2.1.1 (a-f) - Microscopy & Staining

Question 1

1. How is a dry mount prepared?
2. How is wet mount prepared?
3. How is a smear slide prepared?

Answer 1

1. Specimens are sectioned (cut into thin slices) and placed on the slide, place a cover slip on top
2. Suspend the specimen in a liquid. At an angle place the cover slip on (to keep bubbles out)
3. Prepare a wet mount, then use the edge of a slide to smear the sample and place a cover slip on top

Question 2

What is the purpose of staining and what are the 2 types?

Answer 2

• Some cells let light fall through, not enough contrast to see the ultrastructure
• Some stains are specific to different tissues/organelles
  
  • Such as acetic orcein stains chromosomes red

Differential Staining Techniques

Gram Staining

Acid Fast Technique

Question 3

How does gram staining work?

Answer 3

• When stained with iodine or crystal violet, gram positive bacteria shows up blue/purple under a microscope
• Gram negative bacteria will lose the original iodine/crystal violet stain, so they are counterstained and appear red/pink

Question 4

How does the acid fast technique work?

Answer 4

• Cells are dyed in carbolfuchsin
• They are washed in dilute acid/alcohol
• Mycobacterium are not affected, retain the carbolfuchsin dye and appear red
  
  • ​Other bacteria need a counterstain and appear blue as they lose the carbolfuchsin

Question 5

What are the pros and cons to using light or electron microscopes?

Answer 5

Light

• Cheap, user-friendly
• Can use living samples
• However, has limited resolution

Electron

• High resolution
• SEM shows 3D images
• TEM shows detailed ultrastructure
• Require trained operators
• Sample must be dead and dried (or an artefact may be present)
• In black and white

Question 6

What are the pros and cons to using laser confocal scanning microscopes?

Answer 6

• Can use living samples
• Relies on computers to piece together information from dots of light
  
  • ​The image is an interpretation rather than a real image

Question 7

Define

magnification

resolution

Answer 7

Magnification: How many times larger an image is than the actual object

Resolution: The ability to see individual objects/structures as separate entities

Question 8

How do you calibrate a microscope using an eyepiece graticule?

Answer 8

1. Put the Stage Micrometer and Eyepiece Graticule in place
2. Get the scale of the micrometer in clear focus
3. Align the micrometer scale with the eyepiece scale, take a reading.
4. 100 small divisions are 1mm, so 1 division is 10um.
5. Work out the magnification of the lens.
6. Replace the stage micrometer with a specimen and measure using the eyepiece graticule.