Chapter 3
Microscopes and staining
Procedures
• Measurement of microbes – units known
as micrometers.
• 1000 micrometers = 1 millimeter
• Length of bacteria – 2 um to 7 um
• Diameter 0.2 um to 2 um
• Bright field microscope
• Total magnification = magnification by the
objective lens X magnification by the
ocular lens
• Resolving power (resolution)
clarity/sharpness of the image
• Oil immersion improves the resolving
power
• Dark-field microscope – cells are not
stained.
• Objects are bright/light. Background is
dark.
• Treponema pallidum – spirochete –
syphilis.
• Phase contrast microscope – no staining
• Used to see internal structures –
organelles.
• Fluorescent microscope UV light is used to
illuminate the object.
• Cells are stained with fluorescent dyes.
• Auramine O is used to stain
Mycobacterium tuberculosis.
• Cells show up as glowing yellow objects
against a dark background.
• Electron microscope
• Transmission electron microscope (TEM)
• Scanning electron microscope (SEM)
• Beam of electrons are used in place of
light.
• TEM - thin sections of the specimen are
obtained and placed on a copper mesh
grid. Magnifies the object 10,000X to
100,000X. It has the resolving power of
.0025 um. This used to observe internal
structures.
• SEM – used to observe structures found
on the surface of microbes. Magnifies the
object 1000X to 10,000X.
• Resolving power of 0.02 um.
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Dyes are salts
Basic dye – positive ion has the color.
Methylene blue chloride.
Acidic dye – negative ion has the color.
Sodium eosinate
Basic dyes are used to stain the cell.
Bacterial cell is negatively charged. It is
attracted to the positive ions. Ionic bond is
formed between the cell and the stain.
• Negative cell is repelled by the negative
ions. They are used to stain the
background.
• Nigrosin is used – background is black
and cells are bright. Image is similar to
what is seen in the case of dark-field.
• Simple staining – a basic dye is used to
stain the cell to determine the shape and
arrangement of the cells.
• Gram staining is a differential staining. It
places bacteria into 2 groups.
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Gram staining
Crystal violet – primary stain
Iodine – mordant
Alcohol-acetone - decolorizer
Safranin – counterstain
• Gram + are purple
• Gram – are pink
• Gram staining is based on the cell wall
structure.
• Gram positive cells have thick cell walls.
They hold on to the primary stain.
• Gram negative cells have thin cell wall.
• One or two layers of peptidoglycan. They
also have an outer membrane – lipids.
• Alcohol causes damage to the lipids.
Primary stain leaks out.
Acid-fast staining
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Differential staining
Two genera are acid-fast
Mycobacterium and Nocardia
They have a waxy substance known as
mycolic acid in their cell walls.
• Carbolfuchsin – primary stain
• Acid-alcohol – decolorizer
• Methylene blue – counterstain
• Acid-fast – red
• Nonacid-fast - blue
Capsule staining
• A capsule is a gelatinous substance found
around the cell wall.
• It cannot be stained
• Stain the background using nigrosin.
• Stain the cell with crystal violet.
• Background is black.
• Capsule shows up as a clear ring around
the stained cell.
Endospore staining
• Two genera of bacteria that make
endospores are Bacillus and Clostridium.
• Endospores are resistant to hostile
environmental conditions.
• Heat, UV light, disinfectant, desiccation.
• Endospores are formed within the
vegetative cell. Once the formation is
complete, endospores are released into
the environment.
Endospore Staining
• Malachite green - primary stain
• Water – decolorizer
• Safranin – counter stain