Chapter 2.1 Microscopy

Question 1

Definition of microscope

Answer 1

Instrument which enables you to magnify an object hundreds, thousands or even hundreds of thousands of times.

Question 2

Function of microscopes

Answer 2

They make the individual cells of multicellular organisms visible, allowing us to discover details of the structures and how these details relate to their functions.

Question 3

1st type of microscope

Answer 3

Light microscope. Developed in 16th to 17th century

Question 4

When could individual cells be seen

Answer 4

Mid 19th century

Question 5

3 parts of cell theory

Answer 5

• both plant and animal tissue is composed of cells
• cells are the basic unit of all life
• cells only develop from existing cells

Question 6

Benefits of light microscopy

Answer 6

Easily available
Relatively cheap
Can be used out in the field
Observe living and dead prepared specimens

Question 7

History of light microscope

Answer 7

Romans experimented with glass
Objects looked bigger when viewed through glass that’s thicker in the middle than at the edges
13th century - invention of spectacles
Dutch spectacle makers invented telescope
Galileo Galilei developed compound microscope
Galileo instrument first to be named a microscope

Question 8

Stage 1 of development of cell theory

Answer 8

1665 - Robert hooke observed the structure of thinly sliced cork
Used early light microscope
Described compartments as cells
Was observing dead plant tissue so he could only see cell walls

Question 9

Stage 2 of development of cell theory

Answer 9

1674-1683
Leeuwenhoek developed technique for creating powerful glass lenses
Used handcrafted microscopes to examine samples of pond water
First to observe bacteria and protoctista ‘little animals’
Now called microorganisms
Observed red blood cells, sperm cells and muscle fibres

Question 10

Stage 3 of development of cell theory

Answer 10

1832 - Dumortier observed cell division in plants
Disproved theory that New cells arise from within old cells
Disproved that cells formed spontaneously from non-cellular material
Virchow published these findings in 1855

Question 11

Stage 4 of development of cell theory

Answer 11

1833 - Brown described plant cell nucleus

Question 12

Stage 5 of development of cell theory

Answer 12

1837 - 1838
Schleiden- all plant tissues are composed of cells
Purkyně - used microtone to make ultra-thin slices of tissue for microscopic examination
Animals are composed of cells
Basic cellular tissue of animals is clearly analogous to that of plants
Schwann - all living things are composed of cells and cell products

Question 13

Stage 6 of development of cell theory

Answer 13

1844 (1855) - Remak observed cell division within animal cells
Disproved that new cells originated from within old cells
Virchow plublis

Question 14

Stage 7 of development of cell theory

Answer 14

1860 - Pasteur disproved spontaneous generation of cells.

Demonstrated that bacteria would only grow in sterile nutrient broth after being exposed to air

Question 15

How does a compound light microscope work?

Answer 15

2 lenses - configuration allows for higher magnification, reduced chromatic aberration
objective lens (placed near specimen) produces magnified image,
eyepiece lens (where specimen is viewed) magnifies again
Illumination usually provided by light underneath sample
Opaque specimens an be illuminated from above

Question 16

Types of sample preparation

Answer 16

Dry mount
Wet mount
Squash slides
Smear slides

Question 17

Steps for a dry mount

Answer 17

Solid specimen can be viewd whole
Can be sectioned (cut into thin slices using a blade)
Specimen placed on centre of slide
Cover slip placed over the top
( used on hair, pollen)

Question 18

Steps for a wet mount

Answer 18

Specimen suspended in liquid (water or immersion oil)
Cover slip placed on from an angle
(aquatic samples)

Question 19

Steps for squash slides

Answer 19

Wet mount prepared
Lens tissue gently pressed down on cover slip
Depending on material, potential damage can be avoided by squashing the sample between 2 microscope slides
(good for soft samples)
(root tip squashes used to look at cell division)

Question 20

Steps for smear slides

Answer 20

Edge of a slide smears sample
Creates thin and even coating
Cover slip placed over sample
(used to view cells in blood)

Question 21

Types of basic light microscopy

Answer 21

Brightfield microscopy -
Sample is illuminated from below with white light
Observed from above
Wide-field microscopy - 
Whole same illuminated at once

Question 22

Use of stains

Answer 22

Images have low contrast as most cells don’t absorb much light
Cytosol and other cell structures are often transparent
Different components in cells take up stains to different degrees
Stains increase contrast
Increased contrast allows components to become visible so they can be identified

Question 23

Prepare sample for staining

Answer 23

Place on slide
Air dry
Heat-fix by passing thrugh a flame
Specimen will adhere to micrscope slide and will take up stains

Question 24

What is differential staining?

Answer 24

Using multiple stains. It can distinguish between two types of organisms that would’ve been hard to identify. Can also differentiate between different organelles of a single organism within a tissue sample.

Question 25

How do crystal violet and methylene blue work as stains?

Answer 25

Positively charged. Attracted to negatively charged materials in cytoplasm. Results in staining of cell components.

Question 26

Negative stain technique

Answer 26

When you stain the background. Can use nigrosin or congo red Negatively charged dye is repelled by negatively charged cytosol. Dyes stays outside of cell, cell remains unstained Cell stands out against stained background

Question 27

Gram stain technique

Answer 27

Separates bacteria into gram-positive and gram-negative Apply crystal violet to bacterial specimen (on a slide) Apply iodine to fix the dye Wash slide with alcohol Gram+ retains crystal violet stain - appears blue/purple under microscope Gram- has thinner cell walls so loses stain. Stain gram- with safranin dye (counterstain) bacteria appears red.

Question 28

Difference between gram+ and gram-

Answer 28

Gram+ is susceptible to antibiotic penicillin (inhibits formation of cell walls) whilst gram- has thinner cell walls that are not suscetible to penicillin.

Question 29

What is acid-fast technique used for

Answer 29

Differentiate species of mycobacterium from other bacteria

Question 30

Steps for acid-fast technique

Answer 30

Lipid solvent carries carbolfuschsin dye into cells being studied Cells washed with dilute acid-alcohol solution Mycobacterium are not affected by acid-alcohol solution They retain the carbolfuchsin stain Appear bright red Other bacteria lose stain and are exposed to methylene blue stain (appear blue)

Question 31

Stages for preparing slides

Answer 31

Fixing - chemicals (formaldehyde) used to preserve specimens in near-neutral state Sectioning - specimen dehydrated with alcohols. placed in mould with wax/resin to form hard block. Sliced thinly with microtone Staining - specimen treated with multiple stains to show different structures Mounting - specimen secured to microscope slide and cover slip placed on top

Question 32

Risk management

Answer 32

Stains can be toxic or irritants Identify any procedures that may cause harm CLEAPSS provides support for practical work in schools Advice provided to schools and employees Many slides are preprepared due to toxic stains and long complex process of preparing

Question 33

Rules for scientific drawings

Answer 33

``` Title State magnification Sharp pencil White, unlined paper Use majority of paper Smooth continuous lines No shading Clearly defined structures Proportions correct Label lines don't cross or have arrowheads Label libes parallel to top of the page and drawn with ruler ```