Microscopy

What is cell theory?

• all living organisms consist of cells

• cells are formed by division of pre-existing cells

• cells contain DNA that acts as instructions for growth

How long do temporary mounts last?

A few hours

How are permanent specimens prepared?

• specimen is dehydrated 

• fixed in wax

• thinly sliced

Dry mount preparation

• solid specimens can be viewed as a whole

• or cut into very thin slices (sectioning)

• placed on slide and covered with a coverslip

Wet mount preparation

• specimens suspended in liquid

• coverslip placed at an angle to avoid bubbles

• eg, aquatic life, living organisms

Squash slide preparation

• wet mount prepared and coverslip pressed on

• sample is squashed between two slides

Smear slide preparation

• edge of slide used to smear a sample along another 

• coverslip then placed on top 

• eg. blood slide

Why use a stain?

Provides contrast - different components within a cell take up stains to different degrees

Magnification =

Image size ÷ actual size

What do positively charged dyes stain?

Cell components 

What do negatively charged dyes stain?

Stain outside of the cells - the background

Why use a differential stain?

• provides contrast

• only some organelles take up stain

Gram stain technique 

Separates bacteria into two groups:

• gram positive 

• gram negative

Definition of magnification

the number of times larger an image is compared with the real size of the object

Definition of resolution

the ability to distinguish between two separate points

What are the two types of electron microscopes?

1. Transmission electron microscope 

2. Scanning electron microscope 

Transmission electron microscope

• beam of electrons passes through the sample

• electrons are detected by a sensor

• imaged is 2D

• magnification x500 000

• resolution = 0.5nm

Scanning electron microscope 

• scans the surfaces of the sample

• electrons are reflected

• sensor detects electrons and an image is created

• produces a 3D image

• magnification x500 000

• resolution = 3-10nm 

Light microscope

• specimen can be viewed whilst still alive

• can be stained to add contrast

• color can be seen

• light is shone through specimen then passes through two lenses

• lenses magnify the image

• magnification x1500

• resolution = 200nm

Disadvantages of electron microscopes

• only produce black and white images 

• specimen must be in a vacuum - must not be living 

• specimen must be dehydrated

• very expensive 

• very large

Artifact definition

a visible structural detail caused by processing the specimen and is not a feature of the specimen eg. air bubbles trapped under a slide when using a light microscope

Eyepiece graticule

• small ruler that is inserted into the eyepiece of a microscope

• scale is arbitrary

• the eyepiece graticule remains constant at all magnifications

• must be calibrated using a stage micrometer

Stage micrometer

• used to calibrate eyepiece graticule 

• it is 1cm long and is divided into 100 division 

• 1 division = 100 micrometers