Microscopy and Staining Techniques

• 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 characteristic is easily observable

  
• Basic flow of procedures in diagnosis of infectious diseases

  1. Direct examination of patient's specimen for the presence of etiologic agents

  2. Growth of cultivation of the agents from these same specimens

  3. Analysis of the cultivated organisms to establish their identification and other pertinent characteristics such as susceptibility to antimicrobial agents
• Role of Microscopy

  • It is the most common method used both for the detection of microorganisms directly in the clinical specimens and for the characterization of organisms grown in culture

  
• Types of Microscope

  • Bright-Field Light Microscopy
  • Phase Contrast Microscopy
  • Fluorescent Microscopy
  • Dark-Field Microscopy
  • Electron Microscopy

  
• Bright-Field Microscopy


  Visible light is passed through the specimen and then through a series of lenses that reflect the light in a manner that results in magnification of the organisms present in the specimen
• Bright-Field Microscopy


  • Maximum Magnification - 2000x
  • Resolution - 0.2 μm (200 nm)
  • Common multipurpose microscope for viable and stained samples; provides fair cellular details

  
• Phase Contrast Microscopy


  Beams of light pass through the specimen and are partially deflected by the different densities or thickness of the microbial cells or cell structures in the specimen. This microscopy translates differences in phases within the specimen into differences in light intensities that result in contrast among objects within the specimen being observed.

  
• Phase Contrast Microscopy

  • Maximum Magnification - 2,000x
  • Resolution - 0.2 μm (200 nm)

  
• Fluorescence Microscopy


  Microorganisms in a specimen are stained with a fluorescent dye. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained or tagged.

  
• Fluorescence Microscopy

  • Maximum Magnification - 2,000x
  • Resolution - 0.2 μm (200 nm)

  
• Dark-Field Microscopy

  The condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle. Only light that hits objects will be deflected upward into the lens for visualization.
• Dark-Field Microscopy

  • Maximum Magnification - 2,000x
  • Resolution - 0.2 μm (200 nm)
• Transmission Electron Microscopy (TEM)


  Passes the electron beam through objects and allows visualization of internal structures.
• Transmission Electron Microscopy (TEM)

  • Maximum Magnification - 100,000x
  • Resolution - 0.5 nm
• Scanning Electron Microscopy (SEM)

  Uses electron beams to scan the surface of objects and provides three-dimensional views of surface structures
• Scanning Electron Microscopy (SEM)

  • Maximum Magnification - 650,000x
  • Resolution - 10 nm
• Fluorescence microscopy helps distinguish disease-causing organisms that are too small to observe using ordinary staining techniques

  
• Dark-field Microscopy 


  Most effective in visualizing viable, unstained samples that would be distorted by drying or heat, as well as microorganisms that are difficult to stain and spirochetes
• If a dark-field microscope is not available, a light microscope can be converted into one by placing a "dark-field stop" on the condenser
• Magnification

  Objective lens, which is closest to the specimen, magnifies object 100X. Ocular lens, which is nearest the eye magnifies 10X. The combination of the two lenses is called TOTAL MAGNIFICATION.
• Resolution

  defined as the extent to which detail in the magnified object is maintained. Resolving power is the closest distance between two objects that when magnified still allows the two objects to be distinguished from each other.
• Contrast

  needed to make objects standout from the background, most commonly achieved by STAINING TECHNIQUES, that highlight organisms and allow them to be differentiated from one another and from background material and debris.
• Smear preparation of bacterial colonies in broth medium

  1. Hold the broth tube securely, flame the mouth, dip the inoculating loop in the broth

  2. Hold the broth tube securely, flame the mouth, dip the inoculating loop in the broth
• Smear preparation of plated bacterial colonies

  Hold the culture plate flat and securely, lift the lid, use an inoculating loop to fish out colonies, place a drop of NSS on a slide, spread the colonies evenly and air-dry, fix the slide with the appropriate fixative
• Always carry the microscope with two hands-one hand holding the neck and the other supporting the base
• Clean all microscope lens with lens paper and lens cleaner only, particularly when the objectives have come in contact with the immersion oil
• Proper focusing of the microscope

  1. Secure the LPO, use the coarse adjustment knob to focus

  2. Switch to HPO, use the fine adjustment knob to focus

  3. Without disturbing the stage adjustment, remove the HPO and add a drop of immersion oil on the smear area, switch to OIO and use the fine adjustment knob to focus
• Staining techniques

  Used to produce color contrast, routine methods involve dying and fixing smears to kill bacteria
• Objectives of staining

  • To determine the morphology of bacteria
  • To differentiate groups of bacteria
  • To identify organisms with special structures

  
• Basic dyes


  Commonly used in bacteriology, they are cationic dyes with positively charged groups (pentavalent nitrogen) that adhere to negative charges in bacterial cells
• Objectives of the light microscope


  • LPO (10x) - Observation of large structures
  • HPO (40x) - Detailed observation of those identified in LPO
  • OIO (100x) - Assessment of type of bacteria present based on size, shape, gram stain reaction, and intracellular and extracellular location

  
• Structures observed under LPO (10x)

  • Nematode larvae
  • Curschmann's spirals
  • Large granules
  • Grains
  • Bacterial colony-forming units
  • Protozoans
  • Fungi

  
• Staining techniques


  Used to produce color contrast
• Routine methods of staining of bacteria involve dying and fixing smears - procedures that kill them
• Bacteria


  Have an affinity to basic dyes due to the acidic nature of their protoplasm
• Objectives of staining


  • To determine the morphology of bacteria
  • To differentiate groups of bacteria
  • To identify organisms with special structures

  
• Kinds of dyes

  • Basic dyes
  • Acidic dyes
• Basic dyes


  Commonly used in bacteriology, cationic dyes with positively charged groups that adhere to negatively charged molecules like nucleic acids and proteins
• Acidic dyes


  Anionic dyes with negatively charged groups that bind to positively charged cell structures
• Live bacteria do not show structural detail under the light microscope due to lack of contrast