2.1 Microscopy

Question 1

What does the cell theory state?

Answer 1

• Plant and animal tissues are composed of cells
• Cells are basic units of life
• Cells develop from existing cells

Question 2

What are the benefits of light microscopes?

Answer 2

• Easily available
• Cheap
• Can be used outside in the field
• Can be used to observe both living organisms and dead (prepared) specimens

Question 3

How does a compound light microscope work?

Why is a compound light microscope better than a simple light microscope?

Answer 3

Two lenses:

1. Objective lens - placed near specimen
2. Eyepiece lens - through which specimen is viewed

How it works:
Objective lens produces a magnified image which is magnified again by the eyepiece lens
- Illumination is provided by light underneath the sample
- Illumination provided from the top if the specimen is non-transparent

Why it’s better:
The combination of eyepiece lens + objective lens produces images with higher magnification and reduced blurring when compared to a simple light microscope

Question 4

What are the 4 methods of preparing a sample?

Answer 4

• Dry Mount
• Wet Mount
• Squash Slides
• Smear Slides

Question 5

How do you prepare a dry mount slide? Give examples.

Answer 5

1. Either:
   - Solid specimens viewed as whole
   - Solid specimens cut into thin slices (known as sectioning)
2. Specimen is placed onto centre of slide + cover slip is placed over sample

Example:

1. Viewing hair, pollen, insect parts or dust as whole
2. Muscle/plant tissue as sectioned

Question 6

How do you prepare a wet mount slide?

Answer 6

1. Specimens are suspended in a liquid e.g. water or immersion oil
2. A cover slip is placed at an angle:

Example: Aquatic sample

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Question 7

How do you prepare a squash slide?

Answer 7

1. Wet mount first prepared
2. Lens tissue is used to press down cover slip
   {damage to cover slip can be prevented by squashing the sample between 2 slides}

• Good for soft samples
  E.g. Root tip squashes to look at cell division

Question 8

How do you prepare a smear slide?

Answer 8

1. Edge of a slide used to smear sample, creating thin, even coating on another slide.
2. A cover slip is placed over the sample

Example: A sample of blood is a smear slide

Question 9

Why are images at low contrast with a basic light microscope?

Answer 9

Most cells don’t absorb a lot of light

Question 10

What limits resolution in light microscopy?

Answer 10

• Wavelength of light

- Diffraction of light

Question 11

What is diffraction?

Answer 11

Bending of light as it passes close to the edge of an object

Question 12

1. Why do stains increase contrast of a cell’s components? 2. Why is this useful?

Answer 12

1. Increase contrast as different components of cells absorb stains to different degrees
2. Increases contrast which allows components to become visible so they can be identified

Question 13

How do you prepare a sample for staining?

Answer 13

1. Sample is placed on a slide + allowed to air dry
2. Sample then heat fixed by passing through a flame
3. The specimen will adhere to the slide and absorb the stains

Question 14

How do positive dyes work (in terms of staining)?

Answer 14

Positive dyes (i.e. Crystal violet/methylene blue, positively charged) get attracted to negatively charged materials in cytoplasm, causing staining of cell’s components

Question 15

How do negative dyes work (in terms of staining)?

Answer 15

Negative dyes (i.e. Nigrosin or Congo Red, negatively charged) get repelled by negatively charged cytosol.

These dyes stay outside of cells, leaving the cells unstained, which then stand out against the stained background (Called negative stain technique)

Question 16

What is differential staining?

Answer 16

Ability to distinguish between two types of organisms, that would otherwise be hard to identify

Can differentiate between different organelles of a single organism in a tissue sample

Question 17

What is gram stain technique used for?

Answer 17

To separate bacteria into two groups :

• Gram-positive bacteria
• Gram-negative bacteria

Question 18

How do you carry out the gram stain technique?

Answer 18

1. Crystal violet is applied to a bacterial specimen on a slide
2. Iodine is applied, which fixes the dye
3. Slide is washed w/ alcohol

• If Gram-positive, bacteria retain crystal violet/methylene blue + appear blue/purple under microscope

1. Gram-negative bacteria have thinner walls so lose stain
2. They’re then stained w/ safranin dye, called a counterstain

If Gram-negative being tested

1. Stain will be lost
2. Stained w/ safranin dye, a counter dye
3. Bacteria will appear red under microscope

Question 19

How do you carry out the acid-fast technique?

Answer 19

1. Lipid solvent used to carry carbolfuchsin dye into cell being studied
2. Cells are washed w/ dilute acid alcohol solution {Mycobacterium not affected by acid-alcohol + retains carbolfuchsin stain, which is bright red}
3. Other bacteria lose stain + get exposed to methylene blue stain
   * This technique is used to differentiate species of mycobacterium from other bacteria

Question 20

What are the 4 stages involved in preparation of slides?

Answer 20

1. Fixing
2. Sectioning
3. Staining
4. Mounting

Question 21

How is a microscope slide prepared during: Fixing

Answer 21

Chemicals e.g. formaldehyde used to preserve specimens in as natural state as possible

Question 22

How is a microscope slide prepared during: Sectioning

Answer 22

• Specimens are dehydrated w/ alchols
• Then placed in a mould w/ wax/resin to form a hard block
• This can be thinly sliced with a microtome (special knife)

Question 23

How is a microscope slide prepared during: Staining

Answer 23

• Specimens treated w/ multiple stains to show different structures

Question 24

How is a microscope slide prepared during: Mounting

Answer 24

• Specimens secured to slide + cover slip is placed on top

Question 25

*Learn how an onion cell looks and how to draw it*

Answer 25

N/A