Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10⁸ cfu/ml of Escherichia coli and 10⁵ cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.     

Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni

Nuclear polyhedra obtained from diseased cabbage looper, Trichoplusia ni, were digested with sodium carbonate-saline buffer, pH 11.0. The dissolved polyhedra formed 3 general zones when subjected to density gradient centrifugation. The slowest sedimenting component (Zone 1) had an ultraviolet absorption curve typical of protein and a sedimentation coefficient of 11 S. Capsids, 310 × 40 nm, were located in Zone 2. Virus particles were found in 1–3 bands (Zone 3); those with envelopes measured 300 × 72 nm, and those without envelopes measured 300 × 33 nm. Virus preparations stained with phosphotungstic acid at pH 7.0 exhibited extensive disruption whereas preparations stained at pH 3.0 did not. Virus particles in the sodium carbonate-saline-digested polyhedra had a sedimentation coefficient of 1228 S. Virus particles isolated by high speed centrifugation had a sedimentation coefficient of 1530 S.     

Structures of ribosomal subunits from Saccharomyces cerevisiae

The large (60S) and small (40S) ribosomal subunits were isolated from yeast (Saccharomyces cerevisiae), and preparations stained with uranyl acetate were imaged by transmission electron microscopy. Averages of three different projections of each subunit were obtained by computerized image processing with reproducible spatial resolutions approaching 3.0 and 3.5 nm, respectively. Similarities between the structures of these particles and those of other eukaryotic ribosomal subunits contribute to the development of a concensus model for the eukaryotic ribosome.     

The Number of Chromosomes in SCHIZOSACCHAROMYCES POMBE: Light Microscopy of Stained Preparations

Chromosomes have been counted with the light microscope in fixed and stained preparations of Schizosaccharomyces pombe. The least ambiguous images were seen in zygotes fixed at meta-, ana- and telophase of meiosis II. They suggest that the haploid number of chromosomes in S. pombe is three.     

Penetration of Crotalaria juncea,Dolichos lablab,and Sesamum indicum Roots by Meloidogyne javanica

Penetration of Crotalaria juncea (PI 207657 and cv. Tropic Sun) Dolichos lablab cv. Highworth, and Sesamum indicum by juveniles (J2) of Meloidogyne javanica was assessed to investigate the mechanism by which these plants may reduce nematode numbers in the field. Growth chamber experiments were conducted at 25 C, with vials containing 90 g sand infested with 450 J2; tomato (UC 204 C) was included as a susceptible host. Fifteen days after inoculation, roots were stained and the nematodes within stained roots were counted. Both C. juncea lines were highly resistant to penetration, as they contained significantly fewer nematodes per cm of root and per root system than the other plants. Although containing more nematodes per cm of root than C. juncea, S. indicum and D. lablab had significantly fewer nematodes per root system and per cm of root than tomato. Roots were significantly longer in the plants with the lowest nematode penetration. Although C. juncea, D. lablab, and S. indicum may have potential utility as cover or rotation crops in soil infested with M. javanica, further quantitative information on the reproduction of M. javanica and other nematodes in these plants is needed.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Increased vascularity in cervicovaginal mucosa with Schistosoma haematobium infection

Background

Close to 800 million people in the world are at risk of schistosomiasis, 85 per cent of whom live in Africa. Recent studies have indicated that female genital schistosomiasis might increase the risk of human immunodeficiency virus (HIV) infection. The aim of this study is to quantify and analyse the characteristics of the vasculature surrounding Schistosoma haematobium ova in the female genital mucosa.

Methodology/Principal Findings

Cervicovaginal biopsies with S. haematobium ova (n = 20) and control biopsies (n = 69) were stained with immunohistochemical blood vessel markers CD31 and von Willebrand Factor (vWF), which stain endothelial cells in capillary buds and established blood vessels respectively. Haematoxylin and eosin (HE) were applied for histopathological assessment. The tissue surrounding S. haematobium ova had a higher density of established blood vessels stained by vWF compared to healthy controls (p = 0.017). Immunostain to CD31 identified significantly more granulation tissue surrounding viable compared to calcified ova (p = 0.032), and a tendency to neovascularisation in the tissue surrounding viable ova compared to healthy cervical mucosa (p = 0.052).

Conclusions/Significance

In this study female genital mucosa with S. haematobium ova was significantly more vascularised compared to healthy cervical tissue. Viable parasite ova were associated with granulation tissue rich in sprouting blood vessels. Although the findings of blood vessel proliferation in this study may be a step to better understand the implications of S. haematobium infection, further studies are needed to explore the biological, clinical and epidemiological features of female genital schistosomiasis and its possible influence on HIV susceptibility.     

Ribosome structure: hand determination by electron microscopy of 30 S subunits.

Negatively stained Escherichia coli 30 S ribosome subunits have been examined by electron microscopy at tilt angles of ?45 °, 0 ° and +45 °. The tilted views, together with images of metal shadowed particles are consistent with an asymmetric model for the structure of the 30 S subunit and enable the absolute hand of the particle to be determined.     

Location of repetitious DNA in the chromosomes of the desert locust (Schistocerca gregaria)

A modified Giemsa staining technique and the in situ hybridisation technique, have been used to investigate the localisation of highly repeated sequences in the karyotype of the locust Schistocerca gregaria. The centromeric regions are stained densely with Giemsa and further Giemsa-stained bands occur at the telomeric region of the short (S) chromosomes. RNA complementary to repetitious DNA hybridised to loci scattered along the whole length of the chromosomes, with concentrations of grains at the centromeric regions of all the chromosomes and also at the telomeric regions of the S chromosomes.     

Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.     

Temporal germ cell development strategy during continuous spermatogenesis within the montane lizard, Sceloporus bicanthalis (Squamata; Phrynosomatidae)

Sceloporus bicanthalis is a viviparous lizard that lives at higher elevations in Mexico. Adult male S. bicanthalis were collected (n = 36) from the Nevado de Toluca, Mexico (elevation is 4200 m) during August to December, 2007 and January to July, 2008. Testes were extracted, fixed in Trumps, and dehydrated in a graded series of ethanol. Tissues were embedded, sectioned (2 μm), stained, and examined via a light microscope to determine the spermatogenic developmental strategy of S. bicanthalis. In all months examined, the testes were spermiogenically active; based on this, plus the presence of sperm in the lumina of seminiferous tubules, we inferred that S. bicanthalis had year-round or continuous spermatogenesis, unlike most reptiles that occupy a temperate or montane habitat. It was recently reported that seasonally breeding reptiles had a temporal germ cell development strategy similar to amphibians, where germ cells progress through spermatogenesis as a single population, which leads to a single spermiation event. This was much different than spatial development within the testis of other derived amniotes. We hypothesized that germ cell development was temporal in S. bicanthalis. Therefore, we wanted to determine whether reptiles that practice continuous spermatogenesis have a mammalian-like spatial germ cell development, which is different than the typical temperate reptile exhibiting a temporal development. In the present study, S. bicanthalis had a temporal development strategy, despite its continuous spermatogenic cycle, making them similar to tropical anoles.     

酿酒酵母FFC2146 胞内蛋白及胞外蛋白双向电泳条件优化及图谱建立

通过对酿酒酵母(Saccharomyces cerevisiae)的培养基、培养条件及蛋白质提取方案的优化,建立了酿酒酵母胞外和胞内蛋白双向电泳图谱制作方法。在YNB培养基中培养20 h,经过离心取上清-超滤-冻干可得到酿酒酵母胞外蛋白质样品;用SDS缓冲液悬浮酵母细胞-煮沸-超声-增溶,得到了酿酒酵母胞内蛋白质样品。经过双向电泳分离、硝酸银染色和PDQuest图像分析可以检测到了200多种酿酒酵母胞外蛋白和500多种酿酒酵母胞内蛋白。     

Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis

The multisegmented ovoidal inclusion of Bacillus thuringiensis subsp. israelensis was found to be composed of two structurally and biochemically distinct components. Electron microscopy of the inclusion revealed it to be composed mainly of osmiophobic or lightly stained segments crystallized in a lattice showing a repeat of approximately 4.3 nm. These light segments of the inclusions were shared by osmiophylic darkly stained segments with a crystal lattice repeat of approximately 7.8 nm. The lightly stained segments were soluble at pH 9.2 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The extracts of lightly stained segments were lytic to mammalian erythrocytes, and the precipitate obtained by lowering the pH to 5.2 was toxic to the larvae of Aedes egypti. The dark inclusion segments remaining, besides being much less toxic to larvae, were nonlytic to erythrocytes and were soluble at pH 10.5 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The light segment was composed of two major polypeptide doublets with molecular weights of 145,000 and 135,000, and 27,000 and 26,500, and the dark segments were composed of a single major polypeptide with a molecular-weight of 70,000. Hence, the inclusion of B. thuringiensis subsp. israelensis is more complex than previously reported, and we conclude that the toxin may be the polypeptide with a molecular weight of 27,000 and 26,500.     

Serological Diversity Within a Terrestrial Ammonia-Oxidizing Population

Fluorescent antibodies (FAs) prepared against 16 ammonia-oxidizing nitrifying bacteria were examined as to cross-reactivity in heterologous FA staining tests. Virtually all cross-reactions to the seven Nitrosomonas FAs were confined to the Nitrosomonas isolates. The five Nitrosospira, three Nitrosolobus, and one Nitrosovibrio FAs stained isolates only within their respective genera. Within each genus shared antigens were more common among Nitrosomonas and Nitrosolobus than among Nitrosospira isolates. Isolates obtained from a single soil were stained to examine the effectiveness of the suite of FAs for study of a given ammonia-oxidizing population. Of the seven Nitrosomonas isolates, five were stained effectively through the use of four FAs; six of twelve Nitrosospira isolates were identifiable through use of four different FAs. The one Nitrosolobus isolate was stained well only by one (its homologous) FA.     

Application of the Fluorescent-Antibody Technique for the Detection of Sphaerotilus natans in Activated Sludge

Sphaerotilus natans, one of the most widely reported causes of bulking in activated sludge, can exist both within and outside of a sheath. It can easily be confused with similar activated sludge bacteria and thus can be overlooked when present in low numbers. Fluorescent antiserum was successfully prepared against the nonfilamentous form and was shown to be highly specific, showing no reaction with either pure cultures of similar filamentous bacteria or entirely unrelated organisms. It did, however, show a lack of strain specificity since it reacted with S. natans isolates from the Federal Republic of Germany and the United States and with filamentous bacteria in South African activated sludges. Fluorescent antibody is capable of penetrating the filaments of S. natans to stain the cells individually. The use of fluorescent antiserum in the identification of S. natans filaments obscured by activated sludge flocs and other suspended matter was simple since the cells stained brightly and could be observed through the less dense matter, while the use of other microscope techniques would be hampered by these obstructions. The use of fluorescent antibody will facilitate ecological studies of S. natans in activated sludge and other aqueous environments.     

Morphological and Phylogenetic Characterizations of Freshwater Thioploca Species from Lake Biwa, Japan, and Lake Constance, Germany

Filamentous, gliding, sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 μm; the diameters of complete Thioploca filaments ranged from 18 to 75 μm. The cell lengths ranged from 1.2 to 3.8 μm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of the Thioploca from Lake Biwa was amplified by using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, the Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake Biwa Thioploca. Positive signals were obtained with the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake Biwa Thioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, the Thioploca from Lake Biwa and the Thioploca from Lake Constance are closely related to T. ingrica and to each other.     

Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection

An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.