MICROPARA

• Two major types of microscopes:

  • Light microscope
  • Electron microscope
• Light microscope:

  • Visible light is passed through the specimen and then through a series of lenses that reflect the light, resulting in the magnification of the organism
  • Human cannot focus on objects nearer than about 25cm or 10 inches
  • Parfocal
  • Forms a dark image against a brighter background
  • Magnification:
  • Ocular lens – 10x
  • Objective lenses: scanner, HPO, LPO, OIO
  • Ocular lens/eyepiece re-magnifies the image formed by the objective lens
  • Body tube transmits the image from the objective lens to the ocular
  • Arm is used for holding the microscope
• • Nosepiece holds the objectives
  • Objective lenses are primary lenses that magnify the specimen
  • Mechanical stage holds the microscope slide in position
  • Condenser focuses light through the specimen
  • Diaphragm controls the amount of light entering the condenser
  • Coarse and fine adjustment knob can move either the stage or the nosepiece to focus the image
  • Illuminator is the light source
  • Base
• Electron microscope:

  • Uses electrons instead of light to visualize small objects
  • Resolution is at 0.5 nm
  • Fixative: glutaraldehyde or osmium tetroxide
  • Dehydrating agents: alcohol or acetone
  • Stains: lead citrate and uranyl acetate
  • Objects smaller than 0.2 um can be visualized using this type of microscope with 100,000x magnification
• Staining:

  • Staining is not part of phase contrast microscopy
  • Permits examination of internal structures in living organisms
  • Used to identify medically important fungi grown in culture
  • Uses fluorochromes to stain microorganisms
  • Involves excitation of fluorochromes by light
  • Stains: acridine orange, auramine and rhodamine, calcofluor white, fluorescein isothiocyanate (FITC)
• Darkfield microscopy:

  • Uses darkfield condenser that blocks light that would enter
  • Directs the light to hit the specimen at an oblique angle
  • Used to detect spirochetes
  • Can also be used for unstained microorganisms
• Key terms:

  1. Refractive index: measure of the relative velocity at which light passes through a material

  2. Resolution: ability of a lens to distinguish small objects that are adjacent or close together

  3. Resolving power: ability of the lenses to separate closely distant objects

  4. Contrast: to make objects stand out from the background
• Microscope slide preparations:

  • Wet mount preparation
  • Hanging drop preparation
  • Staining
• Basic dyes are cationic and bind to negatively charged molecules like nucleic acids and proteins
• Examples of basic dyes include: Methylene blue, basic fuchsin, crystal violet, safranin red, and malachite green
• Acid dyes are anionic and bind to positively charged cell structures
• Examples of acid dyes include: Eosin, rose bengal, and acid fuchsin
• Simple staining involves using a single stain and is important for morphological structure
• Examples of simple staining dyes are: Malachite green, methylene blue, crystal violet, carbolfuchsin, safranin red
• Differential staining divides bacteria into separate groups
• Two important types of staining for differential staining are AFB staining and Gram staining
• Differential staining involves the use of primary stain, mordant, decolorizing agent, and secondary stain/counterstain
• Gram-positive bacteria have thick cell walls containing teichoic acid and retain the crystal violet-iodine complex after decolorization, appearing purple
• Gram-negative bacteria have thin cell walls containing LPS and do not retain the dye complex, appearing pink
• Negative staining demonstrates the presence of a diffuse capsule surrounding some bacteria
• Examples of negative staining dyes include: India ink and Nigrosin dye
• Diagnostic antibody or DNA probe-mediated staining is used for specific identification of selected pathogens
• Examples of pathogens identified using this staining method are: Chlamydia trachomatis, Bordetella pertussis, Adenoviruses, and respiratory viruses
• India Ink and Endospore Stain are used for microscopic visualization of bacterial morphology
• Steps in Gram staining:

  • Primary stain: purple for both Gram-positive and Gram-negative
  • Mordant: purple for both
  • Decolorizer: purple for Gram-positive, colorless for Gram-negative
  • Counter Stain: Safranin red, turning Gram-positive bacteria pink to red
• Intracellular organisms, organisms without cell walls, and organisms with insufficient dimension to be resolved by light microscopy are examples of organisms that may not follow typical staining patterns
• All cocci are Gram-positive, except Neisseria, Veilonella, Moraxella
• All bacilli are Gram-negative, except Mycobacteria, Corynebacteria, Clostridia, Nocardia, Actinomyces, Bacillus, Lactobacillus, Listeria, Erysipelothrix
• Ziehl-Neelsen and Kinyoun's methods are used for acid-fast staining to identify bacteria with high lipid and wax content in their cell walls
• To facilitate acid-fast staining reaction, techniques such as the steaming process, increasing dye and phenol concentration, prolonging contact with the primary stain, and adding Tergitol can be used
• Examples of acid-fast staining methods include: Ziehl-Neelsen (Hot method), Kinyoun’s (Cold method), Pappenheim’s method, Baumgarten’s method
• Primary Stain, Decolorizer, and Counter Stain are used in acid-fast staining methods like Ziehl-Neelsen
• To temporarily remove mycolic acid for staining, a steaming process is used
• Increasing the concentration of dye and phenol, and prolonging contact with the primary stain can enhance the acid-fast staining reaction
• Specific dye concentrations for acid-fast staining methods include:

  • Ziehl-Neelsen: 3g carbolfuchsin + 5% phenol
  • Kinyoun’s: 4g carbolfuchsin + 9% phenol
• Prolonged staining of safranin may lead to the leaching out of the CV-iodine complex from the Gram-positive organism
• If the CV is rinsed too vigorously before iodine is applied, it may leave poor or no staining of the Gram-positive organism
• If the decolorization is prolonged, the normally Gram-positive organism will not stain
• If the decolorization is not enough, the organism may be stained falsely Gram-positive
• Examples of bacteria that can be stained using acid-fast staining methods are Nocardia Spp. and Mycobacterium Spp.