Lesson 1 - Microscopes

Question 1

Cell theory definition:

Answer 1

All living things are composed of cells and cell products.

• Both plant and animal tissues are composed of cells
• Cells are the basic unit of all life
• Cells only develop from existing life

Question 2

What to say instead of clarity of a microscope?

Answer 2

Object has become visibly distinct with increased resolution.

Question 3

Compound Light Microscope:

Answer 3

Two lenses: objective lens and eyepiece lens
Image is magnified first by objective lens, then eyepiece lens. This configuration allows for higher magnification and reduced chromatic aberration.

Question 4

Dry mount

Answer 4

Solid specimens viewed whole or sectioned. Specimen placed in centre of slide and cover slip is placed over.
Pollen, Hair, Muscle tissue, Dust, Insects

Question 5

Squash slides

Answer 5

Wet mount prepared. Lens tissue used to gently press down the cover slip. Good for soft samples.
- Root tips used to look at cell division.

Question 6

Smear slides

Answer 6

Edge of slide is used to smear the sample, creating thin, even coating on another slide. Cover slip placed over sample. Used on blood.

Question 7

Wet mount

Answer 7

Specimens are suspended in a liquid such as water or an immersion oil. A cover slip is placed on from an angle, as shown. Aquatic organisms.

Question 8

Brightfield microscopy

Answer 8

When a sample is illuminated from below with white light, and observed from above.

Question 9

Wide-field microscopy:

Answer 9

The whole sample is illuminated at once.

Question 10

What is the purpose of staining:

Answer 10

• Increase the contrast of sub-cellular organisms
• Stop the cell from absorbing a lot of the light

Question 11

Diffraction:

Answer 11

The bending of light as it passes close to the edge of an object.

Question 12

Which dyes stain the organelles in the cytoplasm and why?

Answer 12

Crystal violet and methylene blue.

They are positively charged and so are attracted to the negatively charged organelles.

Question 13

Negative stain technique:

Answer 13

When the surroundings are died and not the cell

Question 14

Which dies can be used in negative staining?

Answer 14

• Nigrosin and Congo red. They are negatively charged and so repelled from cytosol.

Question 15

Differential staining

Answer 15

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.

Question 16

Gram stain technique

Answer 16

Used to separate bacteria into two groups:
- Gram-positive bacteria
- Gram-negative bacteria

Question 17

Gram-positive bacteria:

Answer 17

Are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls.

Question 18

Gram-negative bacteria:

Answer 18

Thinner cell walls and are not susceptible to penicillin.

Question 19

How to do the Gram stain technique:

Answer 19

Crystal violet applied to bacteria on slide. Then iodine to fix in place.

The slide is then washed with alcohol.

If specimen retains the crystal violet stain and appears blue/purple under a microscope, it is a Gram-positive bacteria.

Gram-negative bacteria lose stain, as they have a thinner cell wall. They are stained with safranin dye (counterstain). Bacteria appears red.

Question 20

Acid-fast technique:

Answer 20

Used to differentiate between species of Mycobacterium from bacteria.

Question 21

How does Acid-fast technique work:

Answer 21

A lipid solvent is used to carry carbolfuchsin dye into cells being studied. Mycobacterium are not effected by acid-alcohol and retain carbolfuchsin stain and are bright red. Other bacteria lose stain and are exposed to a methylene blue stain (blue).

Question 22

Stages in preparing slide:

Answer 22

Fixing: chemicals like formaldehyde are used to preserve specimens in as near-natural state as possible.
Sectioning: Specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly with a knife called a microtome.
Staining: Specimens are treated with multiple stains to show different structures.
Mounting: The specimens are then secured to a microscope slide and a cover slip placed on top.

Question 23

Risk or stains:

Answer 23

• Toxic and irritants

Question 24

How to draw a cell:

Answer 24

• Title
• State magnification
• Sharp pencil
• White and unlined paper
• As much paper as possible for drawing
• Smooth, continuous lines
• No shading
• Draw clearly defined structures
• Proportions are correct
• Label lines should not cross or have arrow heads
• Label lines should be parallel to the top of the page and drawn with a ruler.

Question 25

How to calibrate a microscope?

Answer 25

1. Line up stage micrometer and eyepiece graticule whilst looking through the eyepiece.
2. Count how many divisions on the eyepiece graticule fit into one division on the micrometer scale.
3. Each division on the micrometer is 10um, so this can be used to calculate what one division on the eyepiece is at that current magnification.

Question 26

Gram-positive staining:

Answer 26

Crystal violet and then iodine to fix the stain. Alcohol used to wash away any unbound stain.
Appear purple-blue as stain is retained due to thicker peptidoglycan cell wall absorbing dye.

Question 27

Why do gram-negative bacteria not retain crystal violet dye?

Answer 27

Peptidoglycan cell wall is thin.

Question 28

Counterstain for gram-negative bacteria?

Answer 28

Safranin turns them red

Question 29

Dye used to stain cytoplasm?

Answer 29

Eosin (pink / dark red)

Question 30

Dye used to stain DNA / chromosomes?

Answer 30

Acetic orcein (dark red)

Question 31

Dye used to stain starch?

Answer 31

Iodine (black/blue –> appears violet under microscope)

Question 32

Dye used to stain cellulose?

Answer 32

Iodine in potassium iodine solution (yellow)

Question 33

Dye used to stain DNA/RNA?

Answer 33

Haematoxylin (purple/blue)

Question 34

All purpose stain often used on DNA?

Answer 34

Methylene blue (DNA blue)