, Magnification
A microscope is an instrument that enables you to magnify an object
- describes how much bigger an image appears compared with the
original object
- ability to produce an image that shows fine detail (distinguish between
2 objects). Diffraction of light limits the resolution. Using a beam of electrons
rather than light, increases resolution as electron beams have a much shorter
wavelength
Image size(what you measure)=actual size x magnification
A scale bar shows the actual size
Centimetre (cm) Millimetre (mm) Micrometre (µm) Nanometre (nm)
X10 X1000 X1000
÷10 ÷1000 ÷1000
1. Measure the length of the scale bar
2. Convert the measurement the scale bar represents to the same unit as the
length of the scale bar. Divide the length (image size) by the representational
length (actual size) to find the magnification.
3. Measure the length of the structure you want to find the actual length of and
divide this by the magnification to find the actual length.
, Compound light microscope
Max mag= x2000 Max resolution= 200nm
Has 2 lenses:
Objective lens- placed near the specimen
producing a magnified image
eyepiece lens- magnified objective lens image
of specimen viewed through eyepiece lens.
Illumination is provided by a light underneath
the sample
Positives Negatives
•The objective/eyepiece lens •Structures closer
configuration allows for a higher together than 200nm will
magnification than in a simple light appear as one object-
microscope Ribosomes are too small to
•cheap & easy to use see.
•Portable •Stains are often needed
•Vacuum not required to make the cells visible
•Specimens can be living or dead
•Simple sample preparation
, Staining for light microscopy
In basic light microscopy the images have a low contrast as most cells do not absorb
a lot of light. Stains increase contrast as different components within a cell take up
stains to different degrees. To prepare a sample for staining it is first placed on a
slide & allowed to air dry. This is then heat fixed by passing through a flame. The
specimen will then adhere to the microscope slide & take up stains.
Positively charged dyes are attracted to negatively charged materials in the
cytoplasm e.g. eosin.
Negatively charged dyes are repelled by the negatively charged cytosol. These
dyes stay outside the cells, leaving the cells unstained, which then stand out
against the stained background.
Differential staining can distinguish between 2 types of organisms or organelles that
would otherwise be hard to identify.
Gram stain technique- used to separate bacteria in 2 groups – gram-positive &
gram-negative. Crystal violet is first applied to a bacterial specimen on a slide,
then iodine, which fixes the dye. The slide is then washed with alcohol. The gram-
positive bacteria retain the crystal violet stain. Gram-negative bacteria have
thinner cell walls, so lose the stain. They are then stained with safranin dye, which
is called a counterstain.
Acid-fast technique- used to differentiate species of mycobacterium from
other bacteria. A lipid solvent is used to carry carbolfuchsin dye into the cells. the
cells are then washed with a dilute alcohol solution. Mycobacterium are not
affected by the acid-alcohol & retain the carbolfuchsin stain which is bright red.
A microscope is an instrument that enables you to magnify an object
- describes how much bigger an image appears compared with the
original object
- ability to produce an image that shows fine detail (distinguish between
2 objects). Diffraction of light limits the resolution. Using a beam of electrons
rather than light, increases resolution as electron beams have a much shorter
wavelength
Image size(what you measure)=actual size x magnification
A scale bar shows the actual size
Centimetre (cm) Millimetre (mm) Micrometre (µm) Nanometre (nm)
X10 X1000 X1000
÷10 ÷1000 ÷1000
1. Measure the length of the scale bar
2. Convert the measurement the scale bar represents to the same unit as the
length of the scale bar. Divide the length (image size) by the representational
length (actual size) to find the magnification.
3. Measure the length of the structure you want to find the actual length of and
divide this by the magnification to find the actual length.
, Compound light microscope
Max mag= x2000 Max resolution= 200nm
Has 2 lenses:
Objective lens- placed near the specimen
producing a magnified image
eyepiece lens- magnified objective lens image
of specimen viewed through eyepiece lens.
Illumination is provided by a light underneath
the sample
Positives Negatives
•The objective/eyepiece lens •Structures closer
configuration allows for a higher together than 200nm will
magnification than in a simple light appear as one object-
microscope Ribosomes are too small to
•cheap & easy to use see.
•Portable •Stains are often needed
•Vacuum not required to make the cells visible
•Specimens can be living or dead
•Simple sample preparation
, Staining for light microscopy
In basic light microscopy the images have a low contrast as most cells do not absorb
a lot of light. Stains increase contrast as different components within a cell take up
stains to different degrees. To prepare a sample for staining it is first placed on a
slide & allowed to air dry. This is then heat fixed by passing through a flame. The
specimen will then adhere to the microscope slide & take up stains.
Positively charged dyes are attracted to negatively charged materials in the
cytoplasm e.g. eosin.
Negatively charged dyes are repelled by the negatively charged cytosol. These
dyes stay outside the cells, leaving the cells unstained, which then stand out
against the stained background.
Differential staining can distinguish between 2 types of organisms or organelles that
would otherwise be hard to identify.
Gram stain technique- used to separate bacteria in 2 groups – gram-positive &
gram-negative. Crystal violet is first applied to a bacterial specimen on a slide,
then iodine, which fixes the dye. The slide is then washed with alcohol. The gram-
positive bacteria retain the crystal violet stain. Gram-negative bacteria have
thinner cell walls, so lose the stain. They are then stained with safranin dye, which
is called a counterstain.
Acid-fast technique- used to differentiate species of mycobacterium from
other bacteria. A lipid solvent is used to carry carbolfuchsin dye into the cells. the
cells are then washed with a dilute alcohol solution. Mycobacterium are not
affected by the acid-alcohol & retain the carbolfuchsin stain which is bright red.
