Preparation Of Permanent Slides Research Articles

Wood microstructure and key for identification of selected willow (Salix) species from India and adjacent regions

This paper deals with the wood microstructure of twenty-nine samples of the ten important species of genus Salix L., viz., Salix acmophylla Boiss., S. babylonica L., S. daphnoides Vill., S. denticulata Andersson., S. x fragilis L., S. oxycarpa Andersson., S. tetrasperma Roxb., S. viminalis L., S. disperma Roxb. ex D.Don, and S. sikkimensis Andersson from India and adjacent regions (i.e., Myanmar and Tibet). Internationally acclaimed standard laboratory procedure was followed in the preparation of permanent slides. The qualitative and quantitative wood anatomical characters were studied as per the International Association of Wood Anatomists (IAWA) terminology for hardwoods. Photomicrographs were taken for showing the diagnostic features of each taxon. The selected Salix species had some similar qualitative wood anatomical features along with differentiating characters based on which species can be distinguished from each other. In addition to qualitative anatomical features, quantitative anatomical features also showed significant differences among species. The main differentiating anatomical characters were druses, vessel-ray pits, ray seriation, shape/outline of solitary vessels and vessel frequency based on which identification key was prepared.

Pharmacognostical Study of the Fruit Peels of Punica granatum

This study deals with Punica granatum fruit peels. This plant was selected because of its popularity and being highly used; it was purchased from herbarium markets of Khartoum Sudan. The study is divided into two parts, the main part is a morph-anatomical study of the fruit peels of the plant because it is important in the identification of the medicinal plant when it is in the powder or broken forms. The preparation of permanent slides was done using wax methods. The results of the morph anatomical studies were described and shown in photomicrographs. Transverse section of the peels of the fruits ofP. granatum is formed of one cell thick epidermis covered by a thin cuticle, followed by many layers of small compact parenchyma cells forming pigment layer and several cell thick isodiametric spongy parenchyma cells filling the section. Vascular bundles are small distributed within the ground parenchyma cells; sclerenchyma cells in the form of stone cells were densely distributed on the section of the peel of fruit. Part two includes physiochemical studies of the powder of the fruit peel to determine the percentage values of moisture, ash, acid insoluble ash, alcohol soluble extractives, and water soluble ash.

First new record of two diatoms (Caloneis africana (Giffen) Stidolph and Luticola nivalis (Ehrenberg) D. G. Mann) from South West Coast of India (Cochin backwaters)

Summary The main objective of this paper is to report two diatoms (Caloneis africana (Giffen) Stidolph and Luticola nivalis (Ehrenberg) D. G. Mann) from Cochin backwaters, which prove to be a new finding from Indian waters. Surface water samples were collected monthly from twelve stations covering the ecosystem from May 2015 to April 2016. The analysis of physicochemical parameters and the preparation of permanent slides of diatoms were performed based on standard procedures. Descriptions, world distribution and photographs of the two diatoms are included in this paper. These two new species further enrich the diatom floristic diversity of Cochin backwaters in India.

Additional characters for taxonomic treatment on Chimonanthus praecox (L.) Link (Calycanthaceae)

The detailed study of Chimonanthus praecox investigated to contribute in Calycanthaceae. Especially for the detail, characters are for further phylogeny as proper placement of the plant. The study reveals that presence of trichome, crystals in mesophyll, seed size, pericarp, seed coat, and ovule structure and pollen character are important for present study. Experiment conducted out from the resin method. Leaf, petiole, and flowers are used for the preparation of permanent slides. The valuable additional characters of Chimonanthus praecox are vascular bundles in the petiole, crystals presence in mesophyll, furrow like pollen grain, and U-shape vascular bundle. These anatomical characters are important for taxonomic treatments and phylogeny of Calycanthaceae.

Pharmacognostical Study of the Fruits of Acacia nilotica, Ammi visnaga and Pimpinella anisum

This study deals with Acacia nilotica fruits, Ammi visnaga fruits and Pimpinella anisum fruits, these plants were selected because of their popularity and they are highly used, they were purchased from herbarium markets of Khartoum. The study is divided into two parts, the main part is morph anatomical studies of the fruits of the plants because it is important in the identification of the medicinal plant when it is in the powder or broken forms. The preparation of permanent slides was done using wax methods. The results of the morphanatomical studies were described and shown in photomicrographs. The transverse sections of the fruits of A. nilotica, A. visnaga and P. anesum are formed of many layers. The epicarp is one layer of small cells covered with cuticle. Many trichomes appeared projecting from the epidermal cells. The mesocarp is formed of parenchyma cells filled with reserved materials, tannins in A. nilotica and vittae oil cavities in A. visnaga and P. anesum. Part two includes physichemical studies of the powder of the studied plant fruits to determine the percentage values of moisture, ash, acid insoluble ash, alcol soluble extractives, and water soluble ash.

Protocol identification and preservation of myxozoan parasites for microscopy with silver nitrate (Klein's dry) staining technique.

The present study, discusses a first report of staining techniques with silver nitrate (AgNO3 ) stain for the preservation and identification of myxozoans globally. The silver nitrate stain was used to prepare permanent slide preparation of myxozoans with some adaptations made in our laboratory. Fresh air dried smear were stained with silver nitrate stain ensuing dark brown color polar capsules and light brown color of spore-wall. The stain is everlasting for years differing to other stains like Geimsa, iron-heamotoxylin Zeihl-Neelsen Blue, and trichome stains. RESEARCH HIGHLIGHTS: Stains usually fade with time and fail to disclose the morphological characters of the specimen. Present staining method helps to detect less infection in the tissue locating the myxospores. The Klein dry method (1958) is useful and suitable for long term preservation of the myxozoan slides and morphological description.

Newly devised glycerol-mounted permanent slide preparation for dissected specimens of micro-crustaceans

Newly devised glycerol-mounted permanent slide preparation for dissected specimens of micro-crustaceans

DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides.

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl2 buffer; (D) Protocol with CaCl2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

Reconstrução paleoambiental de um depósito sedimentar por análises multiproxy, Turvo, Estado do Paraná, Brasil

This research presents results of paleoenvironmental analysis that was achieved in a sedimentary record of 44 cm depth, recovered from a remnant of Mixed Rain Forest, located near the urban area in Turvo, Parana. The record was dated ( 14 C) at two depths: 14,553 cal. years BP (44 cm) and 6.090 cal. years BP (22 cm). Besides the dates, five analyzes of δ 13 C were performed and increasing values from base (-19.0 ‰) to top (-22.9 ‰) were obtained. Samples containing 20 g were extracted from the record on 2 cm intervals (22 samples); then the sediment dissolution (2 g) via chemical treatment was processed according to the protocol used in palynology with subsequent preparation of permanent slides (50 ul material). The slides were observed under an optical microscope, where the count of 400 identifiable phytoliths by gap were determined and the concentration of phytoliths was estimated by random observation in 12 optical fields by gap (x 640). The adaptation indexes to drought (Iph%), climate (Ic%) and water stress (% Fs) were calculated. Two paleoenvironmental areas were established: (i) Zone I, between 44 and 32 cm with a prevalence of drier conditions than current ones, that were evidenced by the increase in the rates of drought adaptation and water stress rates and mixture of C 4 and C 3 plants (pro C 4 ); (ii) Zone II, from 30 cm to the top, characterized by a gradual increase in humidity, low rate of drought adaptation and impoverishment of the isotopic signal with C 3 plants predominance, which was interpreted as an improvement in the moisture conditions and/or available high humidity in the soil. The interpretations corroborate studies for the south and west-central region in Brazil.

IDENTIFICATION OF ANTHRACHNOSE DISEASE ON STRAWBERRY FRUIT (Fragraria vesca L.) AND ITS CONTROL BY BETEL (Piper betle L.) LEAF EXTRACT

<p>In Indonesia, strawberry is one of fruits which has high economic values. The growth of strawberry has increased significantly, but its productivity is still low because of disease attack. Disease that attacks strawberry started from nursery to post harvest is anthracnose that can be caused by Colletotrichum spp. Control efforts of anthracnose disease up to now has been done with application of synthetic chemicals which are known to be hazardous to the environment and human health. Betel leaf (Piper betle L.) is natural plant that potentially used as phytofungicide. Essential oils on betel leaf extract is reported can decrease the growth of pathogenic fungi. The objectives of the research were to identify Colletotrichum spp. in strawberry fruit that showed anthracnose symptoms, to test the potency of betel leaf extract as phytofungicide, and to evaluate the effect of betel leaf extract in inhibiting infection and disease severity of Colletotrichum in strawberry. Samples were taken from strawberry nursery in Magelang, Indonesia. The disease symptoms were isolated on Potato Dextrose Agar, followed by identification of fungal colony and morphology by semi permanent slide preparation. Betel leaves were extracted with water to get a concentration of 20, 40, 60, 80 and 100%, respectively. An in vitro experiment was done to evaluate the inhibition rate of betel leaf extract to the growth of Colletotrichum spp. colony. An in vivo experiment was done by submersion of strawberry to betel leaf extract before (preventive method) and after antrachnose attack (curative method), then disease infection and disease severity were measured after 7 days. The result of the research showed that anthracnose disease in strawberry was caused by C. gloeosporioides (Penz.). Twenty percent of betel leaf extract in an in vitro test had potential as phytofungicide and concentration of 100% betel leaf extract was the best concentration to inhibit the growth of C. gloeosporioides (Penz.) colony by 70.26±0,61%. In an in vivo experiment, submersion of strawberry before pathogen infection (preventive) with 100% betel leaf extract decreased disease infection by 40% and disease severity by 16%, respectively. While treatment of submersion of strawberry after pathogen infection (curative) decreased disease infection by 33,4% and disease severity by 17%. </p><p>Keywords: strawberry, anthrachnose, disease, betel leaf</p>

Study of Acid-Base Disturbances in Patients with Liver Cirrhosis

Background and study aim: Diagnostic examination of stools for opportunistic intestinal parasites in HIV/AIDS patients is given less attention than it should be. The suspected opportunistic intestinal parasites such as Cryptosporidium parvum,Cyclospora cayentanensis, Toxoplasma gondii, Isospora belli and the symptom of explosive watery diarrhea they cause as well as others including Strongyloides stercoralis are the threat against the well-being of HIV/AIDS patients. The objective of this study is to demonstrate the indispensable necessity to free HIV/AIDS patients (who are under medical care in 3 different hospitals, Southern Ethiopia), from opportunistic intestinal parasites using diagnostic examination of stools followed by prompt curative treatment during every safety time interval. Patients and methods: Fresh stools samples from a total sample size of 710 HIV/AIDS patients were taken and examined in the parasitology laboratory, Dilla University, for the suspected intestinal opportunistic parasites. The methods employed to identify the intestinal parasites included observations in : wet mount, formalin-ether concentration technique, and permanent slide preparation as well as Baermann apparatus method for Strongyloides stercoralis. Result: Out of 710 HIV/AIDS patients examined 196 were found to be positive for 6 different species of the suspected intestinal parasites (infection rate of these parasites in the population of HIV/AIDS patients of the 3 different hospitals being 196×100/710=27.6% The six species of parasites isolated from fresh stools samples were: Ascaris lumbricoides, Strongyloides stercoralis, Entameba histolytica, Giardia lamblia, Balantidium coli, and Trichuris trichiura. Conclusion: Reasonably planned successive safety time intervals must be attended continuously by HIV/AIDS patients without interruption to utilize the services of medical care in order to avoid/ neutralize the potential opportunistic infections and reinfections; otherwise, the fulminant death can turn to be true. The safety and well-being of those HIV-infected patients who attend all the medical services & advices provided by clinical experts is not different from that of HIV-noninfected individuals without any trace of exaggeration.

Screening for Opportunistic Intestinal Parasites in HIV/AIDS Patients, Attending the Services of Medical Care in Three Different Hospitals, Southern Ethiopia

Background and study aim: Diagnostic examination of stools for opportunistic intestinal parasites in HIV/AIDS patients is given less attention than it should be. The suspected opportunistic intestinal parasites such as Cryptosporidium parvum,Cyclospora cayentanensis, Toxoplasma gondii, Isospora belli and the symptom of explosive watery diarrhea they cause as well as others including Strongyloides stercoralis are the threat against the well-being of HIV/AIDS patients. The objective of this study is to demonstrate the indispensable necessity to free HIV/AIDS patients (who are under medical care in 3 different hospitals, Southern Ethiopia), from opportunistic intestinal parasites using diagnostic examination of stools followed by prompt curative treatment during every safety time interval. Patients and methods: Fresh stools samples from a total sample size of 710 HIV/AIDS patients were taken and examined in the parasitology laboratory, Dilla University, for the suspected intestinal opportunistic parasites. The methods employed to identify the intestinal parasites included observations in : wet mount, formalin-ether concentration technique, and permanent slide preparation as well as Baermann apparatus method for Strongyloides stercoralis. Result: Out of 710 HIV/AIDS patients examined 196 were found to be positive for 6 different species of the suspected intestinal parasites (infection rate of these parasites in the population of HIV/AIDS patients of the 3 different hospitals being 196×100/710=27.6% The six species of parasites isolated from fresh stools samples were: Ascaris lumbricoides, Strongyloides stercoralis, Entameba histolytica, Giardia lamblia, Balantidium coli, and Trichuris trichiura. Conclusion: Reasonably planned successive safety time intervals must be attended continuously by HIV/AIDS patients without interruption to utilize the services of medical care in order to avoid/ neutralize the potential opportunistic infections and reinfections; otherwise, the fulminant death can turn to be true. The safety and well-being of those HIV-infected patients who attend all the medical services & advices provided by clinical experts is not different from that of HIV-noninfected individuals without any trace of exaggeration.

Microscopic Preparation Techniques for Plant Stem AnalysisMicroscopic Preparation Techniques for Plant Stem Analysis[Gärtner, H. and Schweingruber, F. H. : Kessel Publishing House, Remagen, Germany 78 pp., 2013 ; ISBN: 978-3-941300-76-7 . Available at http://www.forestrybooks.com/ for ca. €22 plus shipping

Microscopic Preparation Techniques for Plant Stem Analysis by Holger Gartner and Fritz H. Schweingruber was published in 2013 by Kessel Publishing House (ISBN 978-3-941300-76-7) and is available directly from the publisher. The authors have team-taught a workshop on wood anatomy for almost a decade, and this book clearly benefits from their extensive experience in teaching the basic techniques of microscopic plant stem preparation to researchers from a broad spectrum of disciplines. The book’s structure leads readers through the sequential steps involved in plant stem analysis. It begins with short sections on fieldbased sampling strategies and methods. Then the book transitions to its dominant focus: laboratory methods for masceration and thin-sectioning of plant material. It provides details on how to stain, bleach, fix, and preserve plant materials for viewing with a microscope. The excellent photographs in these sections let readers ‘‘see’’ the steps involved in microscope slide preparation. The book ends with very brief sections on common errors during sample preparation and microscopy tips for improved viewing of prepared samples. This book takes the extensive experience in microscopic preparation possessed by Dr. Gartner and Prof. Schweingruber and presents it to readers in a highly accessible format. The authors intentionally created an image-rich book to make it easier for readers lacking strong English skills to understand the content; however, this format also serves as a source of inspiration for future research projects. There were many pages where we saw a new technique being demonstrated and realized we had the perfect application for that method in one of our current studies. Although the book is aimed at an international audience, at times it falls short in that goal. Sometimes these shortcomings are entertaining, as in the paragraph on how to hold the razor blade during sectioning by hand, where readers are told to ‘‘cut the object like you would cut a salami, not like you would cut butter’’ (p. 21). This recommendation left us laughing, but having no idea how to hold the razor blade. Occasionally, the stilted language becomes annoying for native English speakers to read, but the meaning is always clear even if the noun and verb agreement is incorrect. One area where we wish the authors had spent more time was on the economics of these laboratory techniques. For example, they advocate the use of Canada balsam for embedding microsections and say, ‘‘for all other embedding resins we have no long-term experience’’ (p. 63). From our perspective, we look at having to pay US$210 for 100mL of Canada balsam vs. having to pay US$15 for 100mL of permanent mounting medium and are left wondering whether the extra US$195 is really worth it. Unfortunately, the authors offer no suggestions for alternative techniques for researchers with more limited funds. Overall, this is a useful reference book for any dendrochronology, wood anatomy, or plant sciences laboratory. In our own work, we have already used this book twice in the past week to find the appropriate solvent for a Safranin stain solution and to find suggestions for eliminating air bubbles during permanent slide preparation. In both cases, the Table of

New approach for the quantification of processed animal proteins in feed using light microscopy

A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.

Bemisia tabaci

<i>Bemisia tabaci</i>

Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay.

A modified version of the local lymph node assay (LLNA) is presented, using bromodeoxyuridine to label proliferating lymphocytes. Cell counting is done on mid-sagittal sections of individual nodes under light microscopy. Two irritants (sodium lauryl sulfate and salicylic acid), four allergens of various sensitizing potential (potassium dichromate, 4-chloroaniline, neomycin sulfate and nickel sulfate) and one chemical of unknown sensitizing potential (ethyl 3-aminobenzoate) were tested either in the short protocol using three daily topical applications and/or in the long protocol with a pre-exposure step under an occluded patch. A weak T cell proliferation was noted with both irritants in the short protocol, but not in the long protocol. Potassium dichromate induced a strong proliferative response in the short protocol. A lesser sensitizing potential was detected for 4-chloroaniline, ethyl 3-aminobenzoate and neomycin sulfate but only in the long protocol. Nickel sulfate was negative in both protocols. The long protocol was the most valuable for weak or moderate sensitizers. Histological examination of nodes ruled out intercurrent processes. The present procedure offers several advantages. The use of a non-isotopic marker enables this test to be run in a routine safety assessment department and allows the preparation of permanent slides. An increased specificity is obtained by restricting cell counting to the paracortex. Moreover, the collection of individual data permits statistical analysis of the results. This method is sensitive and reproducible and may be viewed as a useful adjunct to the LLNA.

Kinetochore detection in micronuclei: an alternative method for measuring chromosome loss.

Whole chromosomes within micronuclei (MN) in cytokinesis-blocked human lymphocytes were detected by using anti-kinetochore antibodies obtained from the serum of scleroderma patients. The primary antibody was localized using a peroxidase-labelled second antibody followed by a nickel chloride modification of the diamino-benzidine reaction to give a permanent slide preparation. Between 82 and 92% of colchicine-induced MN were shown to be kinetochore-positive. Results for spontaneously occurring micronuclei in young (20-35 years) and elderly (greater than 65 years) subjects indicated that 42 (+/- 6) and 50 (+/- 6)%, respectively, contained kinetochores. Perhaps the more novel observation was that approximately 12% of X-ray-induced micronuclei were kinetochore-positive and thus could have been the result of whole chromosome loss events. Kinetochore-detection in micronuclei provides a new approach to measure chromosome loss and therefore may be important in identifying aneuploidy-inducing agents.

A Rapid Silver-Free Method for Staining the Myenteric Plexus

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionein were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic "mirroring," low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.

Rapid method for permanent slide preparation of colonies in soft agar cultures.

A method is presented for preparing permanent microscopic slides from colony-bearing agar layers in soft agar cultures. The main advantages of this technique are its simplicity, rapidity and accurate colony preservation. This method could have broad applications in the human tumor clonogenic assay (HTCA), particularly in the quantitative morphological, cytochemical and immunocytochemical assessment of colonies that form in both control and drug-treated cultures. Thus, this method opens up possibilities for using cytopathological criteria as a quantitative endpoint of the HTCA.

A simple method for the preparation of permanent slides from cell cultures.

(1984). A Simple Method for the Preparation of Permanent Slides from Cell Cultures. Stain Technology: Vol. 59, No. 6, pp. 353-354.