Fluorescent Staining Method Research Articles

In vitro susceptibility of oral Candida albicans strains to different pH levels and calcium hydroxide saturated aqueous solution

Candida albicans is present in the oral cavity and in the whole digestive tract of humans and other animals, being frequently related to endodontic treatment failure. The present study determined the incidence of C. albicans in the oral cavity and the susceptibility of isolates to different pH values and saturated calcium hydroxide aqueous solution at pH 12.5. Sixty-five patients attending the Endodontic Clinic at the Sagrado Coração University participated in the study. The collected samples were cultivated in selective media for C. albicans and the isolates were tested in terms of resistance to both alkaline pH and saturated aqueous solution of calcium hydroxide. In relation to time variables, yeast viability was assessed by the Sabouraud's agar culture and fluorescein diacetate and ethidium bromide fluorescent staining method. Results from the different pHs and experimental times, including those from different techniques measuring fungal viability, were compared using the chi-square and Fisher's exact tests (α=0.05). The yeasts became completely inviable after 48 h of contact with the calcium hydroxide solution. On the other hand, when exposed to the alkaline culture broth, the yeasts were found to be viable at pHs 9.5 and 10.5 for up to 7 days. In conclusion, C. albicans can only be completely inhibited by direct contact with saturated calcium hydroxide aqueous solution after 48 h of exposure.

Effectiveness of adsorbed film and gel layer in hydration lubrication as adaptive multimode lubrication mechanism for articular cartilage

With various daily activities, the effectiveness of adsorbed film formed on a gel-like layer at the uppermost superficial articular cartilage in natural synovial joints becomes important to control the friction and wear of articular cartilage in mixed or boundary lubrication regime as an adaptive multimode lubrication mechanism. Furthermore, in the case where the adsorbed film has been removed, the proteoglycan gel layer is expected to preserve low friction and protect against the wear of bulk cartilage tissue with an effective hydration lubrication mechanism. Besides, it is indicated that the biphasic lubrication plays an important role in lowering of friction in articular cartilage containing a large amount of water. At the present stage, however, the detailed relationship between adsorbed films and hydrated gel layers has not yet been elucidated. In this article, the frictional behaviours of articular cartilage on a glass plate were observed in the reciprocating tests with the restarting process after interruption and unloading. The lubricating effectiveness of adsorbed films in hyaluronate (HA) solutions was examined using intact and partially damaged cartilage specimens. The role of albumin and γ-globulin in relation to the surface conditions of gel layer is discussed. The restarting friction immediately after reloading became lower as a result of recovery of the effective interstitial fluid pressurization and hydration and adsorbed films have a significant effect on the frictional behaviour at local contacts. To clarify the molecular phenomena taking place under rubbing condition, in situ observations of the forming adsorbed film were conducted. The measurements were performed using the fluorescent staining method for protein and HA molecules at low contact pressures.

The fate of the larval epidermis in the Desor-larva of Lineus viridis (Pilidiophora, Nemertea) displays a historically constrained functional shift from planktotrophy to lecithotrophy

Pilidiophora constitutes a clade of nemerteans characterized by a peculiar larval type, the pilidium. A characteristic of this larva is the transitory epidermis in which the juvenile develops from imaginal discs. The primary function of this larval envelope is assumed to be feeding and dispersal. When juvenile development is complete, the larval epidermis is ruptured and swallowed by the juvenile. According to recent cladistic and molecular analyses of the Nemertea, the intracapsular Desor-larva of the sibling species Lineus viridis and L. ruber is thought to have evolved from a pelagic pilidium. The general course of development has been demonstrated to be similar to that of the pilidium, in which the juvenile forms from imaginal discs under the larval epidermis. The two Lineus species, however, differ in their mode of larval feeding: L. ruber being ootrophic and L. viridis being lecithotrophic. In order to elucidate the transition from the planktotrophic pilidum to lecithotrophic development, I studied the early cleavage and metamorphosis from intracapsular Desor-larva to juvenile stages in L. viridis from the island of Sylt, using light microscopical, electron microscopical, and fluorescent staining methods. Due to the specific cleavage pattern with equally sized 1st quartet animal blastomeres and vegetal blastomeres in L. viridis, the larval epidermis later contains a considerable amount of the yolk reserve. During metamorphosis, the larval epidermis is ingested by the juvenile thus displaying behavior similar to that of the pilidium larva. In contrast to the pilidium, the function of the larval epidermis of the Desor-larva has shifted from feeding and dispersal to direct food supply. Thus, the development of L. viridis is a perfect example for strong historical constraints that prevent ancestral larval structures from being lost.

Assessment of apoptosis by using ethidium bromide/acridine orange fluorescence staining method in lymphocyte cell cultures

Assessment of apoptosis by using ethidium bromide/acridine orange fluorescence staining method in lymphocyte cell cultures

Starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus cells

Objective To investigate the starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus cells. Methods Rat annulus fibrosus cells in the logarithmic growth phase were cultured with amino acid free EBSS (Earle' s balanced salt) medium instead of the DMEM-F12 medium.Cells were collected at 1, 2 and 3 h after EBSS culture. Autopagosomes in annulus fibrosus cells were detected with monodansylcadaverine ( MDC) fluorescent staining method. Reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting analyses were used to detect the expression of microtubuleassociated protein 1 light chain 3 (LC3 ) and Beclin-1 in annulus fibrosus cells. Results Compared with the control group, MDC positive cells and autopagosomes were increased with the prolongation of starvation.LC3-Ⅱ and Beclin-1 mRNA expression levels in annulus fibrosus cells were increased with the prolongation of starvation (LC3-Ⅱ : 0.21 ±0.02, 0.45 +0. 12, 0.60±0.05, 0. 74 ±0. 03,P＜0. 05; Beclin-1: 0.20 ±0.06, 0. 37 ±0. 11, 0.52 ±0.07, 0. 69 ±0. 07,P＜0. 05). The ratio of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 protein expression was also increased with the prolongation of starvation (LC3-Ⅱ/LC3- Ⅰ : 0. 12 ±0.05,0.32 ±0.09, 0.63 ±0.07, 0. 92 ±0. 04,P＜0.05; Beclin-1: 0.61 ±0. 10, 0. 83 ±0. 11, 1.09 ±0.05,1. 35 ± 0. 10, P ＜ 0. 05 ). Conclusion Autophagy of annulus fibrosus cells can be induced by starvation,which may be related to the procession of intervertebral disc degeneration. Key words: Intervertebral disc degeneration; Annulus fibrosus cell; Autophagy

The value of proper sputum collection instruction in detection of acid-fast bacillus

This study was conducted to assess clinically whether intervention with instruction applying respiratory rehabilitation method for expectorating sputum was useful or not to obtain more suitable sputum for smear examination of acid-fast bacilli. All specimens examined were sputa obtained from 163 patients without the instruction group and 161 patients with the instruction group, who visited our outpatients clinic during one year from September 1, 2000 to August 31, 2001 and the following one year, respectively. Gross appearance of the sputum according to Miller & Jones' classification (M1, M2, P1-P3) and smear positive rate by fluorescence staining method after N-Acetyl-L-cysteine-NaOH treatment and centrifugation were compared between the two groups. M1 and P1 sputa were 21.5% and 21.5% in the no instruction group, while those were 8.1%, 36.6% in the instruction group. Difference in M2, P2 and P3 sputa were not significant between the groups. Smear positive rate was 10.4% in the no instruction group, while it was 21.1% in the instruction group. According to gross appearance of M2, P1 and P2, positive rate was 11.1%, 11.4% and 30.8% in the no instruction group, and 17.7%, 28.8%, and 26.3% in the instruction group. Chest roentogenographic findings judged by type (cavitary and non-cavitary) and extent of the pulmonary lesions of these smear positive cases (17 in the no instruction group and 34 with the instruction group), revealed that the no instruction group consisted of more predominantly severer disease with cavity and moderately or far advanced lesions as compared with the instruction group. CONSIDERATION: We could exclude that the difference in gross appearance and smear-positive rate of the sputum specimen between groups without and with the instruction might be due to differences in disease severity between the two groups with and without the instruction. The instruction for sputum expectoration seems to be useful to increase positive rate in the smear examination of acid-fast bacilli.

Skeletal muscle tissue from the Goto-Kakizaki rat model of type-2 diabetes exhibits increased levels of the small heat shock protein Hsp27

In order to increase our understanding of diabetes-related muscle weakness, we carried out a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the Goto-Kakizaki rat model of type-2 diabetes. Fluorescence difference in-gel electrophoresis was performed to determine potential differences in the global protein expression profile of muscle extracts. Besides changes in contractile proteins and metabolic enzymes, the abundance of the small stress proteins αB-crystallin and Hsp27 was significantly increased. The up-regulation of the low-molecular-mass heat shock protein Hsp27 was confirmed by an alternative fluorescent staining method of two-dimensional gels and immunoblotting. The observed protein alterations in the cellular stress response, distinct metabolic pathways, regulatory mechanisms and the contractile apparatus might be directly or indirectly associated with peripheral resistance to insulin signalling, making these newly identified muscle proteins potential biomarkers of type-2 diabetes. Increased levels of molecular chaperones suggest considerably enhanced cellular stress levels in diabetic muscle fibres.

Antibacterial properties and human gingival fibroblast cell compatibility of TiO2/Ag compound coatings and ZnO films on titanium-based material

Titanium (Ti)-based materials are widely used in biomedical implant components and are applied successfully in various types of bone-anchored reconstructions. However, in dental implants the Ti materials contact not only bone but also gingival tissues, and are partially exposed to the oral cavity that includes bacteria. This study used titania and silver (TiO(2)/Ag) compound coatings and zinc oxide (ZnO) films to enhance the antibacterial activity of the Ti-based implant. The hydrophobicity of each sample was examined by measuring the contact angle. Streptococcus mutans and human gingival fibroblast (HGF) was cultured on the coated samples, and the antibacterial effects and cell compatibility were determined using a Syto9 fluorescence staining and MTT methods. For the TiO(2)/Ag samples, depositing Ag on the plate at a higher power (which increased the proportion of Ag) increased the contact angle and the hydrophobicity. The bacterial count was lowest for the 50 W TiO(2)/Ag sample, which contained 5.9% Ag. The contact angles of the ZnO samples did not show the same tendency. The antibacterial effect was higher on ZnO-coated samples since bacterial count was threefold lower on ZnO samples as compared to control samples (Ti plate). From the MTT assay test, the mean optical density values for TiO(2)/Ag-coated samples after 72 h of HGF adhesion were similar to the value obtained from the uncoated Ti. However, biocompatibility was lower on ZnO films than in control samples. Conclusively, the antibacterial activity was higher but the cell compatibility was lower on ZnO films than on TiO(2)/Ag coatings.

DNA binding, prominent DNA cleavage and efficient anticancer activities of Tris(diimine)iron(ii) complexes

The complexes rac-[Fe(diimine)(3)](ClO(4))(2)1-4, where diimine = 2,2'-bipyridine (bpy) 1, 1,10-phenanthroline (phen) 2, 5,6-dimethyl-1,10-phenanthroline (5,6-dmp) 3 and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) 4, have been isolated, characterized and their interaction with calf thymus DNA studied by using a host of physical methods. The X-ray crystal structure of rac-[Fe(5,6-dmp)(3)](ClO(4))(2)3 has been determined and the packing diagram shows the presence of two enantiomeric forms of the complex cations in the same unit cell. The structures of 1-4 in solution have also been studied using UV-Visible, Cyclic Voltammetry and ESI-MS data and all data available suggests that they retain their solid state structures even in solution. The absorption spectral titrations of the iron(ii) complexes with CT DNA reveal that the DNA binding affinities of the complexes vary in the order, 4 (K(b): 9.0 × 10(3)) > 2 (6.8 × 10(3)) > 3 (4. 8 × 10(3)) > 1 (2.9 × 10(3) M(-1)). The DNA interaction of dpq complex (4) involves partial insertion of the extended phen ring in between the DNA base pairs, which is deeper than that of phen (2). The 5,6-dmp (3) complex is involved in groove binding in the major groove of DNA. The lower DNA binding affinity of 1 is due to electrostatic interaction of the cationic complexes with exterior phosphates of DNA. The EthBr displacement assay and DNA viscosity study support these DNA binding modes and the above trend in DNA binding affinities. The complexes of 1 and 2 show induced CD (ICD) upon interaction with CT DNA while 3 and 4 bound to DNA exhibit inversion in the positive band with the helicity band showing very small changes, which implies that 3 and 4 bind enantiopreferentially to DNA. The DNA cleavage abilities of 1-4 have been observed at 10 μM concentration of complexes in the presence of 100 μM H(2)O(2) and the DNA cleavage efficiency (> 90%) follows the order 3 > 1 > 2 > 4. The anticancer activity of 1-4 against human breast cancer cell line (MCF-7) has also been studied. The IC(50) values of the complexes at different incubation time intervals of 24 and 48 h follow the order, 3 (0.8, 0.6) < 4 (20.0, 17.0) < 2 (28.0, 22.0) < 1 (32.0, 29.0 μM). Interestingly, 3 exhibits anticancer activity more potent than 1, 2 and 4 and cisplatin for both 24 and 48 h. It induces cell death both through apoptosis and necrosis mechanisms, as revealed by morphological assessment data obtained by using AO/EB and Hoechst 33258 fluorescence staining methods.

Inactivation of Cryptosporidium by ozone and cell ultrastructures

The fluorescence staining method and scanning electron microscopy (SEM) were used to study the effect of ozone (O 3) inactivating Cryptosporidium in water and cell ultrastructures variation to shed light on the mechanism of inactivation preliminarily. Results indicated that O 3 had a stronger inactivating capability. When the concentration of O 3 was above 3.0 mg/L and the contact time was up to 7 min, a significant inactivating effect could be achieved. The turbidity on inactivation effects was also found to be statistically significant in artificial water. With increases in turbidity, the inactivating effect decreased. Inactivation rate improved with a temperature increase from 5 to 25°C, but decreased beyond this. The inactivating capability of O 3 was found to be stronger under acidic than that under alkalic conditions. When the concentration of organic matter in the reaction system was increased, the competition between Cryptosporidium and organics with O 3 probably took place, thereby reducing the inactivation rate. In addition, the cellular morphology of Cryptosporidium varied with different contact times. At zero contact time, cells were rotundity and sphericity, at 60 sec they became folded, underwent emboly, and burst at 480 sec, the cell membrane of Cryptosporidium shrinked and collapsed completely.

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time-to-results from 2-5 times shorter than the traditional testing method. The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.

The CyScope® Fluorescence Microscope, a Reliable Tool for Tuberculosis Diagnosis in Resource-Limited Settings

Poor laboratory equipment and few human resources have made it difficult to implement microscopic diagnosis of pulmonary tuberculosis (TB) on a large scale basis worldwide. Three hundred sputum samples from patients in Cameroon were studied by using the CyScope®, a new light-emitting, diode-based, fluorescence microscope, to compare auramine-rhodamine fluorescence with the conventional Ziehl-Neelsen staining method. Five fluorescence protocols were tested to reduce manipulation time. Smear positivity for acid-fast bacilli with the Ziehl-Neelsen staining method was 27.7% (83 of 300) compared with 33.3% (100 of 300) with the fluorescent method. Staining time with the modified fluorescence protocol could be reduced from 21 minutes to 10 minutes. This study confirmed that the fluorescence staining method is more sensitive than the Ziehl-Neelsen staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.

Visual observation of selective elution of components from skin-mimetic lipid membrane

Selective elution of components was visually observed on a mixture of lipids (ceramide III, palmitic acid, and cholesterol) as a mimicry of stratum corneum (SC) which was melted and sandwiched between glass plates. The lipid membrane was exposed to an aqueous solution of sodium dodecyl sulphate (SDS) and observed by an optical microscope. The contact of the lipid membrane with a SDS solution caused the elution of the lipid component as “myelin-form”, and the lipid membrane changed to a sponge structure. An infrared absorption spectroscopic study revealed that the SDS penetrated into the lipid mixture, and the fraction of ceramide in the sponge phase became higher than that in the lipid membrane before SDS treatment. The selective elution behaviour was confirmed by observing the behaviour of each component in lipid membrane by means of a fluorescence-staining method: The cholesterol was eluted with producing visual myelin-form on the contact with a SDS solution, and the following elution of palmitic acid occurred without myelin-form, while the ceramide III resisted the exposure to the SDS solution. These results are valid to elucidate the influence of surfactants on SC.

Diagnostic efficacy of Ziehl-Neelsen method against fluorescent microscopy in detection of acid fast bacilli

To investigate the application of Ziehl-Neelsen (Z-N) and fluorescent microscopy in detection of acid fast bacilli (AFB). Duplicate smears were prepared from 260 sputum samples and stained with Z-N and fluorescent staining (FS) methods. The efficiency of both methods in primary diagnosis of tuberculosis were evaluated. The smears were positive for AFB in 15 (5.77%) samples by Z-N staining method and in 16 (6.15%) samples by FS method. The sensitivity and specificity of Z-N staining method against FS method were 93.75% and 100% respectively. Though lesser cost-effective than Z-N, FS method is a more sensitive and better case finding tool in detection of AFB.

Involvement of reactive oxygen species and calcium in photo-induced membrane damage in HeLa cells by a bis-methanophosphonate fullerene

Photo-excited bioactivities of fullerene derivatives are attracting much attention. In this report, a bis-methanophosphonate fullerene (BMPF) and the other two fullerene derivatives, a bis-malonic acid fullerene (BMAF) and a fullerol were incubated with HeLa cells and irradiated with a green light emitted from a mercury lamp on a fluorescent microscopy. By using DNA fluorescent probe propidium iodide staining method, damage towards cell membrane could be detected when cells were treated by irradiation altogether with BMPF or BMAF at a low concentration (4 μM), and the damage was dose-dependent. The activity of BMPF was much higher than that of BMAF, while fullerol had no effects under the same condition. It was also revealed that different kinds of reactive oxygen species (ROS) correlated to BMPF and BMAF. Additionally, presence of extracellular calcium could promote the activities of both derivatives, while removal of extracellular calcium could not abort their membrane-damaged activities. These results indicated that ROS and calcium were involved in the photosensitization of fullerene derivatives, and BMPF was a superior photosensitizer which would find potential application in biomedical field.

Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions

Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil–C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems.

313 ASSESSMENT OF SPERM DNA FRAGMENTATION IN CANINE EJACULATES USING THE Sperm-Halomax® KIT: PRELIMINARY RESULTS

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for humans and different mammalian species, using a commercial kit based on the sperm chromatin dispersion (SCD) test; however, a descriptive study in canine semen has not been performed. The aim of this work was to assess the sperm DNA fragmentation in canine ejaculates using the SCD test and 2 different staining techniques. For this purpose, ejaculates were collectedby digital manipulation from4 healthy dogs of different breeds (1 German Pointer, 2 Spanish Greyhounds, and 1 Crossbreed). After collection, the sperm-rich fraction of the ejaculates from 3 dogs were pooled each time (n = 4) and then extended in Dulbecco’s phosphate buffered saline. All the pooled semen samples presented physiological values concerning routine semen parameters (motility, morphology, and sperm concentration). The sperm DNA fragmentation was assessed using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain). Two semen aliquots of the diluted pooled semen samples were processed on each pre-treated slide provided in the kit following the manufacturer’s instructions. The last step was the staining technique. We stained each slide with 2 different staining procedures. The first half of the slide was stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) mixed in a proportion 1 : 1 with an antifading solution. The second half of the slide was stained for 15 min in Wright solution (1.01383.0500, Merck, Whitehouse Station, NJ, USA) 1 :1 in Phosphate Buffer pH 6.88 (1.07294.1000, Merck). The stained slides were observed using fluorescence and light microscopy, respectively. Five hundred sperm per slide were counted. Spermatozoa with fragmented DNA showed a large and spotty halo of chromatin dispersion. Unfragmented sperm only showed a small and compact halo. Statistical analyses were performed using the Statistical Package for Social Science version 12.0 (SPSS Inc., Chicago, IL, USA). The sperm DNA fragmentation index was compared between Wright and fluorescence staining methods by ANOVA. Results were expressed as mean ± standard error of the mean. The first report of the sperm DNA fragmentation index in canine ejaculates was 2.26 ± 0.53% for Wright staining and 1.99 ± 0.10% for fluorescence technique. No differences were found between staining procedures. In conclusion, it was possible to assess the sperm DNA fragmentation of canine ejaculates using 2 different staining procedures, expecting that continuous research could be useful in defining the role of DNA fragmentation SCD test in canine semen evaluation and cryopreservation.

Novel Sulfated Glucomannan-Barium-Alginate Microcapsules in Islet Transplantation: Significantly Decreased the Secretion of Monocyte Chemotactic Protein 1 and Improved the Activity of Islet in Rats

The sulfated glucomannan can be used to filter the heparin-binding properties of cytokines. In this study, novel sulfated glucomannan-barium-alginate (SGA) microcapsules were prepared to encapsulate islets with barium-alginate (ABa) and calcium alginate-poly- l-lysine (APA) microcapsules as controls. SD rat islets were purified as donor cells to Lewis rats that had been treated with streptozotocin. Intraperitoneal transplantation was performed with about 3000 islet equivalent (IEQ) rat. At week three after transplantation, the concentrations of monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-1 β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in intraperitoneal fluid were determined using ELISA. At week 8, the islet cell mass in the abdominal microcapsules was excised to test insulin release. The EB-FDA fluorescence staining method was used to observe the functional activity of the islet cells. Compared with ABa and APA microcapsules, SGA microcapsules showed significantly decreased MCP-1 secretion by β-cells. Also, the concentrations of cytokines IL-1β, IFN-γ, and TNF-α were decreased significantly. The activity of the transplanted islets was significantly improved in SGA microcapsules, which shielded against cytokines better than ABa or APA microcapsules and may serve as novel method.

Characterization of neuronal apoptosis in the ventral ganglia of postembryonic silkworms Bombyx mori (Lepidoptera: Bombycidae)

A fluorescent triple staining method was developed to stain the cytoplasm of neurons red, the nuclei of all kinds of cells, including neurons, blue and the nuclei of apoptotic neurons in cyan in the twelve ventral ganglia (VG) of the Bombyx mori ventral nerve cord. This differential staining method was used to distinguish between apoptotic and normal neurons in the suboesophageal ganglion (SOG), thoracic ganglia (TG)1 to TG3 and abdominal ganglia (AG)1 to AG8 and also determine the changes in the num- bers of apoptotic neurons that occur during postembryonic development. In most of the VG tested, neuronal apoptosis was most marked during the period from the end of larval life to the mid pupal stage. The greatest number of apoptotic neurons was found in SOG of day-5 pupae, TG1 to TG3 and AG1 to AG4 of day-1 pupae, and AG5 to AG8 of day-4 pupae. In vivo injection of 20- hydroxyecdysone (20E) into day-8 5th instar larvae resulted in both a considerable increase in the number of apoptotic neurons and cleavage of procaspase-3 into caspase-3, which induced neuronal apoptosis in SOG and AG6 to AG8 in day-1 pupae, and a slight increase in the number of apoptotic neurons in TG1. In TG3 and AG4, however, it had little effect on the number of apoptotic neu- rons or cleavage of procaspase-3. Treatment of the VG of both day-8 5th instar larvae and day-2 pupae with protein synthesis inhibi- tors by in vivo injection triggered a significant inhibition of neuronal apoptosis and procaspase-3 cleavage in most of these ganglia in day-1 pupae and day-4 pupae, but not TG3 and AG4, in which there was little procaspase-3 and caspase-3. In vivo injection of caspase-8 and -3 inhibitors into day-8 5th instar larvae and day-2 pupae led to a substantial inhibition of neuronal apoptosis and of procaspase-3 cleavage in SOG, AG6 and TAG, but not in TG3 or AG4 of day-1 pupae and day-4 pupae. These findings suggest that neurons that die in SOG, TG1 and AG6 to AG8 in day-1 and -4 pupae may undergo apoptosis induced by the synthesis of a new pro- tein and caspase-8- and -3-implicated signal transduction by the increase in titre of 20E in the haemolymph but not the neuronal aopotosis in TG3 and AG4. This study provides neurobiologists with valuable information and a means of studying neuronal apop- tosis in the nervous system of insects.

Evaluation of a Rapid Fluorescent Staining Method for Detection of Mycobacteria in Clinical Specimens

Rapid detection of mycobacterial disease is essential. Using multiple specimen types and concentrations of mycobacteria, we compared two commercial auramine O stains. The more rapid stain permitted consistent acid-fast bacillus quantitation and exhibited less debris staining, and the staining procedure required less time ( approximately 2 min) to perform. These results suggest that the rapid stain may be more cost-effective and efficient for use in clinical laboratories.