Microscopy and Slide Preparations Lab Discussion 2 PDF

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 2: Microscopy and Slide Preparations Trisha Mae M. Cantillano  Microscopy: Principles behind Microscope...

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 2: Microscopy and Slide Preparations Trisha Mae M. Cantillano  Microscopy: Principles behind Microscope Microscopes allow the study of structure of living organisms and the discovery of numerous species that cannot be seen by our naked eye The first microscope uses single lens (Sir Antonie van Leewenhoek) which can magnify 300x the size of the organism. The first microscope that uses double lenses was not invented until the late 16th century T.M.M. Cantillano Microscopy and Slide Preparations  Microscopy: Principles behind Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Light Microscope- use sunlight or artificial light Bright field microscope Dark field microscope Phase contrast Microscope Fluorescence Microscope Electron Microscope – use electrons Transmission Electron Microscope Scanning Electron Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Bright-Field Microscope Produces a dark image against a brighter background Usually have several objective lenses Can either be Simple or Compound T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Dark-Field Microscope Produces a bright image of the object against a dark background Specimen appears bright against dark background Provides contrast to unstained tissue so living cells can be viewed T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Phase Contrast Microscope Produces a brighter image in a dark background Contrast is due to out of phase rays Allow to see internal cellular components and examine growth of living cells T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Samples are usually stained with fluorochromes Shows bright image of the object from the fluorescent light emitted by the specimen T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Electron Microscope Co-invented by Max Knoll and Ernst Ruska in 1931 Uses a beam of highly energetic electrons to examine objects on a very fine scale Transmission Electron Microscope and Scanning Electron Microscoe T.M.M. Cantillano Microscopy and Microbial Morphology  Microscopy: Principles behind Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Microscopy: Principles behind Microscope Eyepiece/ Ocular lens: 10x Scanning: 4x Low-power Objective: 10x High-Power Objective: 40x Oil Immersion Objective: 100x Linear Magnification = (eyepiece) x (objective) = 10 x 10 = 100 x T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry Micrometry is the science in which we have some measurement of the dimensions of a specimen observed under microscope. Two types – Stage Micrometer (calibrated) – Ocular Micrometer (non-calibrated) T.M.M. Cantillano Microscopy and Slide Preparations  1 Div = 0.01mm = 10 µm 10 Div = 0.1 mm = 100 µm 100 Div = 1 mm = 1000 µm  Micrometry: Calibrating the Ocular Micrometer c = (20 stage units) x (10 µm/stage unit) Calibration constant (c): 30 of ocular units c = (no. of stage units) x (µm/stage unit) = 200 µm/30 ocular units no. of ocular units = 6.67 µm/ ocular unit T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry: Measuring a Specimen Calibration constant (c):6.67 µm/ ocular unit Specimen size = (no. of ocular units) x (c) = (39 ocular units) x (6.67 µm/ ocular unit) = 260.13 µm T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry: Magnification of Illustration MI = Size of illustration. Actual size of specimen = 3cm = 30,000 µm = 30,000 µm 260.13 µm = 115.33 x T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry NOTE THAT: You must Calibrate the objectives first (Scanning, LPO, HPO, OIO) before preparing and measuring your specimens. Measure specimen using the most appropriate objective T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Wet Mount, Hanging drop, and Staining What is the principle behind Wet Mount and Hanging Drop Method? 1. Observe cell activities such as motility and binary fission. 2. Observe the natural sizes and shapes of the cells. T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Wet Mount T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Hanging drop T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining WHAT IS GRAM STAINING? Gram Staining is a differential staining procedure employed to differentiate Gram Positive bacteria from Gram Negative bacteria based on their cell wall. T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining VS. Gram Positive Gram Negative ✓ thick cell wall ✓ Thin cell wall ✓ Peptidoglycan is more ✓ Second lipid membrane called than 50% of dry weight lipopolysaccharides ✓ More of a gel than a rigid layer T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining Reagents used: 1. Crystal violet 2. Gram’s Iodine 3. 95% Ethanol 4. Safranin T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining Bacillus subtilis Escherichia coli T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology WHAT IS MORPHOLOGY? In microbiology, the term morphology means cell shape. T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy I. Letter “e” mounting II. Wet Mount III. Hanging Drop Technique IV. Micrometry V. Gram Staining T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Materials Cut out Letter “e” Labeling materials Cut out thin leaf/ decaying fruit Cleaning materials Cake of baker’s yeast (for the Waste bottle whole class) Test tube and test tube rack Lagoon water (freshly collected) Alcohol lamp Distilled water (1L/ group) Wash Bottle NaCl Solution Ocular and Stage Micrometer (2 Yogurt pcs for the whole class) Toothpick Microscope Plastic dropper Stains (Crytal Violet, Gram’s Vaseline (Petroleum Jelly) Iodine, Safranin, Methylene Blue) Glass slides and cover slip T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure I. Each member should prepare a cut-out letter “e”, and place it at the center of a glass slide. With a plastic dropper carefully place a drop of water on the surface of a clean slide Cover the drop with a clean coverslip. View under 4x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure II. Prepare of thin leaf or a part of a decaying fruit and carefully place it at the center of the slides (prepare 2 slides for each group) Carefully drop a right amount of water and cover the it with a clean coverslip. View under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure III Prepare your lagoon water samples Prepare a clean depression slide/ improvised depression slides Using a toothpick, spread a thin ring of Vaseline approximately ¼ inch outside the concavity Transfer 2 loopfull at the central surface of the cover slip Invert the slide and center the depression over the droplet on the coverslip. Press lightly. Quickly turn over the slide View under 4x and10x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure IV Prepare your solution by adding water in a test tube with yeast enough to cause visible clouding (approximately 1 loopful per 15 ml of water) Remove a small amount of the suspension with a plastic dropper and carefully place a drop on the surface of a clean slide Cover the drop with a clean coverslip. View under 40x magnification Using ocular and stage micrometer, measure at least 5 cells (Calibrate the objectives first) T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure IV Put a droplet of methylene blue at the center of the slide Gently scrape the inside of your cheek with a clean cotton swab or toothpick. Transfer the sample onto the center of a clean glass microscope slide by smearing the swab in a thin layer. Carefully put a clean coverslip. View under 40x magnification Using ocular and stage micrometer, measure at least 5 cells (Calibrate the objectives first) T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure Prepare your samples. Add a smear on a clean slide Heat fix the bacterial samples Add stains View under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Expected Output Picture of each slides that you prepared Microscopic image of cut out letter “e” under 4x magnification Microscopic image of your leaf/ portion of decaying fruit under 4x, 10x, 40x magnification Microscopic image of “suspected algae” in your lagoon water samples under 4x and 10x magnification Microscopic image of yeast/ cheek cell under 40x magnification. Calibration constant of Scanning, LPO and HPO Measurement of 5 yeast/ cheek cells under HPO Microscopic image of gram-stained cells under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations