OCR AS/A Level Biology: Microscopy & Staining

Use of microscopy

To observe and investigate different types of cell and cell structure in a range of eukaryotic organisms

Types of microscopy

Light, transmission electron, scanning electron, laser scanning confocal

Types of mount

Wet, dry, squash, smear

How to do a wet mount

Suspend the specimen in liquid, place cover slip on from an angle

Examples of liquids used in wet mounts

Water, immersion oil

Organisms that can be viewed in wet mounts

Aquatic organisms

How to do a dry mount

Section the sample if it is too large, place specimen on centre of the slide, put cover slip on top

Things that can be viewed in dry mounts

Hair, pollen, dust, insect parts, parts of muscle tissue, parts of plant

How to do a smear slide

Use the edge of a slide to smear out the sample on another slide, put cover slip over the sample

How to do a squash slide

Prepare a wet mount, use a lens tissue or two microscope slides to press down on the cover slip

What can you view with a squash slide?

Root tips during cell division

What can you view with a smear slide?

Blood

Why must a sample be thin for light microscopy?

So the light can shine through it and details can be seen.

How a light microscope works

Objective lens produces a magnified image, image magnified again by the eyepiece lens, illumination provided by a light underneath the sample

How to calibrate a microscope

no

Why must the liquid medium used in wet mounts have a similar refractive index to glass?

To prevent diffraction between the liquid and the glass and thus preventing image distortion.

Why are cover slips placed on wet mounts at an angle?

To prevent the trapping of air bubbles.

Purpose of differential staining

To identify different cellular components and cell types

Examples of differential staining

Gram stain technique, Acid-fast technique

Purpose of the Gram stain technique

To differentiate between Gram positive and Gram negative bacteria.

Purpose of the Acid Fast technique

To differentiate between species of Mycobacterium and other bacteria

Stages in pre-preparation of slides

Fixing, sectioning, staining, mounting

Fixing

Using chemicals like formaldehyde to preserve specimens in a near-natural state.

Sectioning

Dehydrating a sample with alcohol and then placing it in a mould of resin or wax to form a hard block before slicing it with a microtome into thin slices.

Staining

Treating specimens with multiple stains to show different structures.

Mounting

Securing a specimen onto a microscope slide under a cover slip.

Advantages of staining

See more detail, increases contrast, allows you to identify different cells and cellular components like organelles

Magnification Formula

Magnification = Image size / Object Size

Difference between magnification and resolution

Magnification is how many times larger the image is than the actual size of the object whereas resolution is how detailed the image is and the ability to distinguish between two points that are close together.

Resolution of light microscopes

200nm

Magnification of light microscopes

1500x

Resolution of a transmission electron microscope

0.5nm

Magnification of a transmission electron microscope

500000x

Resolution of a scanning electron microscope

3-10nm

Magnification of a scanning electron microscope

100000x

How to tell the difference between an SEM image and a TEM image

-Both images are black and white but TEM images can add false color using computer system.

-SEM=3D

-TEM=2D

-SEM has a higher res.

-The SEM image will only allow you to see the outside of the cell rather than the inside cell detail.

Comparison points for Light Microscopes

-Cheap to purchase and operate

-Small and portable

-Simple and easy sample preparation

-Vacuum not required

-Natural colour of sample is maintained

-Magnifies only up to 2000 times

-Specimens can be living or dead

-Stains are often needed to make the cells visible

Comparison points for Electron Microscopes

-Expensive to buy and produce electron beam

-Large and requires a special room

-Lengthy and complex sample prep

-Vacuum is required

-All images black and white

-Magnifies over 500000 times

-Specimens are dead

-Electron beam can damage specimens as they must be stained with an electron-dense chemical.

Why might organelles appear to be different sizes down a microscope?

Cut in a transverse or longitudinal planes, natural variation in shape, some may have just divided or be growing, an artefact may have deformed it

Advantages of filming when doing microscopy

Can use time lapse, continuous record, you don't need to be constantly looking, not dependent on drawing or describing ability, easy to see detail

Positively charged dyes

Crystal violet, methylene blue

Negatively charged dyes

Nigrosin, Congo red

Why do we use staining?

To increase contrast because cells don't absorb much light

The crystal of cells and other cell structures are often transparent.

What is methylene blue?

Is an all-purpose stain

What is differential staining?

Some stains bind to specific cell structures staining each structure differently so the structures can be easily identified.

Acetin Orcein binds to....

DNA + stains chromosomes dark red.

Eosin stains.....

Cytoplasm.

Sudan red stains....

Lipids.

Iodine in KI solution stains....

The cellulose in plant cell walls yellow

+

Starch granules blue/black might look violet under MS