MCB 2000 Lecture Notes - Lecture 4: Atomic-Force Microscopy, Scanning Electron Microscope, Transmission Electron Microscopy
23 May 2018
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Lecture 4
• Microscopy
o Magnification ranges
▪ In order from strongest magnification to weakest
• Atomic force microscope
• Transmission electron microscope
o Can show details of the inside of the microbe
• Scanning electron microscope
o Can only show details of the surface of the microbe
• Light microscope
o Have to use electron microscope to see viruses
o Brightfield = use visible light as source of illumination (light
colored background)
o Darkfield = illuminates background, uses special
condenser with opaque disk, ligh is reflected by specimen
(black background)
o Phase-contrast = shows more details than other light
microscopes
• Unaided eye
o Other types of microscopes
▪ Confocal = uses a single photon to illuminate one plane of a specimen at
a time
• Will have a black background with many different colors
o Immunofluorescence = use antibodies made by the immune system to recognize
pathogens
▪ Diagnostic tool
▪ Tope of Y shape of antibodies binds to infection
▪ Tails of antibodies are dyed and illuminate the pathogens they attach to
▪ Antibodies are produced in the spleen
▪ Monoclonal antibody = technique used to produce one type of antibody
• Gram staining
o Process
▪ Application of crystal violet (purple dye)
• Gram positive bacteria stain purple because they have a thick cell
wall that this dye sticks to
• Initially stains all bacteria
▪ Application of iodine (mordant)
▪ Alcohol wash (decolorization)
• Washes out any bacteria that can’t hang (gram negative)
▪ Application of safranin (counterstain)
• Stains gram negative bacteria a red/pink color because they have
a thin cell wall and will not retain purple dye after alcohol wash
o Gram pos and neg bacteria have to be treated differently in healthcare so this is
very important for identification of the bacteria type
• Medically important structural stains
o Capsule stain = stains the capsule around the bacteria and allows for ID of the
capsule
▪ Bacteria that have an extra layer around them
▪ Are harder to kill because they have more protection
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