Light microscopes

What is the dry mount method of preparing samples?

-Solid specimens are viewed whole or cut into very thin slices with a sharp blade. This is called sectioning

-The specimen is placed on the centre of the slide and a cover slip is placed over the sample

What is the dry mound method used for?

-Hair, pollen, dust and insect parts can be viewed whole

-Muscle tissue or plants can be sectioned and viewed

What is the wet mound method?

-Specimens are suspended in a liquid - water or an immersion oil

-A cover slip is placed on from an angle

What is the wet mound method used for?

-aquatic samples

-living organisms

How are squash slides prepared?

-A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip

-Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides

-It spreads the cells out into one layer which ensures that light can pass through so the cells are visible

What are squash slides used for?

-root tips

-Soft samples

How are smear slides prepared?

-The edge of a slide is used to smear the sample, creating a thin, even coating on another slide

-A cover slip is placed over the sample

What are smear slides used for?

-cells in blood

Why should the refractive index of the medium be approximately the same as the glass?

To prevent the light from refracting (which would make the sample not visible)

Why is staining necessary when preparing slides for light microscopy?

-The images tend to have low contrast as most cells do not absorb a lot of light

-Most cell structures are transparent

-Stains increase contrast as different components within a cell take up stains to different degrees

-The increase in contrast allows components to become visible

What are the staining techniques?

-Crystal violet/ methylene blue

-Nigrosin/ Congo red

-Gram staining

-Acid fast techniques

How is crystal blue/ methylene blue used?

Positively charged dyes which are attracted to negatively charged materials in the cytoplasm leading to the staining of cell components

How is Nigrosin/Congo red used?

Negatively charged and repelled by the negatively charged cytosol. These dyes stay outside cells leaving the cells unstained, which then stand out against the stained background

How is gram staining used?

-Used to separate bacteria into 2 groups - Gram positive and gram negative

-Crystal violet is first applied to a bacterial specimen on a slide, then iodine which fixes the dye

-The slide is washed with alcohol

-The gram positive bacteria retain the crystal violet stain and appear blue or purple under a microscope

-Gram negative bacteria have thinner cell walls and therefore lose the stain

-They are then stained with safranin dye (a counterstain) which makes them appear red

How are acid fast techniques used?

-Used to differentiate species of Mycobacterium from other bacteria

-A lipid solvent is used to carry carbolfuchsin dye into the cells

-The cells are then washed with a dilute acid-alcohol solution

-Mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain which is bright red

-Other bacteria lose the stain and are exposed to a methylene blue stain

What is differential staining?

It can distinguish between two types of organisms that would otherwise be hard to identify. It can also differentiate between different organelles of a single organism within a tissue sample

What are the stages of preparing a slide for a light microscope?

-Fixing - chemicals like formaldehyde are used to preserve specimens in as near-natural state as possible

-Sectioning - specimens are dehydrated with alcohols then placed inn a mould with wax or resin to form a hard block. This can be sliced thinly with a knife called a microtome

-Staining - specimens are often treated with multiple stains to show different structures (differential staining)

-Mounting - the specimens are secured to a microscope slide and a cover slip placed on top

What is cell theory?

-Both plant and animal tissue is composed of cells

-Cells are the basic unit of all life

-Cells only develop from existing cells