Microscopy and staining Flashcards
Four main microscopes
light:
compound and confocal (laser scanning)
electron:
transmission and scanning
Magnification of light and electron microscopes
light - 2000x
electron - 500 000x
Advantages of light microscope (5)
Cheaper, smaller, portable Specimens living or dead Simple slide preparation Can see colour of sample (or stain) Doesn't require vacuum
Advantages of electron microscope (2)
Higher magnification
Higher resolution/ Very small resolving power
- images are black and white but can be coloured digitally
Resolution of a light microscope - compound
200nm
Resolution of a TEM
0.2nm (highest resolution of microscopes, so number is smallest)
Resolution of a SEM
5nm - often given as 0.2nm in exam questions, the same as TEM
Which microscope produces a 3D image?
Scanning electron microscope
Which microscope produces a virtual image using two convex lenses?
Compound light microscope
What is resolution? definition
The smallest distance between two particles/lines/points which allows them to be distinguished from one another
What happens to an image if magnification is increased but resolution isn’t?
As magnification increases, the limited resolving power gives blurred images
What is resolution limited/determined by?
Wavelength of the light
Objective length aperture (larger gives better resolving power)
Order of units of measurement
metre, centimetre, milimetre, micrometre, nanometre, picometre
Why is staining used?
Images tend to be low contrast as most cells do not absorb lot of light
Cytosol (aqueous interior) of cells and cell structures are often transparent
Gram stain technique:
What does it distinguish?
What substances are used?
What are the results?
-distinguishes gram positive and gram negative bacteria
-Crystal violet then iodine is applied. Slide is washed with alcohol.
Gram positive: retain stain so appear blue/purple
Gram negative: lose stain so stained with safranin dye (a counterstain). Bacteria appear red
Acid fast technique:
What does it distinguish?
What substances are used?
What are the results?
Differentiates different species of Mycobacterium
Liquid solvent carries carbolfuchsin dye into cells. Cells washed with dilute acid-alcohol solution
Mycobacterium retain carbolfuchsin stain and so are bright red.
What type of staining distinguishes between two types of organisms that would otherwise be hard to identify, or between different organelles of a single organism?
differential staining
Two positively charged and two negatively charged dyes? Which one stains the cell components?
+ crystal violet and methylene blue
- nigrosin and Congo red
Positively charged dyes stain cell components, negatively charged dyes stay outside of the cell
Four types of sample slide preparation
dry mount, wet mount, squash slides, smear slides
