Foundations in Biology Module 2.1 -2.3 Microscopy, magnification and calibration Flashcards
Microscopy
What did the cell theory that was developed state
Both animal and plant tissues are composed of cells
Cells are the basic unit of all life
Cells only develop from existing cells
What are the benefits of using a light microscope
Easily available
Relatively cheap
Can be used out on the field
Observes both living and dead cells prepared specimens
Coloured images
What do the objective lens and eyepiece lens do
Increase the magnification of the image and reduce chromatic aberration(distortion)
What is the drawback of light microscopes
Very low resolutions so viewing images at high magnifications aren’t helpful because they are blurred
What is a benefit of using a electron microscope
more detail of cell ultrastructure
What are the drawbacks of using electron microscopes
Very expensive
Can only be used in a carefully controlled environment in a dedicated space
Artefacts can be produced because of very complex sample preparation process
Vacuum required so only dead specimens can be used
Black and white images
What are the two types of electron microscopes
Transmission electron microscope(TEM)
Scanning electron microscope (SEM)
How does a TEM produce an image
Beam of electrons transmitted through specimen and focused to form image
How does a SEM produce an image
Beam of electrons are sent across surface of a specimen and reflected electrons collected
What is the maximum magnification of electron microscopes
x500 000
What is the maximum magnification of light microscopes
x2000
What is the maximum resolution of a TEM
0.5nm
What is the maximum resolution of a SEM
3-10nm
What dimension of images does SEM produce
3D image which shows valuable information about the appearance of different organisms
What are the 4 types of light microscope sample preparation
Dry mount
Wet mount
Squash slides
Smear slides
Method of Dry mount
solid specimens viewed whole or cut into thin slices with a sharp blade
Specimen placed on the centre of the slide
Coverslip placed on top of specimen
When solid specimen is cut into thin slices what is this process called
Sectioning
What are examples of Dry mount (whole)
hair, pollen, dust and insect parts
What are examples of Dry mount ( slices)
muscle tissue and plants
Method of Wet mount
specimen is suspended under a liquid and coverslip is placed over specimen at an angle
What are the examples of liquids used as an suspension for Wet mount
water and an immersion
Method of Squash slides
Wet mount prepared
Lens tissue used to gently press down coverslip
What is a potential prevention of damage to coverslip when producing a Squash slide
Squash specimen between 2 microscope slides
Squash slides a good technique for what samples
Soft samples
Examples of squash slides
root tip which is used to observe cell division
Method of smear slides
Use the edge of the slide to smear the sample
Thin, even coating created on another slide
Place coverslip over specimen
Example of smear slide
Blood
Specimens that are illuminated by white light like a light microscope, the images often have
low contrast
What limits the resolution of light microscopes
the wavelength of light and diffraction of light as it passes through sample
Definition diffraction
bending of light as it passes close to the edge of an object
Cytosol (cytoplasm) of cells and other structures are usually
Transparent
What does stains increase on cells
Contrast as different components within the cell take up stains to different degrees
What does an increase in contrast allow
components to become more visible so they can be identified
Method of preparing a sample for staining
Place sample on slide and leave to air dry
Heat-fixed by passing through a flame
Specimen adheres to microscope slide and will take up stains
Name two positively charged dyes
Crystal violet and methylene blue
What are the dyes crystal violet and methylene blue attracted to
negatively charged materials in cytoplasm resulting in staining of cell components
Name two negatively charged dyes
Nigrosin and congo red
What do the dyes nigrosin and congo red cause in the cell
repulsion of negatively charged cytoplasm
Name the technique is taking place when dyes stay out of cells, leaving them unstained, allowing them to stand out against the stained background
Negative stain technique
What does differential staining do
distinguishes between two types of organisms that would otherwise be hard to identify.
As well as differentiates between different organelles of a single organism within a tissue sample
Why is the gram staining technique used
Separate bacteria into two groups:
- Gram positive bacteria
- Gram negative bacteria
Method of the gram stain technique
- Crystal violet added to bacterial specimen on slide
- Iodine applied which fixes the dye
- Slide washed with alcohol
- Gram positive bacteria - retain stain and gram negative bacteria lose stain
- Safranin dye applied - making bacteria appear red
Gram stain technique: once slide washed with alcohol what happens to the two groups of bacteria
- Gram positive bacteria - retain the crystal violet stain
- Gram negative bacteria - have thinner cell walls so they lose crystal violet stain
What colour is crystal violet dye
Blue/purple under microscope
What colour is safranin dye
Red
What is safranin dye in gram stain technique
It is a counterstain
What is gram positive bacteria susceptible to and what this cause
Antibiotic penicillin - inhibits the formation of cell walls
Why is gram negative bacteria not susceptible to penicillin
They have thinner cell walls than gram positive cells
What is the use of the acid-fast technique
differentiate species of Mycobacterium from other bacteria
Method of acid-fast technique
- Carbolfuchsin dye carried into cells by a lipid solvent
- Cells washed with dilute acid-alcohol solution
Result:
-Mycobacterium not affected by acid-alcohol so retains the carbolfuchsin stain
-Other bacteria lose stain and are exposed to the methylene blue stain which is blue
What is the colour of the carbolfuchsin dye
Bright red
What are the 4 parts of the production process of pre-prepared slides
Fixing
Sectioning
Staining
Mounting
What happens in the fixing part of the production of pre-prepared slides
Chemicals, like formaldehyde are used to preserve specimens in as near-natural a state as possible
What happens in the sectioning part of the production of pre-prepared slides
Specimens are dehydrated with alcohols and then placed in a mould with resin/wax to form a hard block
The hard block is sliced thinly with a microtome
What happens in the staining part of the production of pre-prepared slides
Specimens often treated with multiple stains to show different structures
What happens in the mounting part of the production of pre-prepared slides
Specimens are then secured to a microscope slide and a coverslip is placed on top
What features make a good scientific drawing
- Title
- Magnification
- Sharp pencil for drawings and labels
- White/unlined paper
- At least half the paper provided
- Smooth/continuous lines
- No shading
- Clearly defined structures
- Correct proportions
- Label lines parallel to top of the page
- Label lines - no crossing and no arrow heads
Define magnification
how many times larger the image is than the actual size of the object viewed
Define resolution
ability to distinguish between two points which are close together on an object
What is the magnification formula
magnification = image size/actual size
Measurements from metres - nm
m x1000 = mm x1000= um x1000 = nm
nm/1000 = um/1000 = mm/1000 = m
Features of an eyepiece graticule
- Glass disc
- scale of 1-100
- no units
- remains unchanged under different objective lens
- Relative size of divisions increase as objective lenses increase
Feature of a stage micrometre
- microscope slide
- Very accurate scale in micrometre
- 100 divisions = 1mm
- 1 division = 10um
- 1/100 = 0.01mm
Calculation of 1 graticule division
no of micrometres/no of graticule division
How to calculate actual size of a specimen with after calibration
graticule division x magnification factor
Define artefact
A visibile structural detail caused by processing the specimen and not a feature of the specimen.
- found on both light and electron microscopy