DOCSLIB.ORG

Microscopy 7

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

7 MICROSCOPY Authors: Reviewer: Jeppe L. Nielsen Jiři Wanner Robert J. Seviour Per H. Nielsen 7.1 INTRODUCTION One widely applied method for studying the microbiology dihydrochloride, Neisser, Gram, Nile Blue) for detecting of activated sludge is microscopy. It provides an insight cell viability and intracellular accumulation of storage into the hidden world of microbes, which cannot be seen compounds including poly-hydroxy-alkanoates (PHA) otherwise by the naked eye. In this chapter we describe and poly-phosphate granules (poly-P) and in situ protocols for the microscopic examination of activated identification of targeted microbial populations using sludge samples. These include staining techniques to give Fluorescence in situ Hybridization (FISH). Combinations an understanding the huge taxonomic and functional of these techniques provide powerful tools for elucidating diversity found among the microbes in activated sludge. metabolic features of cells at a single cell level. Combining Direct observations and staining can enable the differences FISH with staining techniques is often problematic, and so between bacterial, fungal and protozoan populations to be careful planning is required to ensure that the distinguished. Studying these microorganisms effectively interpretation of this information is unequivocal. This requires the correct use of the microscope to reveal chapter provides the basis for carrying out standard differences in their shape and size and to diagnose cellular protocols. structures. Standard light microscopes with appropriate performance for routine purposes are available from 7.2 THE LIGHT MICROSCOPE several manufacturers, but using more sophisticated microscopic techniques can enhance the level of The purpose of the microscope is to provide sufficient information generated. magnification to distinguish between the objects examined. The most commonly used microscope is a In this chapter the basic principles of the light and bright field microscope that projects a focussed beam of fluorescence microscope are explained and methodologies light onto the image on a glass slide. Nowadays, almost all for relevant staining techniques and data interpretation are these microscopes are (i) binocular (Figure 7.1), meaning provided. The aim of the experimental protocols outlined that both eyes are used to view the object, making here is to serve as a user-friendly guide for cell persistent use less tiring, and (ii) compound, where more morphological examination characterization, staining than one lens system is used to achieve the required sample techniques (e.g. DAPI: 4',6-diamidino-2-phenylindole resolution. © 2016 Jeppe L. Nielsen et al. Experimental Methods In Wastewater Treatment. Edited by M.C.M. van Loosdrecht, P.H. Nielsen, C.M. Lopez-Vazquez and D. Brdjanovic. ISBN: 9781780404745 (Hardback), ISBN: 9781780404752 (eBook). Published by IWA Publishing, London, UK. 264 EXPERIMENTAL METHODS IN WASTEWATER TREATMENT Camera Lamp housing Camera adapter (light source for incident Eyepiece or reflected illumination) Binoculair photo tube Objective + Reflector filter holders for epifluorecense Condenser Filter receptable (e.g. polarizer) Objective α Light collector 2 α Rotating nosepiece aperture angle focus control Oblective(s) Specimen Stage Lamp housing plane Condenser diaphragm (light source for transmitted illumination) Condenser focus Condenser α Field diaphragm aperture angle Filters Course/fine focus control Condenser (b) Figure 7.1 The compound microscope. The eyepiece (ocular) lenses and the objective lenses provide the resolving and magnifying power of the Figure 7.2 The light path in microscopy illustrating the concept of the microscope, while the condenser lens system, by focussing numerical aperture and condensers. the light source onto the specimen (Figure 7.2), maximises the resolution of the systems by increasing the numerical aperture or light-capturing ability of the objective lenses. At the highest magnifications used (100×), because it Its position is critical for optimization of the performance, has a low light-capturing ability (high NA), it is necessary and therefore it should supply a cone of light capable of to increase the refractive index (RI) of the medium filling the objective lens aperture. The resolution reflects between the specimen and the objective lens. For light the ability of the microscope to discriminate between two microscopes, the highest resolution is obtained when the closely positioned entities. If they are closer than the condenser aperture angle matches the objective. Lenses resolution distance then they appear blurred in the in air as a medium cannot have NA that exceed 0.65, microscope image. Actual size, image size and whereas in water and certain immersions such as oils the magnification are related as follows: objective lens NA can be increased theoretically to ca. 1.515, and the resolution of the microscope is then approx. Image size = Actual size · Magnification Eq. 7.1 0.2 µm, suitable for viewing/studying most bacteria, but The distance between two distinct objects is referred not viruses. to as the resolution d and its relationship to numerical Reflections and loss of illumination intensity can be aperture (NA) and wavelength (λ): eliminated by using immersion oil with a RI that matches 1.22 · λ 1.22 · λ the RI of the lens glass. The NA of an objective is also d = or d = Eq. 7.2 N · sin α NAobjective+NAcondenser partially dependent upon the amount of correction for any optical aberration. Highly corrected objectives have Where, much larger NA for the respective magnification (Table λ is the light wavelength; 7.1). α equals one-half of the objective's opening angle and; N is the refractive index of the immersion medium used below the objective lens. MICROSCOPY 265 Table 7.1 Examples of objectives and their numerical aperture (NA) and 7.2.2 Low power objective optical correction. At the first setup of the microscope for optimal Magnification Plan Achromat Plan Fluorite Plan Apochromat performance, one should always start using a low (NA) (NA) (NA) magnification, e.g. 10× objective or lower. The eyepieces 2× 0.06 0.08 0.10 should be adjusted to a position suitable for viewing with 4× 0.10 0.13 0.20 both eyes. Then the next steps should be followed: 10× 0.25 0.30 0.45 • Lower the stage and insert the prepared slide so that 20× 0.40 0.50 0.75 the specimen is centred in the cone of light after 40× 0.65 0.75 0.95 passing through the condenser. 40× (oil) n/a 1.30 1.40 • Lower the lens until it is located a few millimetres 63× 0.75 0.85 0.95 from the slide surface. Look down the ocular while 63× (oil) n/a 1.30 1.40 carefully turning the coarse focus knob until the 100× (oil) 1.25 1.30 1.40 image becomes visible, and then by adjustment with the fine focus knob bring the specimen image into sharp focus. 7.2.1 Standard applications of light • Adjust the iris diaphragm to optimise the contrast to where the image is sharp. With the eyepiece taken microscopy 2 3 out, check that approx. 3 to 4 of the back lens is light-filled. If not, adjust the condenser position until The microscope must be set up and used properly to the iris diaphragm is sharply focussed and, if generate high quality images. The routine for setting up necessary, readjust the iris diaphragm until it the microscope as described below is recommended. superimposes on the circle of light. The best More experienced users may omit some of the steps in resolution is when the condenser is raised to near its the procedure as they may have gained familiarity with maximum height. the microscope and the samples under examination • Reinsert the eyepiece and examine the preparation already. Microscopes are precision instruments, sensitive using the fine focus adjustment and adjustable and expensive to repair or replace. Thus, they require mechanical stage. careful handling and maintenance. The following advice should be observed: • Avoid large temperature variations, store the 7.2.3 High power objective equipment in a cool and dark place, and protect it from dust. Always use a dust cover when it is not in use. In a good quality microscope the objectives should be • Objective lenses must be cleaned using a lens tissue. parfocal, meaning that all the objectives have very similar Do not dry wipe any lens as this may lead to focus settings. • scratching. Begin by blowing off dust or any loose Rotate the nosepiece to the high power objective and material with a pressurized optical duster. then the fine adjustment to sharpen the image. • • Be careful not to spread residual immersion oil onto Adjust the iris diaphragm to increase the illumination other oculars or clean objectives. Use a commercial and optimise contrast and then re-examine the cleaner or solvent (e.g. 70 % ethyl alcohol - EtOH) specimen. to remove any oil or grease. Flooding or placing solvents directly onto the lens is discouraged, and a 7.2.4 Immersion objective lens tissue must always be used. • Keep the microscope clean at all times. Check the required medium designated for the high • Objectives immersed in oil must be cleaned magnification objective (usually written on the objective immediately after use by wiping with a lens tissue. If or indicated by a black (oil) or blue (water) ring). Add a not, the oil may dry and thus need removal with a small drop of immersion oil or water (< 2 cm in diameter) suitable lens cleaning solution. Do not use any on the slide while rotating the objective lens into place. solvents (e.g. alcohol) as this might lead to lens The space between the slide and the lens should be filled damage. with the immersion medium. Some microscopes are only equipped with oil or water objectives. These should be used by adding a small drop 266 EXPERIMENTAL METHODS IN WASTEWATER TREATMENT of the required medium onto the slide and slowly lowering • Use the correct thickness for the cover slip.
Recommended publications
  • Gst Gram Staining Learning Objectives the Student Will  Use Aseptic Techniques in the Safe Inoculation of Various Forms of Media

    Gst Gram Staining Learning Objectives the Student Will  Use Aseptic Techniques in the Safe Inoculation of Various Forms of Media

    GSt Gram Staining Learning Objectives The student will Use aseptic techniques in the safe inoculation of various forms of media. Follow oral and written instructions and manage time in the lab efficiently. Use the bright field light microscope to view microbes under oil immersion, make accurate observations and appropriate interpretations and store the microscope according to lab procedures. Properly prepare a bacterial smear for accurate staining and describe the chemical basis for simple staining and negative staining. Background/Theory Differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. This link to the OpenStax Microbiology text provides more detail on these differential staining techniques. (OpenStax CNX, 2018) The Gram stain is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria containing the two most common types of cell walls. (OpenStax CNX, 2018) One type consists of an inner plasma membrane and a thick outer layer of peptidoglycan. The other type consists of a double phospholipid Figure 1 Simplified structures of Gram negative cells (left) and Gram positive bilayer with a thin layer of cells (right) peptidoglycan between the two. The Gram Staining technique remains one of the most frequently used staining techniques. The steps of the Gram stain procedure are listed below and illustrated in Figure. (OpenStax CNX, 2018) 1.
  • Small Wonder Acetone Vaporizer

    Small Wonder Acetone Vaporizer

    Acetone Vaporizers and Clearing Supplies: 84-85 Microscope Slides & Cover Glass: 86-87 Sample Collection and Storage Bags: 88 Respirator Fit Test Kits: 89-91 Small Wonder Acetone Vaporizer The Wonder Makers Small Wonder Acetone Vaporizer was developed as an economical and quick way to clear asbestos PCM slides. Specifically designed to meet NIOSH 7400, 7402, or ORM requirements for preparation of PCM samples. Features: ■ Heat control and thermometer built into vaporizer unit ■ Padded carrying case protects equipment and supplies ■ Detailed instruction manual ■ Enough supplies for the analysis of 1/2 gross of slides ■ Lightweight but solidly built ■ Designed in strict compliance with the NIOSH 7400, 7402, or ORM requirements for preparation of microscope slides for PCM analysis Specifications 4-7/8” L x 4-1/2” W x 2-3/4” H (Small Wonder vaporizer) DIMENSIONS 13” L x 11” W x 4” H (Complete Analysis Kit) Plastic case, ABS plastic cover on vaporizer, clear MATERIALS plastic bottles, metal and plastic tools. 2-1/2 lbs. (Small Wonder vaporizer) WEIGHT 8 lbs. (Complete Analysis Kit) Black vaporizer cover with FINISH gray cord. Case color may vary. Complete Kit Shown Includes everything needed to clear slides for PCM analysis PRODUCT DESCRIPTION Small Wonder Analysis Kit WM-42 Includes acetone vaporizer, 1 box microscope slides, 1/2oz Triacetin, 2oz Acetone, 1 microdropper, 1 syringe, 1 scalpel, tweezers, padded carrying case and instruction manual. WM-44 Acetone reagent, 2oz, 99% pure WM-45 Triacetin reagent, 1/2oz, 99% pure WM-47 1cc Syringe WM-48 Rounded Edge Scalpel WM-49 Microdropper 81 The Professionals Choice for Air Sampling Equipment Perm-O-Fix Acetone Vaporizer A simple, easy to use, actone vaporizer for use in preparing asbestos PCM samples as per NIOSH 7400.
  • Lysochrome Dyes Sudan Dyes, Oil Red Fat Soluble Dyes Used for Biochemical Staining of Triglycerides, Fatty Acids, and Lipoproteins Product Description

    Lysochrome Dyes Sudan Dyes, Oil Red Fat Soluble Dyes Used for Biochemical Staining of Triglycerides, Fatty Acids, and Lipoproteins Product Description

    FT-N13862 Lysochrome dyes Sudan dyes, Oil red Fat soluble dyes used for biochemical staining of triglycerides, fatty acids, and lipoproteins Product Description Name : Sudan IV Other names: Sudan R, C.I. Solvent Red 24, C.I. 26105, Lipid Crimson, Oil Red, Oil Red BB, Fat Red B, Oil Red IV, Scarlet Red, Scarlet Red N.F, Scarlet Red Scharlach, Scarlet R Catalog Number : N13862, 100g Structure : CAS: [85-83-6] Molecular Weight : MW: 380.45 λabs = 513-529 nm (red); Sol(EtOH): 0.09%abs =513-529nm(red);Sol(EtOH):0.09% S:22/23/24/25 Name : Sudan III Other names: Rouge Sudan ; rouge Ceresin ; CI 26100; CI Solvent Red 23 Catalog Number : 08002A, 25g Structure : CAS:[85-86-9] Molecular Weight : MW: 352.40 λabs = 513-529 nm (red); Sol(EtOH): 0.09%abs =503-510nm(red);Sol(EtOH):0.15% S:24/25 Name : Sudan Black B Other names: Sudan Black; Fat Black HB; Solvent Black 3; C.I. 26150 Catalog Number : 279042, 50g AR7910, 100tests stain for lipids granules Structure : CAS: [4197-25-5] S:22/23/24/25 Molecular Weight : MW: 456.54 λabs = 513-529 nm (red); Sol(EtOH): 0.09%abs=596-605nm(blue-black) Name : Oil Red O Other names: Solvent Red 27, Sudan Red 5B, C.I. 26125 Catalog Number : N13002, 100g Structure : CAS: [1320-06-5 ] Molecular Weight : MW: 408.51 λabs = 513-529 nm (red); Sol(EtOH): 0.09%abs =518(359)nm(red);Sol(EtOH): moderate; Sol(water): Insoluble S:22/23/24/25 Storage: Room temperature (Z) P.1 FT-N13862 Technical information & Directions for use A lysochrome is a fat soluble dye that have high affinity to fats, therefore are used for biochemical staining of triglycerides, fatty acids, and lipoproteins.
  • Using Microscopes

    Using Microscopes

    Using Microscopes Grade Level: 6, 7, 8, 9, 10 Duration: 30-45 minutes Classification: Classroom Subject(s): Biology, Microbiology Categories (STEM): Science, Technology Keywords: Staining, Microscopes, Microbes Introduction ● Summary: Students will learn how to properly use microscopes and view different cells and organisms. ● Description: Microscopes are often used to view different types of organisms, cells, and organelles. Microscopes can be used to visualize changes in cell structure and identify unknown organisms. Students will learn all the parts of microscopes and how to use and adjust them. Another important step of using microscopes is to learn how to properly prepare slides and wet mounts. Different organisms, cells, and organelles will be viewed under a microscope. In case of school policy limitations, note that student role models are required to cut vegetables at schools before the visit starts. *Note - School is responsible for providing microscopes for this activity. Online Resources: https://www.youtube.com/watch?v=SUo2fHZaZCU\ Vocabulary Organisms: an individual animal, plant, or single-celled life form Cell: smallest structural unit of an organism Organelle: organized structures (compartments) within a living cell Nucleus: dense organelle (generally in the middle of a cell) that contains genetic information Wet Mount: microscope slide holding a specimen suspended in a drop of liquid Materials Materials Quantity Reusable? Nail Polish 1 red per classroom Yes Microscopes (provided by school) TBD by school Yes Dirty
  • Student Safety Sheets Dyes, Stains & Indicators

    Student Safety Sheets Dyes, Stains & Indicators

    Student safety sheets 70 Dyes, stains & indicators Substance Hazard Comment Solid dyes, stains & indicators including: DANGER: May include one or more of the following Acridine orange, Congo Red (Direct dye 28), Crystal violet statements: fatal/toxic if swallowed/in contact (methyl violet, Gentian Violet, Gram’s stain), Ethidium TOXIC HEALTH with skin/ if inhaled; causes severe skin burns & bromide, Malachite green (solvent green 1), Methyl eye damage/ serious eye damage; may cause orange, Nigrosin, Phenolphthalein, Rosaniline, Safranin allergy or asthma symptoms or breathing CORR. IRRIT. difficulties if inhaled; may cause genetic defects/ cancer/damage fertility or the unborn child; causes damages to organs/through prolonged or ENVIRONMENT repeated exposure. Solid dyes, stains & indicators including Alizarin (1,2- WARNING: May include one or more of the dihydroxyanthraquinone), Alizarin Red S, Aluminon (tri- following statements: harmful if swallowed/in ammonium aurine tricarboxylate), Aniline Blue (cotton / contact with skin/if inhaled; causes skin/serious spirit blue), Brilliant yellow, Cresol Red, DCPIP (2,6-dichl- eye irritation; may cause allergic skin reaction; orophenolindophenol, phenolindo-2,6-dichlorophenol, HEALTH suspected of causing genetic PIDCP), Direct Red 23, Disperse Yellow 7, Dithizone (di- defects/cancer/damaging fertility or the unborn phenylthiocarbazone), Eosin (Eosin Y), Eriochrome Black T child; may cause damage to organs/respiratory (Solochrome black), Fluorescein (& disodium salt), Haem- HARMFUL irritation/drowsiness or dizziness/damage to atoxylin, HHSNNA (Patton & Reeder’s indicator), Indigo, organs through prolonged or repeated exposure. Magenta (basic Fuchsin), May-Grunwald stain, Methyl- ene blue, Methyl green, Orcein, Phenol Red, Procion ENVIRON. dyes, Pyronin, Resazurin, Sudan I/II/IV dyes, Sudan black (Solvent Black 3), Thymol blue, Xylene cyanol FF Solid dyes, stains & indicators including Some dyes may contain hazardous impurities and Acid blue 40, Blue dextran, Bromocresol green, many have not been well researched.
  • Microscope Slide-Making Kit

    Microscope Slide-Making Kit

    Microscope Slide-Making Kit 665 Carbon Street, Billings, MT 59102 Phone: 800.860.6272 Fax: 888.860.2344 www.homesciencetools.com Copyright 2005 by Home Training Tools, Ltd. All rights reserved. Viewing Slides Scan a slide at low power (usually 40X) to get an overview of the specimen. Center the part of the specimen you want to view at higher power. Adjust your lighting until the slide specimen has clear, sharp contrast. Then switch to medium power (usually 100X) and refocus to observe tissue and cell variations. Repeat at high power (usually 400X). Making Your Own Slides Whole Mounts: Whole mounts are made by placing small objects or specimens whole on a blank slide and then covering them with a coverslip. If your specimen is thick, a concavity slide will work better. You can make whole mounts of many things. Here are some ideas: Colored thread Feather (piece) Thin plant leaf Print (letters) Cloth fibers Hair strand Small insects Dust Algae Pond water Thin paper Insect parts Whole mount specimens must be thin enough to allow light to pass through them. Do not use large, hard objects like rocks, as they can break your slides and microscope lenses. Sections: Section mounts are made by slicing a very thin section of specimen. A cross section is made by slicing across the width or diameter of the specimen. A longitudinal section is made by slicing across the length of the specimen. It is difficult to make sections thin enough without an instrument called a microtome. You can make a simple microtome with a thread spool, a 1/4” diameter bolt at least 2” long, and a 1/4” nut.
  • An Antique Microscope Slide Brings the Thrill of Discovery Into a Contemporary Biology Classroom

    An Antique Microscope Slide Brings the Thrill of Discovery Into a Contemporary Biology Classroom

    ARTICLE An Antique Microscope Slide Brings the Thrill of Discovery into a Contemporary Biology Classroom FRANK REISER ABSTRACT that few others share my interests), I add them to my growing collection. The discovery of a Victorian-era microscope slide titled “Grouped Flower Seeds” began When I found skillfully prepared Victorian-era microscope slides listed an investigation into the scientific and historical background of the antique slide to under the category “Folk Art” in an antique dealer’s online catalog, it led develop its usefulness as a multidisciplinary tool for PowerPoint presentations usable in not only to my acquiring them but also to the beginning of a journey contemporary biology classrooms, particularly large-enrollment sections. The resultant involving Internet and library research, trips to herbaria, field collecting presentation was intended to engage students in discussing historical and contempo- excursions, and, finally, to the development of a PowerPoint lecture for rary biology education, as well as some of the intricacies of seed biology. Comparisons between the usefulness and scope of various seed identification resources, both online community college biology students. The slide that triggered the snow- and in print, were made. balling project is titled “Grouped Flower Seeds” (Figure 1). Having spent most of my life as a biology educator, I view most of Key Words: Grouped Flower Seeds; Watson and Sons; antique microscope slide; what I encounter in life from a science-oriented perspective. Finding pre- online seed identification resources; seed identification manuals; seed biology; pared microscope slides described as “folk art” sharply conflicted with large-enrollment lecture. my sense of the correct order of disciplines – particularly for items that I would reverently classify as the tools of scientists.
  • Electron Microscopy and the Investigation of New Infectious Diseases

    Electron Microscopy and the Investigation of New Infectious Diseases

    Review Electron microscopy and the investigation of new infectious diseases Alan Curry@) Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258 INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology.
  • GRAM STAIN REAGENTS - for in Vitro Use Only - Catalogue No

    GRAM STAIN REAGENTS - for in Vitro Use Only - Catalogue No

    GRAM STAIN REAGENTS - For in vitro use only - Catalogue No. SG51-55 Our Gram-Stain Reagents are intended to be The last step is the application of a counterstain. The used as a differential stain for the microscopic most common counterstain is safranin, which colors examination of bacterial cultures and laboratory decolorized cells pink. An alternate counterstain is basic specimens. fuchsin, which gives the decolorized cells more of a Gram staining is the single most useful test in bright pink or fuchsia coloration. The basic fuchsin the microbiology laboratory given its simplicity and counterstain works particularly well for anaerobic ability to differentiate bacteria into two main bacteria, but poorly for Legionella and Bordetella groups: gram-positive organisms and gram negative species. organisms. Hans Christian Joachim Gram first devised the original procedure in the late 19 th century, and although modifications have since been Formulation per Litre made, the basic principles and results remain the same. Our formulation is often referred to as the SG51 Gram Crystal Violet Hucker Modification. The staining spectrum includes almost all Crystal Violet ................................................ 20.0 g bacteria, many fungi, and parasites such as Ammonium Oxalate ....................................... 8.0 g Trichomonas , Strongyloides , and protozoan cysts. Methanol ................................................. 200.0 mL The notable exceptions include intracellular pathogens such as Chlamydia and Rickettsia , and SG52 Gram Iodine those organisms lacking a true cell wall such as Mycobacterium , Mycoplasma , and Ureaplasma . Iodine Crystals .............................................. 3.33 g The differential properties of the staining Potassium Iodide ........................................... 6.67 g process are attributed to the differences in composition between gram-positive and gram- SG53 Gram Decolorizer negative cell walls.
  • How to Construct and Use a Simple Device to Prevent the Formation of Precipitates When Using Sudan Black B for Histology

    How to Construct and Use a Simple Device to Prevent the Formation of Precipitates When Using Sudan Black B for Histology

    Acta Botanica Brasilica 29(4): 489-498. 2015. doi: 10.1590/0102-33062015abb0093 How to construct and use a simple device to prevent the formation of precipitates when using Sudan Black B for histology João Marcelo Santos de Oliveira1 Received: April 17, 2015. Accepted: July 1, 2015 ABSTRACT The present work aims to demonstrate the stages of fabrication and use of a simple device to avoid the formation or fixa- tion of precipitates from Sudan Black B solution on tissues. The device consists of four coverslip fragments attached to a histology slide, which serve as points of support for the histological slide under analysis. To work properly, the histology slide with the sections should be placed with the sections facing downwards the device. A small space between the device and the histology slide is thereby created by the height of the coverslip fragments. When Sudan Black B is applied, the solution is maintained within the edges of the device and evaporation is minimized by the small space, thereby reducing the consequent formation of precipitates. Furthermore, by placing the sections facing downward the device, any sporadically formed precipitates are prevented from settling on and fixing to the sectioned tissues or organs. By avoiding the formation of precipitates, plant cells, tissues and organs can be better observed, diagnosed and photomicrographically recorded. Keywords: histochemical tests, histology, lipids, plant anatomy, Sudan Black B Introduction for organic solvents, printer ink, varnishes, resins, oils, fats, waxes, cosmetics and contact lenses. Sudan reagents, including the traditional Sudan III, IV Lansink (1968) isolated two pure fractions of Sudan and Sudan Black B (SBB), are widely utilized to determine Black B, in addition to impurities, and denominated them lipids (Horobin 2002) in animals, plants and hydrophobic SBB-I and SBB-II.
  • Second Harmonic Imaging Microscopy

    Second Harmonic Imaging Microscopy

    170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison.
  • Applications of Microscopy in Bacteriology

    Applications of Microscopy in Bacteriology

    Microscopy Research, 2016, 4, 1-9 Published Online January 2016 in SciRes. http://www.scirp.org/journal/mr http://dx.doi.org/10.4236/mr.2016.41001 Applications of Microscopy in Bacteriology Mini Mishra1, Pratima Chauhan2* 1Centre of Environmental Studies, Department of Botany, University of Allahabad, Allahabad, India 2Department of Physics, University of Allahabad, Allahabad, India Received 28 September 2015; accepted 2 January 2016; published 5 January 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Bacteria are smallest primitive, simple, unicellular, prokaryotic and microscopic organisms. But these organisms cannot be studied with naked eyes because of their minute structure. Therefore in search for the information about the structure and composition of bacterial cells, cell biologist used light microscopes with a numerical aperture of 1.4 and using wavelength of 0.4 µm separa- tion. But there are still certain cellular structures that cannot be seen through naked eyes, and for them electron microscope is used. There are certain improved types of light microscope which can be incorporated to improve their resolving power. Hence microscopy is playing a crucial role in the field of bacteriology. Keywords AFM, SEM, TEM, Microscopy, Bacteriology 1. Introduction To get acquainted with the world of bacteria like small organisms, very effective and advanced technique is re- quired. The size of bacteria ranges between 0.5 - 5.0 micrometer in length; the smallest of them are members of mycoplasma which measures 0.3 micrometers [1].