Microscopy Flashcards
Key terms: Magnification
Magnification is the number of times larger an image appears to be compared to the actual specimen which enables further detail to be seen. Magnification is limited by the resolution of the microscope.
Total magnification of the specimen
total magnification of the specimen=eye piece magnification (x10) x objective magnification (x4,x10,x40)
Key terms: Resolution
Resolution is the ability to distinguish between 2 separate points (determines the clarity of the image)
Why does an electron microscope have higher resolution?
EM have higher resolution as they use a beam of electrons which has shorter wavelength.
Why do we preserve specimens?
-enables them to be cut into sections to observe under a microscope
-enables them to be treated with a variety of different stains to distinguish between different types of cells, living and dead cells and different chemicals or metabolic processes.
-enables different structures to be revealed
Function of eye piece on light microscope
-magnifies specimen
-can be dismantled to insert an eye piece graticule to enable specimens to be measured accurately
Function of barrel on light microscope
-passes light from objective lens to eyepiece
Function of turret
-holds objective lens
-rotates to enable selection of objective lens
Function of objective lens
-magnifies and resolves specimen
Function of stage
-supports slide in correct position at 90 degrees to illuminate source
-enables light to pass through specimen
Function of condenser
-focuses light from illuminator source onto specimen
Function of iris diaphragm
-controls the level of light reaching specimen
-best definition achieved with lower light intensity
function of substage illumination
-source of illumination
-blue light bulb can be used to use light of shorter wavelength to improve resolution
How to prepare a temporary slide for observing using a light microscope?
-fixation: use 70% alcohol
-staining: use few drops of appropriate differential stain
-mounting: cover with cover slip to exclude dust and air
Advantages of preparing a temporary slide
-rapid and simple procedure (no complex apparatus or skill required)
-can mount specimen in glycerine to prolong examination period
How to prepare a permanent slide for observing under a light microscope? 1
1) Fixation
-preserves specimen in lifelike condition
-minimises distortion
-chemicals such as alcohol or acetic acid are added to make proteins and nucleic acids insoluble, fixing them in position
Preparation of permanent slide 2
2) Dehydration
-removes traces of water from fixed material
-achieved by placing the specimen in increasing concentration of alcohol
Preparation of permanent slide 3
3) Clearing
-addition of xylol removes dehydrating alcohol
-ensures material is made transparent
Preparation of a permanent slide 4
4) Embedding
-supports material so it is firm enough for sectioning
-can be resin, plastic or wax
Preparation of a permanent slide 5
5) Sectioning
-use a microtome to cut fine slices embedded specimen
so that light can pass through the specimen
Preparation of a permanent slide 6 7
6) Differential staining
-improves contrast between different tissues and structures
-can be permanent or temporary
7) Mounting
-embeds and protects material
ensures material can be observed over a long period of time
Iodine-KI solution
-for plant specimen
-temporary stain
-colour is blue-black
-used in starch
Aniline sulfate
-for plant specimen
-temporary stain
-yellow colour
-used in lignin
Toluidine
-plant specimen
permanent stain
red/purple
lignin and cellulose
Eosin
-for plant specimen
permanent stain
red colour
used in cellulose
Methylene blue
-animal specimen
-permanent stain
-blue colour
-used in Nuclei reticulocytes
Leishman’s stain
-animal specimen
permanent stain
red pink blue
red blood cells, nucleus of white blood cells
Haematoxylin
-animal specimen
permanent
blue
nuclei of animal cells
Advantages of using differential stains
-most biological specimens are colourless and almost transparent so differential stains make it easier to observe tissues/cells/chemicals
-when observing plant tissue= allows observer to distinguish chemicals enabling the identification of different tissue types such as xylem vessels from phloem
-when observing animal tissue, it allows observer to distinguish between different types of white blood
cell
-improves contrast between structures
Advantages of LM
-low skill set needed by user
-can be transported to use in field work
-can observe living organisms
-relatively inexpensive so available for schools and colleges