microbiology full

• Microscope


  • An important tool used to observe microorganisms
  • Crucial for the development of Microbiology as a Science
  • Consists of a system of lenses of sufficient magnification and resolution
• Procedure


  1. Review of the parts and function

  2. Magnification and Resolution

  3. Micrometry and measurements
• Magnification


  1. A microscope creates a magnified virtual image of the specimen

  2. Each objective has a magnifying factor

  3. Ratio of the size of digital/photo image to the actual size

  4. The magnification on a microscope must be adjusted carefully in proportion to (working) distance
• Magnification factor depends on the curvature of the bi-concave glass lens
• Objectives suffer from pronounced field curvature and project images that are curved rather than flat, an artifact that increases in severity with higher magnification
• Functions of the Objectives

  • Gather the light coming from each of the various parts or points of the specimen
  • Must be constructed to focus close enough to the specimen and project a magnified, real image up into the body tube
  • Short focal lengths cause magnification
• Functions of the Eyepiece

  • Further magnify the real image projected by the objective
  • Fitted with scales, markers, or crosshairs as pointers/measurements
• Focal Length/Point


  • In a finite optical system, light passing through the objective converges at the image plane to produce an image
  • Focal length refers to the amount of distance required between the objective lens and the top of your object, in order to be able to view an image through the microscope that is in-focus
• Total Magnification formula: Mtotal = Mocular X Mobjective
• Resolving Power/Resolution is the ability of a lens to show two adjacent objects/points as discrete
• Resolution of the microscope is the smallest distance at which two points can be seen as separate when viewed through the microscope
• Resolution depends on wavelength of light, angular aperture of the light cone captured by the objective, and refractive index of medium between the objective front lens and the specimen
• Numerical Aperture (NA) = N
• Numerical Characteristics of Objectives

  • LPO: Linear Magnification 10, Working Distance 5mm, Focal Length 16mm, Resolution 0.9 microns, Numerical Aperture 0.25
  • HPO: Linear Magnification 40, Working Distance 0.46mm, Focal Length 4mm, Resolution 0.35 microns, Numerical Aperture 0.65
  • OIO: Linear Magnification 100, Working Distance 0.3mm, Focal Length 1.8mm, Resolution 0.18 microns, Numerical Aperture 1.25
• MICROMETRY is a technique used to measure the dimensions of microorganisms
• MICROMETRY


  1. Ocular micrometer measures the dimensions of microorganisms

  2. Stage micrometer calibrates the ocular micrometer
• Formula used to compute the calibration factor or ocular units: XO = YS
• Enhances contrast between bacteria and the surrounding material
• Permits observation in great detail and better resolution in microscopy
• Types of dyes


  • Colored ions, chromophore
• Examples of dyes


  • Basic dye: basic fuchsin, crystal violet, malachite
• Types of dyes


  • Basic dye
  • Acidic dye
• Dyes selection


  Selected based on the chemical properties and the specimen being observed, which determine how the dye will interact with the specimen
• Positive staining


  A dye is absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background
• Negative staining


  Dyes are repelled by the negatively charged cell parts and absorbed by the background
• Simple staining


  A single dye is used to emphasize particular structures in the specimen
• Simple positive staining


  Intends to show cell structures by stain absorption
• Simple negative staining


  Intends to show cell structures by stain repulsion (only the background is colored)
• Differential staining


  Intends to differentiate structures based on their differential color retention (rooted in the cell chemistry)
• Positive staining with basic dye crystal violet


  Negatively charged cell
• Negative staining with acidic dye

  Negatively charged cell
• How to prepare a smear

  1. Label your slide with a pencil

  2. Use a clean slide: manipulate by the edges

  3. Using a sterilized loop spread of drop of bacterial culture on the slide

  4. If solid culture - put a drop of water on the glass slide then add a little bit of bacteria (using loop)

  5. A thin film is better

  6. Dry faster, distribution of dye and decolorizer more even

  7. No coverslip on! let dry (until most water evaporated)
• Fixation


  1. Pass the slide through the flame of the bunsen burner

  2. Moving in a circular motion (to avoid localized overheating)

  3. The slide should not be too hot to touch but slightly warm

  4. If too hot, over-fixed/burnt bacteria will be black

  5. If under-fixed - bacteria do not stick to slide and will be washed away during the staining procedure
• Gram staining is the most commonly used technique to stain and differentiate bacteria based on cell wall organization
• Gram staining is almost always the first step in the identification of bacteria
• Gram-negative bacteria stain red-pink, Gram-positive bacteria stain blue-purple
• Gram staining procedure


  1. Crystal violet

  2. Iodine

  3. Ethanol

  4. Safranin
• Gram staining characteristics


  • Thin peptidoglycan decolorize with ethanol
  • Alcohol dehydrates the thicker cell wall in gram-positive bacteria
  • Prevent diffusion of the violet-iodine complex
  • Safranin colors pink
• Gram staining steps


  1. Enhance the leaching of the primary stain, causing decolorization

  2. Flood the slide for 30 seconds

  3. Rinse gently with water and shake off the excess water from surface

  4. Slides can be air dried or blotted

  5. Put between 2 sheets of absorbent paper (bibulous paper is often used)

  6. DO NOT RUB THE SMEAR (bacteria will come off)…just blot between papers
• Older bacterial cells may have damages to their cell walls that cause them to appear gram-negative even if the species is gram-positive. It is best to use fresh bacterial cultures for Gram staining