BACTE PRELIMS TO MIDTERMS

• Beginning of microscopy


  1665
• Hooke published his treatise Micrographica, which included illustrations of mold forms and the anatomy of the flea
• Simple microscope


  Contains a single bi-convex magnifying lens which is thicker in the center than at the periphery, produces a magnified image that is in the same orientation as the original object

  
• Simple microscope


  • Have limited resolution and magnifying power because of their low NA, most commercial magnifiers are able to produce a ×2 to 30 magnification, and the better lenses will have a resolution of about 10 μm

  
• Simple microscope


  • Jeweler's loupes, photographic slide viewers, and simple magnifying or reading glasses
• First compound microscopes were constructed around 1590 by Dutch spectacle makers Zaccharias Janssen and Hans Janssen
• Compound microscope


  The Janssen microscope consisted of an object lens (objective) that was placed close to the specimen and the eye, or an ocular lens that was placed close to the eye, the lenses were separated by a body tube, the objective lens projects a magnified image into the body tube and the eyepiece magnifies the projected image, it produces a two-stage magnification

  
• Light microscope


  Instrument for visualizing fine detail of an object, it creates a magnified image through the use of a series of glass lenses, which first focus a beam of light onto or through an object, and convex objective lenses to enlarge the image formed, uses visible light to detect small objects, used in microbiology, pathology, cell biology, metallurgy, materials science, computer chip design, and micro-surgical applications
• Types of light microscope


  • Bright field
  • Dark field
  • Phase contrast
  • Fluorescence

  
• Bright field microscope


  An ordinary type of microscope, it forms a dark image against a brighter background

  
• Parts of the bright field microscope


  • Ocular lens (eyepiece)
  • Body tube
  • Arm
  • Nosepiece
  • Objective
  • Mechanical stage
  • Condenser
  • Diaphragm
  • Coarse adjustment knobs
  • Fine adjustment knob
  • Illuminator
  • Base
• Resolution


  The ability of a lens to separate or distinguish between small objects that are close together

  
• Abbe sine equation


  Formulated by Ernst Abbe, shows the maximum resolution for a microscope, d = 0.5 λ / n sin θ, as d becomes smaller, the resolution increases

  
• Wavelength


  Important factor, must be shorter than the distance between 2 objects, the shorter the wavelength, the higher the resolution, use blue end of visible spectrum (450 – 500 nm)
• Numerical aperture (NA)


  θ is half the angle of the cone of light entering an objective, n is the refractive index, no lens working in air can have NA of greater than 1, to increase the refractive index, use immersion oil

  
• The maximum theoretical resolving power of a microscope with an oil immersion objective (NA 1.25) and blue green light is approximately 0.2 um
• Working distance


  Distance between 2 front surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus, objectives with large numerical apertures and great resolving powers have short working distances

  
• Dark field microscope


  Uses darkfield condenser that blocks light that would enter the objective directly, directs the light to hit the specimen at an oblique angle, all the other light that passes through the specimen will miss the objective, thus making the background a dark field, organisms will appear extremely bright against a dark field, used to view spirochetes (Treponema pallidum) and examine unstained microorganisms suspended in liquid, living organisms that are invisible in ordinary light
• Phase contrast microscope


  Converts slight differences in refractive index and cell density into easily detected variations in light intensity, the phase differences are seen through the microscope as different degrees of brightness, staining is not part of phase-contrast microscopy, permits detailed examination of internal structures in living organisms, used to identify medically important fungi grown in culture and detect bacterial components, condenser has an annular stop - opaque disk with a thin transparent ring which produces a hollow cone of light, the background is bright while the unstained object appears dark as well as well-defined (Dark Phase microscopy)

  
• Fluorescence microscope


  When some molecules absorb radiant energy, they become excited and release much of the trapped energy as light, exposes the specimen to UV light, violet or blue light and forms an image of the object with the resulting fluorescence light, fluorochromes - stains used in this technique that fluoresce brightly upon exposure to light of a specific wavelength
• Electron microscope


  One of the most advanced and important types of microscopes, has the highest magnifying capacity, uses electrons to illuminate the tiniest particles, more powerful tool than light microscopes
• How the electron microscope works


  Energy source is a beam of electrons, electrons have exceptionally short wavelength, strike most objects in their path, travel in a vacuum to avoid contact with deflecting air molecules, magnets focus the beam on the object to be viewed, an image is created on a monitor and viewed by the technologist, increases the resolution of the microscope significantly

  
• Types of electron microscope

  • Transmission electron microscope (TEM)
  • Scanning electron microscope (SEM)
  • Scanning tunneling microscope (STM)

  
• Transmission electron microscope (TEM)


  The more traditional form of electron microscope, ultra-thin slices of microorganisms or viruses are placed on a wire grid, stain: gold or palladium, the densely coated parts of the specimen deflect the electron beam, and both dark and light areas show up on the image

  
• Scanning electron microscope (SEM)

  The more contemporary form electron microscope, gives lower magnification than the TEM, permits three-dimensional views of microorganisms and other objects, whole objects are used, stain: gold or palladium
• Scanning tunneling microscope (STM)


  A type of electron microscope, shows three-dimensional images of a sample, the structure of a surface is studied using a stylus that scans the surface at a fixed distance from it, electrons tunnel between the surface and the stylus, producing an electrical signal, stylus tip is formed by one single atom, it scans across the surface at a distance of only an atom's diameter, the vertical movement of the stylus is recorded
• Microbiology

  Study of organisms and agents too small to be seen by the unaided eye, study of microorganisms

  
• Branches of microbiology


  • Bacteriology - study of bacteria
  • Mycology - study of fungi
  • Virology - study of viruses
  • Phycology - study of algae
  • Protozoology - study of protozoans

  
• Roman Philosopher Lucretius suggested that diseases were caused by "invisible living creatures"


  98 – 55 BC
• Girolamo Fracastoro


  1478 – 1553
• Aristotle suggested that simpler invertebrates could arise from spontaneous generation

  384 – 322 BC
• Francesco Stelluti made the earliest microscopic observations on bees and weevils using a microscope probably supplied by Galileo


  1625 and 1630
• Francesco Stelluti demonstrated that maggots do not arise spontaneously from decaying meat, his results were a serious blow to the long-held belief that large forms of life could arise from non-life


  1626 – 1697
• Anton van Leeuwenhoek, the "first true microbiologist", was the first person to observe and describe microorganisms accurately, he described the microorganisms from the pond water as "animalcules", he used his self-made single lens microscope with 50 – 300x magnification

  1632 – 1723

  
• John Needham observed that boiled mutton broth (tightly sealed) eventually became cloudy with microorganisms, he proposed that organic matter possessed a "vital force" that could give rise to life


  1748
• Lazzaro Spallanzani improved the previous experiments of Needham by using sealed boiled water and seeds, he observed that no growth took place as long as the flasks remained sealed, he proposed that air carried microorganisms to the culture medium and that external air might be essential for the growth of animals present already in the medium

  1729 – 1799
• Rudolf Virchow challenged spontaneous generation with the concept of biogenesis


  1858
• Theodore Schwann observed that no growth occurred in a flask containing nutrient solution after allowing air to pass through a red-hot tube

  1810 – 1882
• John Tyndall showed that dust carry germs which contaminates sterile broth, he developed "tyndallization"– a form of sterilization for three consecutive days

  1820 – 1893
• Louis Pasteur resolved the issue of spontaneous generation, he stated that microorganisms are indeed present in the air and can contaminate seemingly sterile solutions, however the air itself does not create microbes, he showed that microorganisms can be present in non-living matter, he stated that microbial life can be destroyed by heat (basis of the aseptic technique – techniques to prevent contamination by unwanted microorganisms), he provided evidence that microorganisms cannot originate from mystical forces present in nonliving materials


  1822 – 1895