian blue, Safranin-O, Fast green and Tartrazine) staining method has been developed (Leung V.Y.L. et al., 2009). This method provides additional information unavailable through conventional methods such as differentiation of the structures within the disk and identification of degenerative matrix remodeling through changes in the staining pattern. Although the FAST method has proved helpful in animal models to assess alteration of glycosaminoglycan content, its usefulness to assess human disk histopathology remains unknown. Materials and Methods Human disks were obtained (a) surgically from chronic LBP patients with moderate-severe disk degeneration or (b) postmortem from transplant donors. Disks were cut into quadrants, fixed in 4% paraformaldehyde with 14% (v/v) saturated picric acid for 3 days, embedded in OCT and stored at −80°C. Tissue samples were cut on a cryostat and transverse sections of 14 um and 20 um were obtained for H&E and FAST staining, respectively. Slides were coverslipped with DPX and scanned using a Zeiss MIRAX Scan Digital Slide Scanner for further examination. Results The FAST protocol creates high-resolution and polychromatic images of the general disk structure, and reveals the overall organization of the disk. The transition from the nucleus pulposus (NP) to the inner annulus fibrosus (IAF) and the outer annulus fibrosus (OAF) was clearly visible through color boundaries. Signs of degeneration could be observed with the FAST staining method in both IVDs obtained postmortem with no history of LBP and from chronic LBP patients. For example, FAST staining uncovered the loss of a clear boundary between the NP and the IAF that was not visible with H&E. These data suggest that relevant pathological details might be missed if assessing histological changes with the use of H&E only. Conclusion The combined use of H&E and FAST methods provides a more complete picture of the anatomy and physiology of human IVDs. The FAST profile distinguishes IVD compartments and shows matrix remodeling events within the disk, while H&E provides complementary information regarding the cellular content of the tissue. These two staining methods complement each other to give a clearer differentiation of IVD sub-regions of both normal and degenerated disk architecture. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Leung V.Y.L. et al. Journal of Histochemistry and Cytochemistry 2009;57:249

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

Introduction Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy. Materials and Methods Degenerated lumbar disks (grade 3, Schneiderman's classification) ( n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks ( n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG ( n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination. Results Human fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks. Conclusion Human fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Yang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121 Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

IntroductionCadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy.Materials and MethodsDegenerated lumbar disks (grade 3, Schneiderman's classification) (n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks (n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG (n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination.ResultsHuman fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks.ConclusionHuman fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredYang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

Evaluation of demographic, clinic and treatment features of patients and a cross-sectional survey of cyclosporiasis in patients with diarrhea in Southeastern Turkey

In this research, we aimed at reporting the results of a cross-sectional epidemiological scanning performed on an outbreak of cyclosporiasis, occurring in a family and patients' socio-demographic epidemiological, clinical, diagnostic and therapeutic features, after detecting Cyclospora oocysts on stool sample of a person admitted to gastroenterology polyclinic. Scanning was performed in the neighborhood of the patient. The investigation group consisted of 75 individuals with diarrheal occurring from neighbor and family of patients. A questionnaire was performed for information on epicrisis of diarrheal persons and the samples were collected in stool containers. The samples were examined with native-lugol, sedimentation and modified acid fast staining methods. The stools were cultured in Salmonella-Shigella agar medium to investigate their bacteriological properties. The different vegetables from the mobile market place (peddler) founded in the neighborhood and water samples from house were collected to detect the infection source. Parasitosis (single or mix parasite) were encountered in 20 out of 75 persons in the examined samples (26.6%) and C. oocysts were detected in 13 out of 75 persons (17.3%). Out of the total number of patients having cyclosporiasis, none has immunodeficiency and chronic diseases. All cases were determined in the month of July. Oocysts were detected in six different families. Bacteria were not cultivated in stool cultures and occult blood was negative. The agent was not encountered in green vegetables, though water samples were examined to detect infection resource. Examination of the samples for Cyclospora was not neglected in diarrhea individuals; as such an examination was performed for the source of transmission of the infection.Cyclospora may generate family infection in individuals and if detected in one individual of a family, all the family individuals were examined for this infection. Key words: Cyclospora cayetanensis, diarrhea, familial transmission, Southeastern Turkey.

Intestinal Protozoa in Immunosuppression: A Medical Hassle

Introduction: Immunity forms the backbone of our very existence and opportunistic infections have plagued the immunosuppressed since long. A significant morbidity and mortality is attributable to intestinal parasitosis in the immunocompromised population. Objectives: This study aims to study the prevalence of intestinal protozoa in various immunosuppressed groups and to correlate degree of immunosuppression with protozoal infections. Material and Methods: The study was conducted on 400 patients divided into 4 groups: Group I (HIV patients), Group II (patients on chemotherapy/chemotherapy and radiotherapy for various malignancies), and Group III (diabetes patients) and Group IV included children presenting with diarrhea. Group I, II and III included patients with and without gastrointestinal symptoms. Stool samples were investigated microscopically for cysts/trophozoites after concentration with formol-ether method. Iodine wet mount and Modified acid fast staining methods were used. Result: Intestinal protozoa were detected in 40.75% (163/400); more commonly in patients with higher degree of immunosuppression (CD4 count 7: 95.3% in diabetics and moderate to severe malnutrition: 61.8%. Most common protozoa associated was Cryptosporidium in 66 patients (40.5%) followed by, Entamoeba histolytica in 48 (29.4%), Giardia lamblia in 35 (21.5%), Isosporabelliin 9 (5.5%), Blastocystis hominis in 4 (2.5%), Cyclospora cayetenensis in 1 (0.61%). Conclusion: High prevalence of intestinal protozoa was seen in all groups of immunosuppressed patients and a significant association seen between degree of immune suppression and protozoal infection. Focus needs to be on routine screening of all immunosuppressed patients and their immune reconstitution.

English

The aim of the present study was to evaluate the diagnostic value of nested PCR in genitourinary tuberculosis (GUTB) compared with acid fast staining and culture method. In total 200 urine samples from suspected cases of GUTB were collected during the period of study. Urine pellets were used for smear preparation, culture and DNA extraction by ether-chloroform method. Nested PCR was performed according to standard protocol using primers based on IS6110 gene fragment. The results obtained by PCR were compared with those obtained by standard acid-fast bacilli stain and culture method. Based on obtained results, the positivity rate of urine samples in this study was 5.0% by using culture and PCR methods and 2.5% for acid fast staining. Four out of total samples showed positive results in all three methods (2%). The sensitivity of PCR in this study was estimated as high as culture equal to 100%, while the sensitivity for direct smear staining was 41.6%. In conclusion, the obtained rate of GUTB in our study was 5.0%. Since the detection rate of culture and nested PCR was identical, we could suggest PCR as a rapid alternative to culture especially for confirmed cases of GUTB.

Cold acid fast staining method: Efficacy in diagnosis of Mycobacterium tuberculosis

Ziehl Neelson, a carbol fuschin based stain, using the 3 component hot process is a standard staining technique for staining sputum smears for detection ofMycobacterium tuberculosis. We evaluated a 2 step cold staining method for detection of acid fast bacilli in sputum smears. 1836 sputum samples from patients of pulmonary tuberculosis were studied. Smears were prepared from each sample and slides were simultaneously stained by both methods and examined. The 2 step cold stain method was found to be equally sensitive as the ZN method detecting 20% of cases, when the primary stain was kept for a period of 20 min. The CS method also has the advantage of being simple, economical and less cumbersome and in the procedure of staining, heating, a much too precise step in ZN method for large scale application, is eliminated. The CS method may therefore prove as a good alternative for diagnosis of M.tuberculosis, particularly in smaller laboratories and peripheral centers where there is limitation of the funds and equipments. Key words: Cold stain, acid fast bacilli, Mycobacterium tuberculosis.

Effects of Proteins from Culture Medium on Surface Property of Silanes- Functionalized Magnetic Nanoparticles

Monodisperse magnetic nanoparticles (MNPs) were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2), short-chain poly(ethylene glycol) (PEG), and carboxylic group (COOH). Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs) were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs.

Matrix Remodeling During Intervertebral Disc Growth and Degeneration Detected by Multichromatic FAST Staining

Various imaging techniques have been used to assess degeneration of the intervertebral disc, including many histological methods, but cartilage-oriented histological stains do not clearly show the comparatively complex structures of the disc. In addition, there is no integrated method to assess efficiently both the compartmental organization and matrix composition in disc samples. In this study, a novel histological method, termed FAST staining, has been developed to investigate disc growth and degeneration by sequential staining with fast green, Alcian blue, Safranin-O, and tartrazine to generate multichromatic histological profiles (FAST profiles). This identifies the major compartments of the vertebra-disc region, including the cartilaginous endplate and multiple zones of the annulus fibrosus, by specific FAST profile patterns. A disc degeneration model in rabbit established using a previously described puncture method showed gradual but profound alteration of the FAST profile during disc degeneration, supporting continual alteration of glycosaminoglycan. Changes of the FAST profile pattern in the nucleus pulposus and annulus fibrosus of the postnatal mouse spine suggested matrix remodeling activity during the growth of intervertebral discs. In summary, we developed an effective staining method capable of defining intervertebral disc compartments in detail and showing matrix remodeling events within the disc. The FAST staining method may be used to develop a histopathological grading system to evaluate disc degeneration or malformation.

Comparison of Multiplex PCR and Acid Fast and Auramine -Rhodamine Staining for Detection of Mycobacterium tuberculosis and Non tuberculosis Mycobacteria in Paraffin- Embedded Pleural and Bronchial Tissues with Granulomatous Inflammation and Caseous Necrosis

Objective The aim of this study was to compare the sensitivity and specificity of Acid fast and Auramine-Rhodamine staining and Multiplex PCR for the detection of Mycobacterium tuberculosis complex and non tuberculosis Mycobacteria on formalin fixed paraffin embedded tissues (FFPE) Materials and Methods Forty cases of FFPE pleural and bronchial tissue with chronic granulomatous inflammation and caseous necrosis and 10 cases with bronchogenic carcinoma as controls were investigated. We designed a Multiplex PCR DNA amplification method with two targets: 123bp DNA fragment from IS6110, which is present only in mycobacterium tuberculosis complex and 162bp DNA encoding Ag 85complex which is present in all of mycobacteria. The FFPE also stained by Acid fast and Rhodamine-Auramine staining method. Results In 26 samples (65%) 123 bp and 162 bp DNA fragments were detected together (12 in bronchial samples and 14 in pleural samples).The 162 bp fragment wasn't detected alone. The sensitivity of PCR was 65% and the specificity was 100%. Eleven cases were positive for Acid fast staining. There was 27.5% sensitivity and 100% specificity. Thirteen cases were positive for Auramine-Rhodamine staining (A-R-S); there was 32.5% sensitivity and 100% specificity. All of the 10 controls were negative for 123 bp, 162 bp DNA fragments, for Acid fast and Auramine-Rhodamine staining. Conclusion Multiplex PCR is a sensitive, specific and rapid method for detection of M. tuberculosis in FFPE tissues.

A simple staining method for observation of germinatedStrigaseeds

AbstractIn vitrotechniques are essential forStrigaresearch and the development of appropriate control methods. In the laboratory, pre-screening of non-host or false-host plants ofStrigafor trap cropping or the screening of hosts for resistance involves visual evaluation ofStrigaseed germination that may be stimulated by plant parts or root exudates. This technique is presently laborious because the smallStrigaradicles are colourless. A number of solutions were evaluated to visualize the radicles ofStriga hermonthicato obtain a reliable, simple and fast staining method yielding good contrast for visual observation, with readily available, inexpensive and minimally toxic dyes and staining solutions. The inks Brilliant Blue (Pelikan), Blue (Geha) and Brilliant Green (Pelikan), in either vinegar or lactic acid, produced radicles with excellent contrast, whereas radicles stained with Brilliant Red (Pelikan), Black (Sheaffer), Brilliant Black (Pelikan) gave good contrast.Strigaradicles stained with Aniline Blue in vinegar or lactic acid, or Coomassie Brilliant Blue R250 in lactic acid, showed excellent contrast. Radicles stained with Ink Black (Sheaffer), Cotton Brown, or Rubin S in either vinegar or lactic acid also showed good contrast. With water as the diluent, only Lactophenol Blue showed excellent contrast. For health, safety and environmental concerns, availability and staining time, Blue ink in household vinegar (5% acetic acid) appears to be an excellent dye forStrigaradicles and could be used in routineStrigagermination assays.

A Fast Histochemical Staining Method to Identify Hyaline Droplets in the Rat Kidney

A Fast Histochemical Staining Method to Identify Hyaline Droplets in the Rat Kidney