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