Tortora Ch' 3

1
Tortora Ch. 3

• Observing Microorganisms Through a Microscope
• http//www.az-microscope.on.ca/history.htm

2
Units of Measurement

• Think small!
• Bacteria are measured in micrometers (microns)
• A micron is 1/1000th of a millimeter (.001)
• Viruses are even smaller!
• The nanometer is used to measure viruses
• A nanometer is 1/1000000th of a mm (.000001)

3
Light Microscopy

• A light microscope is any kind that uses visible
  light to observe specimens
• Compound light microscope is what we use in lab
• The resolution (resolving power) is the ability
  of the lenses to distinguish between 2 points
• Total magnification is a combination of the
  powers of the objective and the ocular lenses.

4
Compound microscope

• http//www.scs.sk.ca/cyber/elem/learningcommunity/
  sciences/biology20/curr_content/biology20/unit1/un
  it1_mod2_les1.html
• Go to the above link for a diagram of a compound
  microscope

5
Other types of microscopes

• Darkfield microscope is used to examine lives
  specimens that are invisible in ordinary light,
  cant be stained by standard methods, or are
  distorted by stain
• A darkfield condenser blocks direct light and
  only allows reflected light which makes the
  specimen appear lighter
• Phase contrast microscope permits details
  examination of internal structures in living
  specimens

6
Other continued

• Fluorescence microscopes use fluorescence, the
  ability of substances to absorb short wavelengths
  of light and give off light at longer wavelengths
• If the specimen doesnt naturally fluoresce it is
  stained with a special fluorescent dye
• See examples of the different types on page 62
  and 63

7
Electron microscope

• Used on objects smaller than 2 microns like
  viruses or to observe internal structures
• Uses an electron beam instead of light
• Images are black and white
• Transmission electron microscope (TEM) has a high
  resolution and is valuable for examining thin
  sections (layers)
• Scanning electron microscope (SEM) provides 3
  dimensional views and is used to study structures
  in intact cells

8
Preparing specimens for microscope

• Staining is necessary to improve visual when
  using a microscope
• First prepare a smear..(details in class)
• Next use either a basic dye or an acidic dye
• Basic dye has a positive ion that is attracted to
  the bacteria
• Acidic dye has a negative ion that is repelled by
  the bacteria

9
Types of stains

• Simple-uses a single type of dye
• Usually use crystal violet (basic dye) or one
  like it
• Negative stain-uses nigrosin which is an acidic
  dye and sticks to the class
• For an interesting web site on staining and
  unknowns click here http//www.kcom.edu/faculty/
  chamberlain/Website/lab/Main.htm

10
Differential stains

• Use 2 different stains applied in separate steps
• Gram stain-invented by Christian Gram
• Gram positive-retains the primary stain and stays
  purple
• Gram negative-retains the secondary stain and
  turns pink/red
• Youll go over this in class or go here
• http//www.ncl.ac.uk/dental/oralbiol/oralenv/tutor
  ials/gramstain.htm

11
Differential continued

• Acid fast stain-uses carbolfuchsin as primary
  stain, acid alcohol, and methylene blue as the
  secondary stain-must use steam
• Acid fast bacteria retain primary stain and are
  red (Mycobacterium) http//www.nottingham.ac.uk/pa
  thology/protocols/afb.html

12
Differential continued again

• Endospore stain-uses malachite green as primary
  stain and safranin as secondary
• Use steam with endospore stain too
• Spores will be green, bacteria will be red
  (reverse watermelon)
• Go here for a view of the slide
  http//deepti.myweb.uga.edu/staining.htm
• See table 3.3 for details on all

13
Purification of cultures

• One method used to purify or check colonies is a
  streak plate
• http//www.sumanasinc.com/webcontent/anisamples/mi
  crobiology/streakplate.html
• It is used to isolate colonies, for plate counts,
  and other things