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.
• 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.
• 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
• 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