micropara 1

• Metric units are used to express the sizes of microbes.
• The basic unit of length in the metric system is the meter (m);

  it is equivalent to 39.4 inches.
• Metric units


  Used to express the sizes of microbes
• Meter (m)


  Basic unit of length in the metric system, equivalent to 39.4 inches
• Micrometer (µm)


  One millionth of a meter, used to express the sizes of bacteria and protozoa
• A typical spherical bacterium (coccus) is approximately 1 µm in diameter
• A typical rod-shaped bacterium (bacillus) is approximately 1 µm wide x 3 µm long
• Nanometer (nm)

  One billionth of a meter, used to express the sizes of viruses
• Most viruses that cause human diseases range in size from 10 to 300 nm
• The Ebola virus can be as long as 1,000 nm (1 µm)
• When using a microscope, the sizes of microorganisms are measured using an ocular micrometer
• Optical instruments


  Devices that process light waves to enhance an image for a clearer view
• Resolving power


  The limit as to what can be seen using an optical instrument
• The resolving power of the unaided human eye is approximately 0.2 mm
• Resolution


  The ability of an optical system to differentiate between two closely spaced lines
• Microscope


  An optical instrument used to observe tiny objects that cannot be seen with the unaided human eye
• Simple microscope


  • Contains only one magnifying lens
  • Magnifying glass could be considered a simple microscope, with a maximum magnifying power of about 300
• Compound microscope


  • Contains more than one magnifying lens
  • Uses visible light as the source of illumination, also referred to as a compound light microscope
  • Has a resolving power of approximately 0.2 µm, about 1,000 times better than the resolving power of the unaided human eye
• Total magnification


  Calculated by multiplying the magnifying power of the ocular lens by the magnifying power of the objective lens being used
• Photographs taken through the lens system of the compound light microscope are called photomicrographs
• Brightfield microscope


  • Objects are observed against a bright background or "bright field"
  • Used to observe the morphology of microorganisms (≥0.2 μm) such as bacteria, protozoa, fungi and algae in living (unstained) and non-living (stained) state against a bright background
  • Has low contrast, so most of the cells need to be stained to be viewed properly
• Darkfield microscope


  • Illuminated objects are seen against a dark background or "dark field"
  • Ideal for unstained specimen (≥0.2 μm), appearing brightly lit against a dark background
  • Useful for examining spirochetes (spiral-shaped bacteria)
• Phase-contrast microscope


  • Has a contrast-enhancing technique to produce high-contrast images of transparent specimens
  • One of the major advantages is that living cells can be examined in their natural state without previously being killed, fixed, and stained
• Fluorescence microscope

  • Contains a built-in ultraviolet (UV) light source
  • When the UV light strikes certain dyes and pigments, these substances emit a longer-wavelength light, causing them to glow against a dark background
• Electron microscopes


  • Have a much higher resolving power than compound light microscopes
  • Can see extremely small microbes
  • Living organisms cannot be observed as the processing procedures kill the organisms
  • Use an electron beam as the source of illumination, and magnets to focus the beam
  • There are two types: transmission and scanning
• Transmission Electron Microscope

  • Uses an electron gun to fire a beam of electrons through an extremely thin specimen (<1 µm thick)
  • Produces 2-dimensional, black-and-white images
  • Magnifies objects up to 200,000X
  • Has a resolving power of approximately 0.2 nm
• Scanning Electron Microscope


  • Electrons are bounced off the surface of a specimen and the image appears on a monitor
  • Used to observe the outer surfaces of specimens
  • Resolving power is about 100 times less than that of transmission electron microscope
• Staining


  Procedures developed to facilitate visualization of bacteria, as most microorganisms are devoid of color and are therefore difficult to see even with the use of a microscope
• Major categories of staining procedures

  • Simple staining
  • Differential staining (Gram and acid-fast)
  • Structural staining (capsule, spore, flagella)
• Fixation


  The process that serves to kill organisms, preserve their morphology, and anchor the smear to the slide
• Heat fixation


  • Not a standardized technique, excess heat will distort bacterial morphology
• Methanol fixation


  • A standardized technique, the preferred method
• Simple bacterial staining technique

  • Makes use of a single dye (water-based or alcohol-based)
  • Quick and easy way to visualize cell shape, size, and arrangement of bacteria
  • Negatively charged bacterial cells adhere readily to positively charged dye, enabling visualization of bacterial cell morphology
  • Stains: safranin, methylene blue, crystal violet
• Gram staining procedure


  • Divides bacteria into two major groups: Gram-positive (blue to purple) and Gram-negative (pink to red)
  • The final Gram reaction depends on the organism's cell wall structure
• Differences between Gram-positive and Gram-negative bacteria

  • Crystal violet (primary stain): purple or blue for both
  • Gram's iodine (mordant): purple or blue for both
  • Acetone or 95% alcohol (decolorizer): purple or blue for Gram-positive, colorless for Gram-negative
  • Safranin (counterstain or secondary stain): purple or blue for Gram-positive, red or pink for Gram-negative
• Acid-fast stain


  • Used for bacteria with high lipid content in their cell wall, which cannot be stained using Gram stain
  • Mycobacterium spp. are often identified using the acid-fast stain
  • Two methods: Ziehl-Neelsen (hot method) and Kinyoun (cold method)
• Reagents and results for acid-fast staining

  • Carbol-fuchsin (primary stain): red or pink for acid-fast, red or pink for non-acid-fast
  • Acid alcohol (decolorizer): red for acid-fast, colorless for non-acid-fast
  • Methylene blue (Ziehl-Neelsen) or malachite green (Kinyoun) (counterstain or secondary stain): red organism/blue background (Ziehl-Neelsen), red organism/green background (Kinyoun) for acid-fast, blue organism/blue background (Ziehl-Neelsen), green organism/green background (Kinyoun) for non-acid-fast
• Special stains

  • Used to demonstrate specific structures in a bacteria cell, such as metachromatic granules, capsule, cell wall, flagella, spores, and fungal capsule
• Colony morphology


  The appearance of a bacterial colony, which varies from one species to another and includes size, color, overall shape, elevation, and the appearance of the edge or margin of the colony
• Colony morphology is an important "clue" to the identification (speciation) of bacteria