MICROPARA

• Virtual image


  A much larger image seen in the microscope field and represented an apparent size of the object
• Working distance


  Distance between the front (bottom) of the objective lens and the top of the cover glass
• Numerical aperture


  Measurement of the angle of the maximum cone of light that may enter the lens
• Resolving power


  The ability of the microscope to distinguish and clearly see two points or lines individually (reveal small details)
• Magnification


  The ratio of the apparent size of the object as seen through the microscope and the actual size of the object
• Units of measurement


  • Microns (µ)
  • Micrometer (µm)
  • Nanometer (nm)
  • Angstrom (A°)
• Light microscope


  • Consists of series of lenses that magnify the image using visible light
• Electron microscope


  • Widely used for studying detailed structure of cells
• Brightfield microscopy

  • Simplest of all the optical microscopy illumination techniques
  • Dark objects are visible against a bright background
• Darkfield microscopy


  • Light objects visible against dark background
  • Used to enhance the contrast in unstained samples
  • Instrument of choice for spirochetes
• Phase contrast microscope


  • Reveals transparent structures
  • Uses visible light but arrangement of filters and annular diaphragm shows difference in brightness into areas of light and shade (slightly different in optical density)
  • Rays of light emerge out giving a pattern of bright and dark relief
• Fluorescence microscopy


  • Uses UV light
  • Fluorescent substances absorb UV light and emit visible light
  • Cells may be stained with fluorescent chemicals (fluorochromes)
  • Immunofluorescence
• Transmission EM


  • Used to study internal structures of cells
  • Uses beams of electrons with a series of electromagnets
  • Wavelength of electron beam = 0.005 nm, shorter than UV
• Scanning EM


  • Focused study as a very fine probe or spot; use beam of electron
  • Specimen is coated with heavy metal
  • Allows contamination of submicroscopic entities like viruses
  • 50,000x magnification
  • Electron micrograph
• Wet mount or hanging drop

  Allow examination of characteristics of live cells: motility, shape, & arrangement
• Fixed mount


  Made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts

  
• Heat fixation


  • Preserves overall morphology, but not the structures within cells
• Chemical fixation


  • Used to protect fine cellular substructure and the morphology or larger, more delicate microorganisms
  • Chemical fixatives penetrate cells and react with cellular component to render them inactive, insoluble, and immobile
  • e.g., ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde

  
• Simple stains


  • Use a single basic dye
  • A mordant may be used to hold the stain or coat the specimen to enlarge it
• Differential stains


  • Differential staining is used to distinguish one group of bacteria from another
  • Different bacteria have certain fundamental chemical differences in their cell walls, and this is the basis of differential staining
• Gram staining


  • Application of crystal violet (purple dye)
  • Application of iodine (mordant)
  • Alcohol wash (decolorization)
  • Application of safranin (counterstain)
• Acid fast staining


  • The primary stain Carbol fuschin, a red dye
  • The slide is rinsed and then flooded with acid-alcohol, a potent decolorizing agent. This removes carbol fuschin from all cells except from unusual microorganisms (Mycobacterium)
  • Methylene blue is then used as counterstain to make acid-fast organisms visible as they do not take up methylene blue in contrast to other cells
  • Acid-fast appear as bright reddish-pink and other organisms/cells stain blue
• Endospore stain


  • Members of certain Gram-positive genera including Bacillus and Clostridium form a special type of dormant cells, an endospore, that is resistant to destruction and to staining
  • Generally, malachite green is used as primary stain
  • When rinsed with water only endospore retain malachite green
  • Smear is then counterstained with red dye safranin thus spores appear green and a background of pink cells

  
• Capsule stain


  • Colors the background, allowing the capsule to stand out as a halo around an organism. Capsule stain is done as wet mount

  
• Flagellar stain


  • Uses a mordant which allows the staining agent to adhere to and coat thin structures of flagella and increases their diameter so they can be seen with light microscope

  
• Components of culture media

  • Peptone
  • Beef extract
  • Yeast extract
  • Trace elements
  • Growth factor
• Peptone


  Protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources
• Beef extract


  Obtained by extracting the water-soluble components from beef tissue
• Yeast extract


  An aqueous extract of yeast cells containing vitamins and other growth factors
• Trace elements


  Na, Fe, Ca, and Mg
• Growth factor


  Minerals, vitamins
• Types of culture media

  • Solid
  • Liquid
  • Semi-solid
• Whatever medium is used, all vessels must be sterilized before adding microorganisms for investigation
• Capsule stain


  • Colors the background, allowing the capsule to stand out as a halo around an organism
  • Capsule stain is done as wet mount

  
• Flagellar stain


  • Uses a mordant which allows the staining agent to adhere to and coat thin structures of flagella and increases their diameter so they can be seen with light microscope

  
• Types of culture media

  • Solid
  • Liquid
  • Semi-solid
• Agar


  • Added to solidify media
  • Complex polysaccharide
  • Useful in microbiology laboratory
  • Most microbes cannot digest agar; media remain solid when bacteria and fungi are growing
  • Powdered agar dissolves in water at 100°C, a temperature at which most nutrients remain undamaged
  • Solidifies at temperatures less than 40°C
  • Solid agar melts at temperature greater than 100°C

  
• Defined or synthetic culture media

  Exact chemical composition is known
• Example of defined medium for culturing E. coli

  • Glucose: 1.0 g/L
  • Na2HPO4: 16.4 g/L
  • KH2PO4: 1.5 g/L
  • (NH4)2PO4: 2.0 g/L
  • MGSO4: 7H2O 0.2 g/L
  • CaCl2: 0.01 g/L
  • FeSO4: 7H2O 0.005 g/L
• Complex culture media


  Exact composition is unknown because partial digestion releases many different chemicals in a variety of concentrations