Micrometry Study Guide

MICROMETRY  Helps in measurement of the size of microscopic objects.  Achieved by the use of1.STAGE MICROMETER 2.MICROMETER EYE PIECE Ocular micrometre with retaining ring inserted into the eyepiece’s base CALIBRATION OF THE OCULAR MICROMETER Stage micrometre (objective micrometre) has calibration lines or graduations that are separated 0.01 mm (10 μm) apart. Calibration achieved by superimposing the two scales and determining how many ocular graduation coincide with graduations on stage micrometre. Stage micrometre positioning by cantering The small glass disk over the light source After completed calibration, specimen’s slide is positioned for measurement TYPES OF MICROSCOPES 1.SIMPLE MICROSCOPE 2.LIGHT MICROSCOPE A. Bright field microscope B. Darkfield microscope C. Phase contrast microscope D. Fluorescence microscope 3.ELECTRON MICROSCOPE 4.OTHER MICROSCOPES: INVERTED STEREO POLARIZING CONFOCAL SCANNING LASER 5.NEWER MICROSCOPE: SCANNING PROBE 1.SIMPLE MICROSCOPE  The simple microscope consists of a single lens or a set of lenses close to one another.  The image is virtual; i.e. it cannot be projected on a screen as can a real image.  e.g. magnifying lens. 2.LIGHT MICROSCOPE A. Bright field microscope (compound light microscope) B. Dark field microscope C. Fluorescence microscope D. Phase contrast microscope A. BRIGHT-FIELD MICROSCOPE The basic microscopic system used in microbiology laboratory. Called as light microscope / bright-field microscope because visible light passes directly through its lenses until it reaches the eye.  Its also known as compound microscope because of its two – lens system with the objective lens nearer the object and the ocular lens nearer the eye. PRINCIPLE  2 different lens system produce more magnified image.  Objective lens magnifies the object and produces an inverted real image and eye piece magnifies that real image to an inverted virtual image.  The total magnification of microscope = Me X Mo OPTICS OF COMPOUND MICROSCOPE The final image formed is Magnified ,Virtual and Erect.  Broadly categorized into two types- Upright  Inverted ( based on the positions of the light source and the objective).  UPRIGHT MICROSCOPE 1) The objective is pointing INVERTED MICROSCOPE 1) The objective is underneath the downward to view the stage and light is directed from specimen from above & Light above. from below. 2) Suitable for slides. 2) Suitable to see culture plates such as Petri dishes. COMPOUND LIGHT MICROSCOPE UPRIGHT INVERTED Images formed by bright field microscope Gram positive cocci Gram negative bacilli ADVANTAGES  Used to view fixed specimen or live cells.  Simple to use with fewer adjustments required. LIMITATIONS  It has very low contrast and most cells have to be stained.  Staining may destroy or introduce artefacts.  Resolution is limited to 200nm. B.DARK FIELD MICROSCOPE Produces a bright image of the object against a dark black ground. Used to render visible delicate organisms which cannot be seen in unstained preparations in an ordinary microscope. REQUISITES FOR DARK FIELD MICROSCOPY • Dark background • Condenser • Illuminant of sufficient intensity • Funnel stop PRINCIPLE:  A dark field condenser with a central opaque area that blocks light from entering objective lens and a peripheral annular hollow area which allows light to pass through and focus on the specimen obliquely is used.  Only the light reflected by the specimen enters the objective lens whereas the unreflected light doesn’t.  Hence the specimen is brightly illuminated but the background appears dark.  ADVANTAGE: 1) Structure & motility can be seen. 2) Used to identify living unstained, transparent cells.  LIMITATION: 1) Limited colours. 2) Poor depth of field (internal structures not seen).  USES: 1) Extremely slender organism- Spirochetes, Leptospira & Borellia . 2) Sheath and nuclei of microfilaria. 3) Vibrio cholera, Campylobacter jejuni, Endospore. C. PHASE CONTRAST MICROSCOPE PRINCIPLE:  Converts slight differences in refractive index and cell density into easily detectable variations in light intensity.  Specimens appear dark against a bright background -positive contrast  Bright against a dark background -negative contrast.  The borders of images are surrounded by a characteristic bright halo. REQUISITE FOR PHASE CONTRAST MICROSCOPY  Annular Diaphragm or Condenser Annulus : It’s opaque flat-black (light absorbing) plate with a transparent annular ring.  Produces hollow cone of light. Phase Plate: Placed in back focal plane of objective. Function:  Enhances phase difference by retarding diffracted wave front by one quarter of wavelength.  Reduces intensity of direct rays and equalizes it with diffracted rays intensity. ADVANTAGES : Suitable for transparent specimens, living cells (natural state) without being killed, fixed and stained. Ex- Vibrio cholerae in stool specimen and culture.  Most commonly used for Fungal cell identification. Microbial motility can be studied. LIMITATIONS :  Annuli or ring limits the aperture to some extent which causes decrease in resolution.  Not ideal for thick specimen. Shade off and Halo effect may occur. D.FLUORESCENCE MICROSCOPE PRINCIPLE: When fluorescent dyes are exposed to UV rays they become excited and are said to fluoresce i.e. they convert this invisible short wavelength rays into light of longer wavelength. Micro organisms stained with the fluorescent dye and exposed by these exciting rays ,when examined under the microscope appear bright against the dark background.  AUTO FLUORESCENCE: Certain microbes directly fluoresce when placed under UV lamp,eg. Cyclospora.  Certain microbes fluoresce when stained non- specifically by fluorochrome dyes. Based on the composition of fluorescent stain reagents, fluorescent staining technique may be divided in two general categories: a. Fluorochroming- in which a fluorescent dye is used alone. b. Immunofluorescence- in which a fluorescent dye has been linked (conjugated) to specific antibodies. Fluorochroming  In fluorochroming a direct chemical interaction occurs between the fluorescent dye and a component of the bacterial cell as in light microscopy.  The most common fluorochroming methods used are: A) Acridine orange stain: gives orange red fluorescence with RNA and yellow green fluorescence with DNA. Its used for detection of malarial parasite by Quantitative Buffy Coat (QBC) examination. B) Auramine -rhodamine stain: used for tubercle bacilli which appear bright yellow or orange against a greenish background. C) Calcofluor white stain: used to facilitate the visualisation of fungal elements. Immunofluorescence  It uses the principle of antigen-antibody reaction.  Antibodies have high specificity for microbial antigens. That is, antibodies specific for an antigen characteristic of a particular microbial species will only combine with that antigen.  Therefore, if antibodies are conjugated to a fluorescent dye, the resulting dye-antibody conjugate can be used to detect or “tag” specific microbial agents. 4.ELECTRON MICROSCOPE  It uses a beam of electrons instead of beam of light and electromagnets rather than glass lenses to focus beam.  The electrons must travel through vaccum because collision with air molecules would scatter the electrons and produce distorted image. PRINCIPLE: Beam of electrons pass through magnetic condensor and bombard on thin sliced specimen mounted on copper slide. The specimen scatters the electrons passing through it and is focused by magnetic lenses to form a visible image on fluorescent screen. ELECTRON MICROSCOPE  Transmission electron microscope (TEM):Electrons are generated by electron gun which travel in vaccum and then bombarded on the thin spliced specimen.  Scanning electron microscope (SEM):A beam of electrons scan the surface of an opaque object and produce an image of that surface. TRANSMISSION ELECTRON MICROSCOPE(TEM) • Gives a 2-D view. • Thin slices(20-100 nm.) of specimen are suitable. SCANNING ELECTRON MICROSCOPE(SEM) • . • 3-D View. • Resolution< 7nm. FREEZE FRACTURING AND ETCHING TECHNIQUE FREEZE FRACTURING-It is a technique to view the interior of the cell. Here the cell is rapidly frozen, then warmed and then fractured with a knife. The cleaving of the specimen reveals the internal structure of the cell. FREEZE ETCHING-It involves the evaporation of water from the frozen and fractured specimen, that can then expose additional surfaces for examination. COMPARING SEM AND TEM TEM SEM Electron Beam Broad, static beams Beam focused to fine point; sample is scanned line by line Voltages Needed TEM voltage ranges from 60-300,000 volts Accelerating voltage is lower; not necessary to penetrate the specimen Interaction of the beam electrons Specimen must be very thin Wide range of specimens allowed; simplifies sample preparation Imaging Electrons must pass through and be transmitted by the specimen Information needed is collected near the surface of the specimen Image Rendering Transmitted electrons are collectively focussed by the objective lens and magnified to create a real image Beam is scanned along the surface of the sample to build up the image  ADVANTAGE: The limit of resolution is improved by a factor of 1000. So object can be measured up to nanometer.  DISADVANTAGE: 1) Specimen preparation is time-consuming. 2) Only fixed and stained (dead) specimens can be examined.  USES: 1)Virus detection 2) Thin sections Type of microscope Special feature Appearance of structure Magnification Light Visible light illuminates objects Stained microorganisms on clear background 1000X Dark field Special condenser scatters light Unstained microorganisms on dark background 1000X Phase contrast Special condenser throws light “out of focus’’. Unstained microorganisms on dark background 1000X Fluorescence UV light illuminates fluorescence coated object Fluorescing microorganisms on dark background 1000X TEM Short wavelength electron beam penetrates section Alternating light and dark areas reflecting internal structure 20 million X SEM Short wavelength electron beam knocks loose electron showers Microbial surfaces 1-2 million X OTHER MICROSCOPES: INVERTED MICROSCOPE • Used in metallurgy • Examination of cultures in flat bottom dishes • Micro dissection • Examination of parasites • Observation of agglutination in serology STEREO MICROSCOPE • They are constructed like binoculars , enabling specimens to be simultaneously viewed with both eyes in 3-dimension. • The distance between the objective and the stage is long enabling activities such as dissection of tiny insects placed under the objective. POLARIZING MICROSCOPE  Uses two Polariser  Gives information about Birefringence of a sample.  Used in Crystallography, Urine examination  Apple Green Birefringence seen in AMYLODOSIS CONFOCAL LASER SCANNING MICROSCOPE  Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.  It enables the reconstruction of 3D structures from the obtained images. NEWER MICROSCOPE SCANNING PROBE MICROSCOPE  Developed in 1981, by Gerd Binnig and Heinrich Rohrer, Nobel Prize in 1986.  It measures surface features by moving a sharp probe over object surface. Used to visualize atoms and molecules. Scanning Tunnelling Microscope Atomic Force Microscope (STM) (AFM) CARE OF MICROSCOPE 1) Keep the microscope clean before and after use. 2) Moist, living or preserved materials must be observed through a cover slip & maintain a safe distance between the cover slip and the objective lenses. 3) Clean the lenses with lens paper only. DO NOT CLEAN THE LENSES WITH HANDKERCHIEFS, FACIAL TISSUES, PAPER TOWELS, ETC. 4) After use, the microscope should be wrapped properly and with the lowest power objective lens in position. 5) Always carry the microscope in a straight upright position with one hand around the arm and the other hand under the base. 6) Keep in warm and dry condition