Chapter 3 Microscopy and Preparation of Specimen PDF

CHAPTER 3 MICROSCOPY AND PREPARATION OF SPECIMEN Light Properties Types of microscope Preparation of Specimens Staining Techniques & Application MICROSCOPE DEVELOPMENT Hand-colored engravings show the kinds of “animalcules” Leeuwenhoek observed and recorded wi...

CHAPTER 3 MICROSCOPY AND PREPARATION OF SPECIMEN Light Properties Types of microscope Preparation of Specimens Staining Techniques & Application MICROSCOPE DEVELOPMENT Hand-colored engravings show the kinds of “animalcules” Leeuwenhoek observed and recorded with his superior microscope. http://discovermagazine.com/2015/june/21-leeuwenhoeks-lucky-break MICROSCOPE DEVELOPMENT  UNITS OF MEASUREMENT Ranges of sizes which can be detected by unaided human eye & various microscopy  Modern Microscopes Compound light microscope Scanning electron microscope (SEM) Transmission electron microscope (TEM) LIGHT PROPERTIES  LIGHT PROPERTIES Wavelength  Microscope Resolution ability of lens to separate or distinguish small objects that are close together wavelength of light used is major factor in resolution shorter wavelength  greater resolution  EFFECT OF WAVELENGTH ON RESOLUTION Resolution- ability to see two items as a separate and discrete unit (a) rather than as a fuzzy, overlapped single image (b) EFFECT OF WAVELENGTH ON RESOLUTION  ANALOGY FOR THE EFFECT OF WAVELENGTH ON RESOLUTION Light must pass between 2 objects in order to be seen as separate things Key to resolution : short enough wavelength to fit between the objects Target diameter = various wavelength LIGHT INTERACTIONS WITH AN OBJECT IT STRIKES Reflection Transmission Absorption Refraction  Light Properties Refraction – light is refracted (bent) when passing from one medium to another of diff. density – refractive index (RI) a measure of the speed at which light passes through the material  QUESTION Why do you apply immersion oil? How does immersion oil help to improve the quality of image? REFRACTION & IMMERSION OIL Refractive index glass = 1.5 air = 1.0 oil = 1.5 water = 1.3  TYPES OF MICROSCOPE TYPES OF MICROSCOPE 1. LIGHT MICROSCOPY Light microscope Bright-field Dark-field Dual Beam microscope Phase-contrast Differential Interference Contrast Microscope (DIC) Fluorescence microscopy 2. ELECTRON MICROSCOPY Scanning Electron Microscope (SEM) Create images of specimen surface Transmission Electron Microscope (TEM) Create images of internal structure of microbes Scanning Tunneling Microscope (STM) Creates surface image at the atomic level  LIGHT MICROSCOPY Also called compound microscopes – image formed by action of 2 lenses Many types of light microscope Total magnification: ocular lens x objective lens  working distance distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus microscopic field the areas that is visible through a microscope  Microscope Lenses focus light rays at a focal point, F focal length, f - distance between center of lens and focal point strength of lens related to focal length short focal length  more magnification  Bright-Field Microscope produces a dark image against a brighter background can be used to examine stained and unstained samples Water flea Vorticella sp.  Dark-Field Microscope produces a bright image against dark background used to observe living, unstained preparations – For eukaryotes - has been used to observe internal structures – For prokaryotes - has been used to identify bacteria such as Treponema pallidum (causative agent of syphilis)  Phase-Contrast Microscope enhances contrast between intracellular structures that have slight differences in refractive index & density excellent way to observe living cells – useful for detecting bacterial components (e.g: endospores and inclusion bodies) that have refractive indices different from water Amoeba  Differential Interference Contrast Microscope (DIC) creates image by detecting differences in refractive indices and thickness of different parts of specimen excellent way to Phase-contrast observe living cells – Live, unstained cells appear brightly colored and three- dimensional (3D) unsuitable for thick Differential Interference Contrast samples, highly (DIC) Bright-field Dark-field Phase-contrast DIC  Fluorescence Microscope specimens usually stained with fluorochromes exposes specimen to ultraviolet, violet, or blue light shows a bright image resulting from fluorescent light emitted by specimen  Flavivirus NS1 protein Nucleus staining of Vero expressions in Vero cells cells (FITC staining) (DAPI staining) Source : Siti NK Addis, Australian National University ELECTRON MICROSCOPY electron beams pass through object produce high magnification and high resolution image. But how?  How electron microscopy produces higher quality of image? wavelength of electron beam is shorter than light, therefore, gives higher resolution of image ~0.005 nm wavelength (~100,000x shorter than visible light) Resolution Wavelength Types of Electron Microscope 1. Scanning Electron Microscope (SEM) 2. Transmission Electron Microscope (TEM) 3. Scanning Tunneling Microscope (STM) Can you tell which image is from SEM, TEM and STM? 1. Scanning Electron Microscope (SEM) uses electrons reflected from the surface of a specimen to create image produces 3-dimensional image of specimen’s surface Can resolve objects as close as 20nm, giving magnification up to 50,000x Scanning Electron Microscopy Y.K. Yamasaki et al., “Evaluation of Proctophyllodes huitzilopochtlii on feathers from Anna’s (Calypte anna) and Black-chinned (Archilochus alexandri) Hummingbirds: prevalence assessment and imaging analysis using light and tabletop scanning electron microscopy,” PLoS ONE, doi:10.1371/journal.pone.01 91323, 2018. 2. Transmission Electron Microscope (TEM) The technology was first developed by German scientists Max Knoll and Ernst Ruska in 1931 requires thin sections (70-90nm) of a specimen Can resolve objects as close as 1 nm and magnify up to 500,000x Transmission Electron Microscopy Can you tell what is the function of TEM and SEM? TEM An electron micrograph of a thin section of MERS- CoV, showing the spherical particles and cross- sections through the viral nucleocapsid. Source: CDC  The World’s Highest Resolution Electr on Microscope - Hitachi Can you tell the challenges in getting the highest resolution at atomic level? 3. Scanning Tunneling Microscope (STM) An instrument for imaging surfaces at the atomic level Probe is lowered toward the specimen surface Electron flow from the tunnel in the probe STM Operating principle Probe movement while following the surface contours is recorded IMAGES OF STM PREPARATION OF SPECIMEN FOR MICROSCOPY PREPARATION OF SPECIMEN (FOR LIGHT MICROSCOPY increases visibility of specimen enhance specific morphological features preserves specimens e.g: wet mounts, bacterial smear (Prac 3: Preparation of Bacterial Smear & Staining)  WET MOUNTS Provide good view of microbial motility (movement)  BACTERIAL SMEAR organism is killed and firmly attached to microscope slide internal and external structures of the bacteria are preserved and fixed in position Too thick: trouble seeing individual cells Too thin: find no microorganisms FIXATION  Fixation Methods of fixation:  HEAT FIXATION  used for prokaryotes  preserves overall morphology but not internal structures  CHEMICAL FIXATION  used for larger, more delicate microorganisms  protects fine cellular substructure  PREPARATION OF SPECIMEN (FOR ELECTRON MICROSCOPY) Specimens must be cut very thin Specimens are chemically fixed and stained with electron dense material FREEZE-ETCHING – freeze specimen in liquid nitrogen – cleaving of a specimen reveals surfaces of inside a cell. SHADOW CASTING – coating specimen with a thin film of platinum or other heavy metal – area behind the specimen did not receive a coating of metal appear as shadow Scientists Who Developed Cryo-Electron Microscopy Win Nobel Prize  STAINING TECHNIQUES AND APPLICATION PRINCIPLES OF STAINING DYES – Used to stain a specimen by binding to a cellular structure – Make internal and external structures of cell more visible by increasing contrast with background – Two common features: have chromophore* groups able to bind to cells * A chromophore is the part of a molecule responsible for its colour. The colour arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. – Ionizable dyes have charged groups Basic dyes have positive charges -methylene blue, crystal violet, safranin & malachite green Acidic dyes have negative charges -Eosin and rose Bengal, acid fuchsin TYPES OF STAIN Simple stain Differential stain 1. SIMPLE STAINING a single dye is used use can determine size, shape and arrangement of bacteria 2. DIFFERENTIAL STAINING Make use of two or more dyes to distinguishes two kind of organism or btw two different parts of an organism Divides microorganisms into groups based on their staining properties – e.g. Gram stain – e.g. acid-fast stain  GRAM STAINING most widely used differential staining procedure divides bacteria into two groups: Bacillus anthracis Gram positive and Gram negative based on differences in cell wall structure Escherichia coli STEPS IN GRAM STAINING  ACID-FAST STAINING also called Ziehl-Neelsen Staining useful for staining members of the genus Mycobacterium due to high lipid content in cell walls e.g. Mycobacterium tuberculosis e.g. Mycobacterium leprae * Acid-fastness is a physical Acid fast bacteria : red Not acid fast bacteria: Blue property of some bacteria referring to their resistance to decolorization by acids during staining procedures STEPS IN ACID-FAST STAINING STAINING SPECIFIC STRUCTURES Certain specific structures of bacteria can be stained: Flagellar Capsule Endospore  FLAGELLAR STAIN used to stain specific flagellar structure (too thin to be easily seen with the light microscope) mordant applied to increase thickness of flagella *mordant is a substance used to set dyes on tissue sections by forming a coordination complex with the dye which then attaches to the tissue  CAPSULAR STAIN used to visualize capsules surrounding bacteria-usually resist taking up a stain. capsules are colorless against stained background  ENDOSPORE STAIN Bacterial endospore (resistant cells) is green color and vegetative cell is a different color (red) B. subtilis endospores (green) STEPS IN ENDOSPORE STAINING End of Chapter 3