The Microscope: Window on an Invisible Realm
Units of Measurement
All measurements are made using the metric system; review metric units of length
Micrometer (micron) = 10-6 meter = 1 millionth of a meter = 1/1000 mm.
Nanometer - 10-9 meter
Microscopy: The Instruments - See Table 3.5 for Comparison of Types of Microscopy
Light Microscopy
A. Compound Light Microscope - series of lenses (compound) ; visible light for illumination
 Know the names and functions of all the parts listed on Fig 3.15
 Magnification – the ability to enlarge objects
o Magnifying Lenses
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Objectives - 4X, 10X, 40X, 100X (oil immersion)
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Ocular lens (in the eyepiece) – (10X)
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Total magnification – ocular mag X objective mag – see p.70
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Resolution – ability to distinguish fine detail and structure ; to distinguish magnified
objects clearly
 Primarily a function of the wavelength of light that forms the image
 Shorter wavelength = better resolution – Fig 3.17
 Best - about 0.2 um with blue light ( use blue filters)
 Numerical Aperture – relative efficiency of a lens to bend light
 Higher N.A. = better resolution
 Immersion oil – same refractive index as glass ; eliminates refraction
(bending) as light rays pass from glass slide to the air. – Fig 3.18
Brightfield illumination – background is brightly lit
Contrast – the difference between the specimen and the background: To Increase contrast
o decrease light by turning down rheostat or closing the iris diaphragm
o stain specimen
Darkfield Microscopy
o Light is reflected off the sides of the specimen
o specimen is bright against a dark background
o for organisms that stain poorly ( e.g., Treponema pallidum, the syphilis spirochete)
o For living cells; no staining=no distortion ; get good surface detail; doesn’t give good
internal detail
Phase-Contrast Microscopy
o Detailed examination of internal detail in living microorganisms
o Unstained specimen
o Based on differences in light diffraction ( scattering); denser areas in the cell scatter light
more – appear darker
Fluorescence Microscopy
o Uses short wavelength, high-energy ultraviolet (UV) light
o Fluorescence – the ability to absorb short wavelengths of light (UV)and give off that
energy as the longer wavelength (visible) of light
o Fluorochromes – fluorescent dyes – stain specimens
o Fluorescent-antibody technique – antibodies can be tagged with fluorescent dye
o specific antibodies can be applied to patient specimen or to a bacterium
o will specifically bind to matching antigen – used for diagnosis or identification of a
microbe
Confocalo Uses laser to scan and form images at various depths of the cell
o exceptionally clear 2-D images on a single plane; 3-D computer reconstruction of entire
cells
B. Electron Microscopy
 Uses beam of electrons instead of light – shorter wavelength = better resolution
 To see objects smaller than 0.2 um
 Electrons are focused onto a collection plate ( CRT, photographic film, computer)
 Specimens are processed – ultrathin sections, frozen sections, coated with heavy metals (
e.g., osmium, platinum, gold)
 Transmission Electron Microscopy (TEM)
o Electron beam passes through ultrathin sections
o Good internal detail – 2-D image
o 10,000 – 100,000 X
o can be used to see viruses, organelles, large molecules
 Scanning Electron Microscopy
o Electrons bounce off the surface of the specimen
o Good for seeing surface features of cells and viruses
o Image appears to be 3-D
o 1000 – 10,000 X
C. Scanned Probe Microscopy – See Insight 3.2
o Scanning Tunneling microscopy (STM)
o Atomic Force Microscopy (AFM)
o Uses fine probe to scan surface of specimen
o Good for detail at the molecular level – individual small, biological molecules (antibodies,
enzymes, etc) or atomic detail; nanotechnology
Preparation of Specimens for Light Microscopy
Fresh Specimens
 Wet mounts – done in saline, water or broth
 True size, shape motility and arrangement can be seen
 Poor contrast
 Short term – cells dry out
Preparing Smears For Staining
 Developed by Robert Koch – more permanent
 Staining – coloring microorganisms with a dye that emphasizes certain structures
 Smear – the thin film of material containing microorganisms spread on the surface of a slide
 Air dry - air drying can be done on the benchtop or on a slide warmer
 Fixing – heat or chemical; fixes microorganisms to the slide; kills bacteria; preserves cell
structures
Dyes – the colored chemicals that are applied to the cells
 chromophore – the colored ion in a dye
 basic dye – the color is carried on the (+) ion
1. crystal violet; methylene blue; malachite green; safranin; carbolfuchsin
2. attracted to the negatively charged bacterial cell
 acidic dye – the color is carried on the negative ion
1. eosin; acid fuchsin; nigrosin; India ink
2. negative ions are repelled by the negatively charged bacterial surface
3. colors the background instead
Simple Stains
Simple stain – uses 1 dye ; basic shape & structure can be seen - Fig 3.21
o direct (Positive) staining – stains the organism ( uses a basic dye)
o negative staining – technique that prepares colorless bacteria against a colored background ; good
for overall cell size, shape, and seeing capsules
o See Table 3.6 for comparison
Differential Stains – react differently with different kinds of bacteria; can be used to distinguish among them
See – Fig 3.21
Gram Stain
 Most common differential stain done on bacteria ; useful for identification of organisms and diagnosis
of infection
 Can be done on cultured organisms or on clinical specimens ( sputum, pus, urine, etc.)
 Classifies bacteria into 2 groups; Gram positive and gram negative
 Primary stain – crystal violet
 Mordant – iodine
o Mordant – a chemical added to a solution to intensify the stain – increases the affinity of a
stain for a biological specimen ( e.g., iodine in the Gram stain)
 Decolorizing agent – alcohol-acetone
 Counterstain – safranin
 Differences between gram(+) and gram (-) organisms are based on differences in their cell walls
o Gram (+) – thick peptidoglycan layer; resist decolorization; retain crystal violet; appear
purple
o (Gram (-) – thin peptidoglycan layer; thick lipopolysaccharide layer is dissolved by the
decolorizer; cells will accept the counterstain – appear pink
 Best when performed on young, growing bacteria
 Gram (+) and Gram (-) have different sensitivity patterns to antibiotics; can help doctor when initially
choosing an antibiotic to treat disease
Acid-Fast Stain
 Binds strongly to bacteria that have waxy material ( mycolic acids) in their cell walls
 Used to identify Mycobacterium tuberculosis, Mycobacterium leprae and pathogenic strains of
Nocardia sp.
 Ziehl-Neelson method uses heat; Kinyoun method is done at room temperature
 Primary stain – carbolfuchsin
 Decolorizer – acid alcohol; very strong decolorizer
 Counterstain – methylene blue
 Acid Fast organisms – carbol fuchsin is retained in the lipid-rich cell wall – stain red
 Non-acid-fast organisms – decolorize with the acid – accept the counterstain – stain blue
Special Stains - See Fig 3.21
Negative stains for Capsules
 Capsules are virulence factors ; help bacteria cause disease
 Capsules won’t accept most dyes – appear as unstained halos surrounding stained bacteria
Endospore staining
 Endospores – resistant, dormant structure that protects a bacterium from adverse environmental
conditions ; don’t stain by ordinary methods - most dyes do not penetrate the thick spore coat.
 Schaeffer-Fulton endospore stain – uses heat to help the stain penetrate the spore coat
 Primary stain – malachite green applied with heat / rinse w/ D.W. / counterstain – safranin
 Endospores – green / vegetative cells – pink
Flagella staining
 Flagella – motility structures
 Carbolfuchsin and mordant build up layers to make flagella visible under the light microscope
 Arrangement of flagella – see Lab Ex. 7 and chapt 4
 Monotrichous – single polar flagellum
 Amphitrichous – a tuft of flagella at each end of the cell
 Lophotrichous – 2 or more at one pole of the cell
 Peritrichous – distributed over the entire cell