1.0.0 Development of practical skills in biology

Question 1

What is a light microscope?

Answer 1

• A type of microscope used to view cells by producing an enlarged image that shows some of the detail inside the cell.
• Uses different sets of lenses (compound microscope) to magnify the specimen.

- Does not include internal structures (limited detail)

Question 2

What are light microscopes used for?

Include HOW

Answer 2

Examining microscopic organisms, cells or tissue sections using a light source.

Light source: visible light (EM)

Question 3

Do not include the function

Different componenets of a microscope

There are 9 main components

Answer 3

1. Arm
2. Stage
3. Eyepiece (ocular) lense
4. Objective lenses
5. Condenser
6. Iris diaphragm
7. Illuminator
8. Coarse focus
9. Fine focus

Question 4

Functions of the microscope components

Answer 4

1. Supports microscope parts, carries the microscope
2. Slides can be held down by stage clips
3. Used to look down the microspcope to view image (x10)
4. Set of lenses of differing magnifications that rotate into place directly above the microscope slide (x4, x10, x40)
5. Directs the beam of light from the source onto the specimen
6. Alters the amount of light directed onto the specimen
7. Light source used to pass light through the specimen
8. Large focusing dial that is used to generally focus the image; used with low power lens only
9. Small focusing dial that is used to focus the image sharply

Question 5

Equation

Magnification

Answer 5

I
A M
Maginification = image size / actual size

Question 6

Define resolution

Answer 6

The ability to see two distinct points seperately

Question 7

What is the resolution of a light microscope

Answer 7

200 nm is the smallest distance at which two individua points can be seen as seperate objects

Question 8

Conversions

Answer 8

1 nanometre (nm) = 10-9 m
1 micrometre (µm)= 10-6m

Question 9

Produce a set of instructions on how to use a light microscope

Answer 9

1. Place the low power objective lens in position (scanning/shortest lens x4).
2. Place the microscope slide on the stage.
3. Use the coarse focus to raise the stage close to the low power lens. It should lock into place.
4. Use the coarse focus to slowly raise the low power lens until the object is visible and in focus.
5. Use the fine focus to sharpen the focus on the image.
6. Ensure that the part of the slide to be viewed is in the centre of the field of view.
7. Rotate the medium power objective lens (x10) into place. It will be very close to the slide.
8. Using the fine focus only, refocus the image. It should need very little adjustment but if needed go back to step 1.
9. Repeat steps 7-8 with the high power lens (x40).

Ensure that

Always carry the microscope by the arm & support the base.

Question 10

Decribe the setup of a cavity slide

Answer 10

A drop of pond water can be placed into the well in the slide to view live microscopic organisms such as protists (unicellular organisms) or Daphnia (small crustacean)

Question 11

Describe the setup of a flat slide

Relates to thin structures

Answer 11

Used with thin sections (one cell thick) that are usually stained to enable structures to be seen and distinguished from each other.

Question 12

Why is staining of tissues essential?

Answer 12

Cells & their components are transparent making it very difficult to distinguish them and their components as there is no contrast between them.

Question 13

What is methylene blue stain?

Include what it does specifically

Answer 13

• It is used as a general biological
  stain
• It stains the nucleus blue

Question 14

What is gram staining?

Answer 14

It is used to enable bacteria to be seen with the light microscope.
It stains gram positive bacteria violet and gram negative bacteria red

Question 15

Detailed walkthrough of each step

Decribe the process of gram staining

Question 16

What is H&E stain?

Answer 16

H&E stain is a mix of two dyes:
* Haematoxylin (blue)
* Eosin (red)

Question 17

Decribe what each dye does

Answer 17

• Haematoxylin is a positively charged stain that reacts with negatively charged molecules in cells. (it stains acidic structurese blue)
  e.g. it reacts with nucleic acids.
  These stain blue
• Eosin is a negatively charged stain and so reacts with positively charged molecules in cells. (it stains basic structures pink, i.e. cytoplasm)
  e.g. it reacts with amino groups in proteins. These stain pink.

Question 18

What is a stage micrometer?

Answer 18

A type of microscope slide with a scale etched onto it

* Used to calibrate an ‘eyepiece graticule’.

Question 19

What is the eyepiece graticule?

Answer 19

Fixed scale inside the eyepiece lens. It must be calibrated for each objective lens using the stage micrometer.

Question 20

Covert your answer into µm

What is one eyepiece division equivalent to (in mm) in this diagram?

Answer 20

One small division on stage micrometer = 0.1mm
One eyepiece division:
= 2 divisions on stage micrometer scale
= 0.2 mm
= 0.2 x 1000 = 200 µm

Question 21

Different types of cells

Include examples

Relates to microscopy slides light micrographs

Answer 21

• Squamous cells e.g. wall of alveoli, lining of mouth
• Cuboidal cells e.g wall of kidney tubules
• Columnar cells e.g intestine lining
• Ciliated cells e.g lining of trachea
• Red and white blood cells e.g. lymphocytes

Question 22

Name a type of microscope derived from a light microscope

Answer 22

Light microscopes can be used with different techniques to enhance contrast in the image - laser scanning confocal microscope

Magnification - around x10000
Resolution - approx. 1000nm

Question 23

Decribe how it works?

Laser scanning confocal microscope

Answer 23

• A laser beam is passed through the objective lens to illuminate a single point in the specimen.
• Light photons released from the specimen are passed through a photomultiplier tube (PMT). This amplifies the light and converts it into a digital signal.
• Any unfocused light is filtered out by passing the light through a pinhole.
• The laser scans across the specimen point by point at a specific depth to build up an image pixel by pixel on the computer screen.
• Scanning at different depths enables a 3D image to be built up.

Question 24

What is an electron microscope?

Answer 24

Type of microscope that uses a a beam of electrons to obtain an image.

Question 25

Include description

State the two main types of electron microscopes

Answer 25

• Transmission electron microscope (TEM):
  e.g. used to view internal structure of cells, protein molecules, cytoskeletal filaments
• Scanning electron microscope (SEM):
  e.g. used to view the surface of cells or organisms.