CHAPTER 2 - METHODS IN MICROBIOLOGY.pdf

CHAPTER 2: Methods in Microbiology Prepared by: Glaezel Angelique T. Barredo, PhD Expected Learning Outcomes At the end of this chapter, you should be able to: 1. Enumerate and differentiate the types of microscope; 2. Associate the microscope type to their specific applications; and 3. Describe...

CHAPTER 2: Methods in Microbiology Prepared by: Glaezel Angelique T. Barredo, PhD Expected Learning Outcomes At the end of this chapter, you should be able to: 1. Enumerate and differentiate the types of microscope; 2. Associate the microscope type to their specific applications; and 3. Describe the types of staining procedures and associate these with their specific applications. Microscopy Robert Hooke Anton van Leeuwenhoek Galileo Galilei Joseph Jackson Lister 1940 – Electron Microscope https://openstax.org/books/microbiology/pages/2-2-peering-into-the-invisible-world Microscopy Units of measurement: 1 micrometer (µm) = 10-6 m 1 nanometer (nm) = 10-9 m What makes a good microscope? Types of Microscopes 1. LIGHT MICROSCOPES use light waves and mirrors a. Simple - short focal length - only 1 lens - magnification: 300x b. Compound or Complex - 2 sets of lenses - magnification: 1000x  https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Typical Brightfield Microscope Types of Microscopes 2. ELECTRON MICROSCOPES use electron beams as energy source has higher magnification and resolving power for objects smaller than 0.2 mm in diameter in vacuum  Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock biology of microorganisms (15th edition). Harlow: Pearson Education Limited. Electron Microscope Types of Light Microscopes 1. BRIGHT FIELD objects under study are darker Bacillus anthracis in cerebrospinal fluid microscopic field is brightly lit gross morphology https://openstax.org/books/microbiology/pages/2-introduction Credit: modification of work by Centers for Disease Control and Prevention Types of Light Microscopes 2. DARK FIELD microscopic field is dark Treponema pallidum objects under study are luminous for specimens that are: invisible in the ordinary light microscope cannot be stained by standard methods distorted by staining https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: Centers for Disease Control and Prevention Types of Light Microscopes 3. PHASE CONTRAST principle is based on variations in Unstained Epithelial Cells the refractive indices (measure of bending or refracting of a beam of light on entering a denser medium) not necessary to fix or stain cells detailed examination of internal structure https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Types of Light Microscopes 4. DIFFERENTIAL INTERFERENCE CONTRAST Fonsecaea pedrosoi principle is based on variations in the refractive indices advantage: no diffraction halo associated with phase contrast disadvantage: the three-dimensional appearance may not represent reality https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Types of Light Microscopes 5. FLUORESCENCE makes use of fluorochromes visualizes specimens that fluoresce detection of immunological reactions https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Neisseria gonorrhoeae Schistosoma mansoni https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by Centers for Disease Control and Prevention Types of Light Microscopes 6. CONFOCAL useful for thick specimens Roof-dwelling Cyanobacterium Biofilm like biofilms used to visualize structures https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by American Society of Microbiology Types of Light Microscopes 7. TWO-PHOTON useful for examining living Mouse Intestine Cells Stained With Fluorescent Dyes cells within intact tissues currently limited to advanced clinical and research laboratories https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by Alberto Diaspro, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola Ramoino, Cesare Usai Types of Electron Microscopes 1. TRANSMISSION ELECTRON Cells in a Biofilm MICROSCOPE (TEM) ultrastructure in thin section of the cells can project images in a much higher resolution—up to the atomic level of thinner objects examine viruses https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by American Society of Microbiology  https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Bruslind)/02%3A_Microscopes How does TEM work? Types of Electron Microscopes 2. SCANNING ELECTRON MICROSCOPE (SEM) surface features of viruses and Staphylococcus aureus cells reveals a 3-D image https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by Centers for Disease Control and Prevention  https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Bruslind)/02%3A_Microscopes How does SEM work? Scanning Probe Microscope SCANNING TUNNELING ATOMIC FORCE MICROSCOPE (STM) MICROSCOPE (AFM) Pure Gold Surface Nanocellulose https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy https://openstax.org/books/microbiology/pages/2-3-instruments-of-microscopy Credit: modification of work by “Erwinrossen”/Wikimedia Commons Credit: modification of work by “Erwinrossen”/Wikimedia Commons EXAMINATION OF MICROORGANISMS A. Living or Natural State Advantages: observation of unaltered/undistorted characteristics cellular processes can be studied motility can be observed simple to prepare Disadvantage: refractive index of cells almost similar to that of water EXAMINATION OF MICROORGANISMS B. Stained Preparations Advantages: provides contrast slides can be preserved specimens are killed Disadvantages: more complicated and tedious to prepare expensive Staining Three basic steps in staining microorganisms: 1. Smear Preparation 2. Fixation 3. Staining Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock biology of microorganisms (15th edition). Harlow: Pearson Education Limited. STEP 1: Smear Preparation Smear – a thin, dry film of microorganisms Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock biology of microorganisms (15th edition). Harlow: Pearson Education Limited. STEP 2: Fixation Purpose: kills the cells makes the cells sticky increases apparent diameter of cells Types: 1. Heat Fixation through direct flame or steam Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock biology of 2. Chemical Fixation microorganisms (15th edition). Harlow: Pearson Education Limited. using alcohols STEP 3: Staining Staining application of biological dyes Dyes (Stains) organic compounds carrying chromophoric ions make cell’s internal and external structures more visible with the increased contrast with the Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock biology of microorganisms (15th edition). Harlow: Pearson Education Limited. background STEP 3: Staining Types of Stains: 1. Basic or Positively-Charged Dye 2. Acidic or Negatively-Charged Dye 3. Neutral STAINING PROCEDURES 1. SIMPLE STAINING only one dye a. Positive / Direct Positive Staining of Bacillus anthracis cells are the same color as the dye crystal violet, malachite green, methylene blue, safranin https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by Centers for Disease Control and Prevention STAINING PROCEDURES 1. SIMPLE STAINING b. Negative / Indirect Negative Staining of Bacillus megaterium cells are colorless or luminous acid fuchsin, eosin, rose bengal, india ink, nigrosin https://openstax.org/books/microbiology/pages/2-4-staining- microscopic-specimens Credit: modification of work by Anh-Hue Tu STAINING PROCEDURES 1. SIMPLE STAINING b. Negative / Indirect https://www.ruf.rice.edu/~bioslabs/bios318/staining.htm STAINING PROCEDURES 2. DIFFERENTIAL STAINING two or more dyes and/or reagents Examples: Gram Staining gram-positive / gram-negative Acid-Fast Staining diagnosis of tuberculosis STAINING PROCEDURES 2. DIFFERENTIAL STAINING Cheek Cells with Streptococcus https://www.ouhsc.edu/histology/Glass%20slides/91_03.jpg Gram Staining https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Gram Staining Staphylococcus aureus and Escherichia coli https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by Nina Parker Acid-Fast Staining acid-fast and non-acid fast bacteria Mycobacterium tuberculosis used for diagnosis of tuberculosis Methods: Ziehl-Neelsen Technique Kinyoun Technique https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by American Society of Microbiology STAINING PROCEDURES 3. STRUCTURAL STAINING two or more dyes and/or reagents For: Capsules Endospores Flagella Storage Granules Capsule Staining Bacillus cells https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by American Society of Microbiology Endospore Staining Bacillus subtilis https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by American Society of Microbiology Flagella Staining Bacillus cereus https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens Credit: modification of work by Centers for Disease Control and Prevention Storage Granule Staining Metachromatic granules of Corynebacterium diphtheriae http://textbookofbacteriology.net/diphtheria.html References 1. https://openstax.org/details/books/microbiology (Chapters 2.2-2.4) 2. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(OpenStax)/02% 3A_How_We_See_the_Invisible_World (Chapters 2.2-2.4) 3. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Bruslind)/02%3 A_Microscopes (Chapter 2) 4. https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/3%3 A_Microscopy (Chapters 3.1A, 3.1B, 3.2, 3.3) 5. Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D.A. (2019). Brock biology of microorganisms (15th edition). Harlow: Pearson Education Limited. 6. Prescott, L. M., Harley, J. P., & Klein D.A. (2002). Microbiology (5th edition). New York: McGraw-Hill Higher Education.3 7. Todar, K. G., & University of Wisconsin--Madison. (2006). Todar's Online textbook of bacteriology. Madison, WI: Kenneth Todar, University of Wisconsin-Madison Dept. of Bacteriology. http://textbookofbacteriology.net/  Copyright © 2020. Glaezel Angelique T. Barredo. All Rights Reserved. No part of this file may be published without the creators’ consent.