2.1- microscopy

What does cell theory state?

cell theory states that…

1. Both plant and animal tissues are composed of cells

2. Cells are the basic unit of all life

3. Cells only develop from existing cells

What does a ‘compound light microscope’ have?

Compound light microscope have two lenses: objective lens and eyepiece lense

How does a compound light microscope work?

In a light compound microscope, the light traveled through the specimen, the objective lens magnifies the image, then the eyepiece lenses magnifies it again for a higher magnification and reduces chromatic aberration

What is chromatic aberration?

Chromatic aberration is the effect produced by the refraction of different wavelengths of light through slightly different angles, resulting in a failure to focus

What do stains do?

Stains increase the contrast as different parts of the cell can absorb different stains. This allows us to see more details and identify organelles

How do you prepare a sample for staining?

To prepare a sample for staining you…

1. Place sample on a slide and allow it to air dry

2. Heat fixed by passing the slide through a flame

What are examples of positively charged stains?

1. crystal violet

2. Methylene blue

Why does crystal violent and methylene blue stain the cytoplasm?

Crystal violent and methylene blue stain blue stain the cytoplasm because they are attracted to the negatively charged materials on the cytoplasm which leads to those parts becoming stained

Which stains are negatively charged?

1. Nigrosine

2. Congo red

Why do nigrosine and Congo red dye stay outside the cell?

Nigrosine and Congo red are negatively charged stains so they are repelled by the negatively charged cytoplasm, this leaves the cell unstained standing against a stained background

How can stains be used to identify white blood cell from red blood cells?

White blood cells nucleus takes up stains, red blood cells do not contain a nucleus and therefore do not absorb stain to the same degree

What is gram stain technique used for?

Gram stain techniques are used to separate bacteria into two groups

What are the two group of bacteria in regard to gram stain technique?

1. Gram-positive

2. Gram-negative

What are the steps to the ‘gram stain technique’

1. Crystal violent is applied to specimen on a slide, done is used to fix the dye

2. The slide is washed with alcohol

What will happen to gram-positive bacteria when treated?

Gram positive bacteria retain the Crystal violent stain

Why do gram-positive bacteria retain the crystal violent stain?

Gram-positive bacteria retain the crystal violent stain because they have thicker peptidogylcan wall

What colour do treated gram-positive bacteria appear?

Gram positive bacteria appears blue/purple under the microscope

Which stain is used to identify gram-negative bacteria?

Safranin

What colour with gram-negative bacteria appear?

Red

Why are gram-negative infections harder to treat?

Gram-negative infections are harder to treat because they have an extra cell membrane that prevents antibiotics from entering

What is the acid-fast technique used for?

The acid-fast technique are used to tell the difference between species of mycobacterium from other bacteria

What are the steps for acid-fast technique

1. A lipid solvent is used to take carbolfuchsin into the cell

2. The cell is then washed by diluted acid-alcohol solution

3. Mycobacteria are not effected by the acid-alcohol so they will retain the stain which appears red

4. Other bacteria are counter stained with methylene blue

What are the steps to prepare a sample for microscopy?

1. Fixing

2. Sectioning

3. Staining

4. Mounting

What does the step ‘fixing’ involve in preparing a slide?

In fixing, use chemicals like formaldehyde to preserve specimens in a near-natural state

What does the step ‘sectioning’ involve in preparing a slide?

in sectioning, the specimen are dehydrated with alcohol and placed in a wax or resin mould to make a hard block, this can be sliced thinly with a knife

What does the step ‘staining’ involve in preparing a slide?

In staining, the specimen are treated with multiple stains to show different parts

What does the step ‘mounting’ involve in preparing a slide?

In mounting, the specimen are secured to a microscope slide and a cover slip is placed on top to hold in in place and protect the sample

What is ‘dry mount’?

In dry mount, solid specimen are viewed whole/cut into very thin slices. The specimen is placed on the centre of a glass slide and covered over with a coverslip

What is ‘wet mount’

In wet mount, specimen are suspended in a liquid. A coverslip is placed gently over the sample at an angle

What is a ‘squash slide’

In a squash slide, a wet mount is prepared first. Then, a lens tissues is used to gentle press down on the coverslip

What is a ‘smear slide’

In a smear slide, the edge of a slide is used to smear the sample, making a thin, even coating on another slide, a coverslip is placed over the sample.

What’s an example of a smear slide?

A blood sample

What’s an example of when a squash slide will be used?

To look at cell division in a root tip

When is a wet mount more likely to be used?

For aquatic samples and other living organisms

What is a stain?

A dye used to colour parts of a cell to make them easier to see

What is magnification?

Magnification is how much bigger something appears compared with its actual size

What is resolution?

Resolution is the smallest distance between two points that can be seen as two points and not blurred into one point