BACTE M4

• Microscopy


  The use of a microscope to magnify (i.e., visually enlarge) objects too small to be visualized with the naked eye so that their characteristics are readily observable
• Basic flow of procedures involved in the laboratory diagnosis of infectious diseases

  1. Direct examination of patient specimens for the presence of etiologic agents

  2. Growth and cultivation of the agents from the specimens

  3. Analysis of the cultivated organisms to establish their identification
• Microscopic examination


  • Living state
  • Fixed state
• Living state


  Direct wet mount<|>Hanging drop method
• Fixed state


  Physical fixation (e.g. heat)<|>Chemical fixation (e.g. 95% methanol)
• Microscopy for Diagnostic Microbiology

  • Bright-Field
  • Phase Contrast
  • Fluorescent
  • Dark field
  • Electron
• Bright-Field (Light) Microscopy


  Uses visible light as a source of illumination<|>Object appear dark against a light background
• Bright-Field Microscopy


  • Magnification: ocular lens: 10x
  • Total magnification= Ocular lens x Objective lens
  • Resolution– the extent to which detail in the magnified object is maintained
  • Resolving power- the closest distance between two objects that when magnified still allows the two objects to be distinguished from each other
  • Oil (xylene oil and synthetic immersion oil) enhances resolution by preventing light rays from dispersing and changing wavelength after passing through the specimen
  • Contrast - needed to make objects stand out from the background, achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris
  • Simplest way to improve contrast is to reduce the diameter of the microscope aperture diaphragm, increasing contrast at the expense of the resolution
• Essential parts of a microscope

  • Lens system
  • Body
  • Illumination system
• Lens system


  Oculars<|>Objectives<|>Coarse- and fine-adjustment knobs
• Illumination system


  Light source<|>Condenser<|>Iris (aperture) diaphragm - contains a number of leaves that the operator may open or close to increase the amount of light illuminating the object<|>Field diaphragm -may also be present in the light source; may be opened or closed
• Fixation


  The living organisms are no longer alive, they are fixed on the glass slide forever. Their transparency is also diminished.
• Phase Contrast Microscopy


  Phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast<|>Phase-contrast objective lens – contains a phase ring that retards light<|>As light rays are slowed, there is a decrease in the intensity of light producing contrast<|>Enhances visualization of elements with low refractive indices<|>Not commonly used<|>Used to ID fungi<|>Eliminates fixing or staining living cells<|>For observation of viable organisms<|>Contrast can be generated by limiting the amount of background light reaching the image plane, or by creating a constructive interference between background and foreground light
• Fluorescent Microscopy


  Visualization of naturally fluorescent substances or those stained with a fluorochrome<|>Fluorescence is the property by which some atoms absorb light at a particular wavelength and subsequently emit a light of a longer wavelength
• Principles of Fluorochroming and Immunofluorescence

  1. Microorganisms in a specimen are stained with a fluorescent dye

  2. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained (i.e., fluorochroming) or "tagged" (i.e., immunofluorescence)
• Fluorochroming Methods


  • Acridine Orange
  • Auramine-Rhodamine
  • Calcofluor White
• Dark-Field Microscopy


  Used to enhance visualization of specimens not viewed with a bright-field microscope<|>Specimen appears light against a black background<|>Used to detect Spirochettes<|>Treponema pallidum<|>Dark-field condenser with a central circular stop (contains an opaque disk), which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope<|>Uses reflected light instead of transmitted light used in the ordinary light microscope (prevents light from falling directly on the objective lens)➔result that the microorganisms appear brightly stained against a dark background
• Electron Microscopy


  Magnification of 100,000× or more<|>Uses electrons instead of light and, instead of lenses, the electrons are focused by electromagnetic fields and form an image<|>Fixative: glutaraldehyde or osmium tetroxide<|>Dehydrating agent: alcohol or acetone<|>Stain: lead citrate and uranyl acetate
• Two General Types of Electron Microscopy

  • Transmission Electron Microscope (TEM)
  • Scanning Electron Microscope (SEM)
• Transmission Electron Microscope (TEM)

  Passes electron beam through objects<|>Visualization of internal structures
• Scanning Electron Microscope (SEM)


  Electron beam scans surface of a sample and collects the different signals produced using specialized detectors<|>Three-dimensional views of surface structures
• Electron microscope images are ALL black and white indicating presence or lack of electrons. The "colored" images from SEM or TEM are manipulated and "colored" after they acquired.
• Digital Microscopy


  Sophisticated software and unique technology now permits laboratories to acquire microscopic digital images of Gram stains using a web-based interface<|>This interface allows images using a fully automated microscope to be viewed on a single screen
• Staining


  The process of artificially coloring microorganisms with dyes
• Objectives of Staining


  • To determine the morphology of bacteria
  • To differentiate groups of bacteria
  • To identify organisms with special structure
• Staining Techniques


  • Simple Stains
  • Differential Stains
  • Negative Staining
• Simple Stains


  Utilizes only one dye<|>Coloring forms and shapes of the cell<|>Examples: Crystal violet, methylene blue
• Differential Stains


  More than one dye added in several steps<|>Coloring components of the cell<|>Examples: Gram stain, Acid-fast stain
• Negative Staining


  Stains the background rather than the bacteria<|>Demonstrates capsules<|>Example: India ink
• Kinds of Ionizable Dyes Used in Staining Bacteria

  • Basic Dyes
  • Acidic Dyes
• Basic Dyes


  Cationic dyes with positively charged groups<|>Adhere to negatively charged molecules like nucleic acids and proteins<|>Examples: methylene blue, basic fuchsin, crystal violet, safranin, and malachite green
• Acidic Dyes


  Anionic dyes with negatively charged groups (carboxyls and phenolic)<|>Binds to positively charged cell structures<|>Examples: eosin, rose bengal, and acid fuchsin
• Smear Preparation


  1. Patient specimens (Direct Smear)

  2. Culture (Indirect Smear)
• Direct Smear


  Dropped using pipette (liquid) or rolled (swab) onto slide<|>Dry and fixed before staining
• Indirect Smear


  Liquid medium: aspirate sample and apply to slide<|>Solid medium: sterile pipette, add one drop of sterile saline to the center of the microscope slide, aseptically pick a small amount of an isolated colony with a loop and gently mix/emulsify into the drop of sterile saline or water using circular motions
• Squash OR Crush Prep

  1. The aspirate placed in the anticoagulant EDTA tube and inverted several times to mix the contents

  2. A drop of the aspirate is placed on a slide and a second slide is gently placed on top

  3. Press 2 slides together, crushing or squashing any particulate matter

  4. The two slides are then gently slid or pulled apart using a horizontal motion

  5. Air-dried before staining
• Cytocentrifugation or concentration of a sterile body fluid such as cerebral spinal fluid (CSF) enhances the ability to identify cells in a specimen that may contain small numbers of microorganisms.
• Cytocentrifugation


  Load a few drops of sample into the tunnel port, then load fixative into the sample well. As the rotor accelerates, the cells are fixed after they sediment onto the slide.
• Gram Staining


  Divides bacteria into Gram Positive (violet, purple or deep blue) and Gram Negative (red or pink)<|>Gram Positive take up the basic dye, crystal violet<|>Gram Negative don't take up the primary stain, primary stain washed out by the decolorizer, and take up the counterstain
• Gram Staining Procedure


  1. Fix material on slide with methanol or heat

  2. Flood slide with primary stain (Crystal violet), rinse with tap water

  3. Flood the slide with iodine, rinse with tap water

  4. Flood the slide with decolorizer and rinse off with tap water

  5. Flood the slide with counterstain (Safranin)

  6. Rinse with tap water and gently blot the slide dry with paper towels or bibulous paper or air dry

  7. Examine microscopically under OIO for phagocytes, bacteria, and other cellular material