Overview: Unit 17AB is an assignment based assessment where learners will have to learn
and practise microscopy, learn the difference between SEM and TEM and another type of
microscopy method, practise via light microscope practicals, learn how to find actual sizes of
microorganisms, classify microorganisms, apply them into industries and assess their
reliability. Information is strictly AI based so take as prior knowledge.
Types of Microscopy apparatus
Type Description
SEM Scanning electron microscope uses a focused beam of electrons
to scan the surface of a specimen, this creates 3D imagery of the
surface. They have a general magnification of 20x - 1,000,000 x
with a resolution of 1-10 nanometres. Samples are prepared with
metal coating making them conductive.
TEM Transmission electron microscope uses a beam of electrons
transmitted through a thin specimen creating 2D imagery of the
cell ultrastructure. TEM offers a high resolution of 0.1
nanometres with a magnification range of 50x - 2,000,000x.
Samples often use ultra-thin specimens that are stained with
heavy metals
Cryo-EM Cryogenic electron microscopy uses biological samples that are
rapidly frozen, preserving their structure without using stains.
They are used to observe proteins and viruses in their hydrated
state. These microscopes operate at around -180 degrees
celsius as well as offering high resolutions to near atomic levels
(0.2-0.3 nanometres). They capture 3D structures using
computational image reconstruction.
CM Confocal Microscope uses lasers to scan specimens and
produce high resolving (200 nanometres) imagery of sample
layers. They use pinhole aperture to block out-of-focus light to
present optical slices through thick specimens, they are then
combined to create 3D images.They can magnify up to 2000x.
AFM Atomic force microscopy uses a probe (sharp tip) over the
specimen to measure tiny forces between tip and sample. This
then produces highly detailed 3D imagery at atomic level without
the use of light or electrons. AFM can reach a resolution less
than a nanometre, has a magnification of 10x-1,000,000x and
can be used on living samples. AFM does not use optical
magnification but uses topographical data measuring the surface
height at nanoscale.
, Describe oil immersion
You would notice that the highest objective lens (100x) is labelled “oil”, this is for a
microscopy technique known as oil immersion. Oil immersion increases resolution of an
image. You would apply a drop of immersion oil between the cover slip and the specimen,
the oil has a similar refractive index to glass, so the bending of light is reduced when it
passes from slide to lens. This allows more light rays to enter the objective lens which
increases numerical aperture and improves image resolution and brightness.
Describe Gram staining
One way of classifying bacteria is from gram staining. Gram staining is a common
technique that puts bacteria into gram-positive or gram negative based on the cell
wall composition. To do the technique, you first place the sample on the microscope
and then heat fix to kill the cells. Then you apply crystal violet and then wait for a
minute. Then you add iodine solution which forms a crystal violet-iodine complex
trapped inside the cell walls. You then decolourise with acetone to differentiate the
gram. And then finally to counterstain with safranin (this is because, the gram
negative was washed out of colour because of the acetone whereas the gram
positive retains their purple colour, when applied safranin the gram negative is
stained pink/red while the gram positive remains purple). The result can be seen
under the microscope. Gram positive bacteria are composed of a thick peptidoglycan
layer so they will be stained purple. Gram negative is composed of a thinner
peptidoglycan layer with an outer membrane so they will be stained pink/red.
Viroids are infectious agents. Describe what
Viroids are?
Viroids are the smallest known infectious agents, composed of circular short
stranded of single stranded RNA. Virioids are different to viruses due to the absence
of a protein coat or any DNA or protein-coding genes. Viroids are about 250-400
nucleotides long and are stable and compact due to the highly folded RNA
composition. Viroids are known to hijack the host’s RNA polymerase using the
viroids RNA as a template, their replication occurs via rolling circle mechanism
where long chains of RNA are cut and circulated by the host enzymes. Viroids infect
plants only which makes them plant pathogens, they can interfere with the plants
gene expression leading to stunted growth and for farmers; reduced yield. Common
diseases include Potato spindle tuber disease, chrysanthemum stunt disease and
coconut cadang-cadang disease.
and practise microscopy, learn the difference between SEM and TEM and another type of
microscopy method, practise via light microscope practicals, learn how to find actual sizes of
microorganisms, classify microorganisms, apply them into industries and assess their
reliability. Information is strictly AI based so take as prior knowledge.
Types of Microscopy apparatus
Type Description
SEM Scanning electron microscope uses a focused beam of electrons
to scan the surface of a specimen, this creates 3D imagery of the
surface. They have a general magnification of 20x - 1,000,000 x
with a resolution of 1-10 nanometres. Samples are prepared with
metal coating making them conductive.
TEM Transmission electron microscope uses a beam of electrons
transmitted through a thin specimen creating 2D imagery of the
cell ultrastructure. TEM offers a high resolution of 0.1
nanometres with a magnification range of 50x - 2,000,000x.
Samples often use ultra-thin specimens that are stained with
heavy metals
Cryo-EM Cryogenic electron microscopy uses biological samples that are
rapidly frozen, preserving their structure without using stains.
They are used to observe proteins and viruses in their hydrated
state. These microscopes operate at around -180 degrees
celsius as well as offering high resolutions to near atomic levels
(0.2-0.3 nanometres). They capture 3D structures using
computational image reconstruction.
CM Confocal Microscope uses lasers to scan specimens and
produce high resolving (200 nanometres) imagery of sample
layers. They use pinhole aperture to block out-of-focus light to
present optical slices through thick specimens, they are then
combined to create 3D images.They can magnify up to 2000x.
AFM Atomic force microscopy uses a probe (sharp tip) over the
specimen to measure tiny forces between tip and sample. This
then produces highly detailed 3D imagery at atomic level without
the use of light or electrons. AFM can reach a resolution less
than a nanometre, has a magnification of 10x-1,000,000x and
can be used on living samples. AFM does not use optical
magnification but uses topographical data measuring the surface
height at nanoscale.
, Describe oil immersion
You would notice that the highest objective lens (100x) is labelled “oil”, this is for a
microscopy technique known as oil immersion. Oil immersion increases resolution of an
image. You would apply a drop of immersion oil between the cover slip and the specimen,
the oil has a similar refractive index to glass, so the bending of light is reduced when it
passes from slide to lens. This allows more light rays to enter the objective lens which
increases numerical aperture and improves image resolution and brightness.
Describe Gram staining
One way of classifying bacteria is from gram staining. Gram staining is a common
technique that puts bacteria into gram-positive or gram negative based on the cell
wall composition. To do the technique, you first place the sample on the microscope
and then heat fix to kill the cells. Then you apply crystal violet and then wait for a
minute. Then you add iodine solution which forms a crystal violet-iodine complex
trapped inside the cell walls. You then decolourise with acetone to differentiate the
gram. And then finally to counterstain with safranin (this is because, the gram
negative was washed out of colour because of the acetone whereas the gram
positive retains their purple colour, when applied safranin the gram negative is
stained pink/red while the gram positive remains purple). The result can be seen
under the microscope. Gram positive bacteria are composed of a thick peptidoglycan
layer so they will be stained purple. Gram negative is composed of a thinner
peptidoglycan layer with an outer membrane so they will be stained pink/red.
Viroids are infectious agents. Describe what
Viroids are?
Viroids are the smallest known infectious agents, composed of circular short
stranded of single stranded RNA. Virioids are different to viruses due to the absence
of a protein coat or any DNA or protein-coding genes. Viroids are about 250-400
nucleotides long and are stable and compact due to the highly folded RNA
composition. Viroids are known to hijack the host’s RNA polymerase using the
viroids RNA as a template, their replication occurs via rolling circle mechanism
where long chains of RNA are cut and circulated by the host enzymes. Viroids infect
plants only which makes them plant pathogens, they can interfere with the plants
gene expression leading to stunted growth and for farmers; reduced yield. Common
diseases include Potato spindle tuber disease, chrysanthemum stunt disease and
coconut cadang-cadang disease.
