Microscopy Flashcards
What are the two main types of microscopes
- Light Microscopes
- Compound light microscope - Electron Microscopes
- transmission electron microscopy
- Scanning electron microscopy
- Scanning tunneling microscopy
What is a Compound light microscope
A light microscope
- It is any microscope that uses visible light to observe specimens
Compound light microscope uses two lenses to observe specimens
1. Objective Lens
2. Ocular Lens
Objective lens
On a Compound Light microscope
located closest to specimen and magnifies the specimen (primary lenses that magnify the specimen - first to magnify whatever is on the slide)
- 10x-100x
Ocular Lens
Lens located within the eye piece of the compound light microscope
Magnifies the specimen 10x in eyepiece
Calculating total magnification equation
Compound microscope
**Total magnification **= objective lens magnification X ocular lens magnification
example:
ocular lens = 10x
objective lens = 100x
total magnification 10x100 = 1000
You have a microscope that has a 6nm resolving power, what do you see?
If a microscope has a 6nm resolving power than it can distinguish two points if they are atleast 6nm a part
Define Resolution and provide an example
The ability to distinguish fine detail and structure, and to distinguish 2 points a certain distance a part
ex) if a microscope has 6nm resolving power, it can distinguish two points if they are at least 6nm a part
In order for 2 objects to be seen as distinct from one another light must pass in between them
- the light that passes in between the objects must be of a short enough wavelength otherwise, they will be seen as one object
in order for two objects to be seen as distinct from one another, what must happen?
With resolution, it is the ability to distinguish 2 points a certain distance a part (not only for fine detail and structure), so light must past in between the two objects to be seen as distinct from one another
- the light that passes in between the object it must be of short enough wavelength, otherwise they will be seen as one object
What is the general principle of resolution?
The general principle of resolution states that the shorter the wavelength of light, the better the resolution will be
Electron microscopes
compare and contrast to light microscopes
uses beams of electrons instead of light
Like light of light microscopes, the electrons also travel in waves, but the waves are much shorter than light waves
- due to the electrons travelling in smaller waves = greater resolution
Electron microscopes also achieve a much greater magnification (as high as 500,000)
- They added ability of these microscopes allow us to view internal cell structures and viruses
Primary differences b/w the light microscope and the electron microscope to view small objects
A compound microscope can be used to view an object as small as 0.20µm
An electron microscope can be used to view objects as small as 0.20nm
Using the same magnification, an electron microscope will also provide an image w/ a better resolution than a light microscope
0.10mm is the
The smallest object visible with the human eye
mm = 10^-3
0.20µm
A compound microscope can be used to view an object as small as 0.20µm
0.20nm
An electron microscope can be used to view objects as small as 0.20nm
TEM stands for
Transmission electron microscope
What is TEM used for
used to examine internal cell structure
- electron beams do not penetrate the thick cell well which requires the cell to be cut in order to be viewed, this process is called thin sectioning - Stains must be used before
What microscope uses Thin sectioning and why?
What is thin sectioning?
Transmission electron Microscope (TEM) uses the technique thin sectioning because electron beams do not penetrate the thick cell well which require the cell to be cut in order to view the internal structure = Thin sectioning
- these thin sections must be stained before they can be viewed under the TEM
Uranium is an example of?
Is an example of a stain thats used to stain the thin sections of a cell before they can be viewed under the TEM
What is the function of stains of a TEM
Stains function to improve contrast in between different cell structures (to view the slices with just the electron beams is a poor penetration power b/c it doesnt show anything, so you have to stain with heavy metal (=amniotic number is high))
SEM
Scanning electron microscopy
Can only be used to view the surface of an object
- the specimen must be coated w/ a thin film of heavy metal
ex) gold
- Allows a wide range of magnifications to be achieved (15x-100,000x)
STM
Scanning tunneling microscopy
- This is the most powerful of the electron microscopes
it is used to visualize atoms
- uses thin metal probe to scan specimens revealing surface - irregularities such as bumps and depressions
What microscope is the most powerful of the electron microscopes
Scanning tunneling microscopy (STM)
You need to view the internal cell structure of a cell, what type of microscope would you use and why?
Transmission electron microscope
- uses thin sectioning and the thin sections are stained before they are viewed under the TEM (ex. uranium)
You need to view the surface of a cell, what type of microscope are you using
Scanning electron microscopy
- can only be used to view the surface of an object, so the specimen must be coated with a thin film of heavy metal
- allows a wide range of magnifications to be achieved (15x-100,000)
Clinical use of microscopes
They are used for diagnostics (whether if my paitent has something or no)
What must be used to make microorganisms visible through the microscope and why
stains because the microorganisms are colorless
- stains are composed of positvely and negatively charged ions and one type either positive or negative is colored called the chromophore
Chromophore
A type of stain thats either positively or negatively charged (ions)
Staining technique steps
- A thin film of material called a smear contains the microorganism of interest is ‘smeared’ on a slide
- The sample is then fixed by passing it through a flame (trying to dehydrate it thru glass so that when u rinse its not not going anywhere
- The stain is then applied to the sample
- The stain is then removed from the sample by rinsing
- Then the stained sample is now viewed under a microscope (allows you to make a diagnosis, and know whats going on)
What is the protocol of the staining procedure
When the stain is applied to the sample
How do stains works
Outer surface of bacteria carries a net negative charge
- The negative charge always attracts positive charge and repels negative charge and is this principle that stain manipulate
What principle do stains manipulate
The surface of bacteria carries a negative charge
- so stains manipulate the charge of the bacteria, where negative attracts positive and it also repels negative
Positive stain
these positively charged stains will adhere to the negatively charged bacterium
- the bacteria will appear the color of the stain, and the background will appear clear
ex. crystal violet
Crystal violet stain
Is a positively charged stain (=adhere to negatively charged bacterium)
- The negatively charged bacterium and appear the color of the stain and the background will remain clear
What are Negative stains and provide a function
The negative stain will be repelled by the negatively charged bacterium
- bacteria will appear clear and background is colored
- ex. Nigrosin
Nigrosin
a type of negatively charged stain
The stain will repel the negatively charged bacterium
- background will be colored and bacteria is colorless
Simple stains
Require no differentiation
- a single colored basic dye is used
- basic dyes have a positively charged color ion that binds to the organism making it appear in the color of the dye
- Sometimes a mordant is used (ex. iodine or vinegar) = to increase intensity of the stain
Where are they often used Mordants and provide an example
They are often used in simple stains (and the gram stain)
ex. iodine or vinegar will work to increase the intensity of a stain
Iodine or vinegar are examples of
examples of a mordant, which work to increase (bulken up) the intensity of stain
What are differential stains used for?
- Why are differential stains important?
- provide examples of differential stains
used to differentiate in between diff types of bacteria (ex. behave differently b/c of cell wall structure)
- stains react differently will different types of bacteria
Differential stains are very important for diagnostics
- the stains exploits differences in cell wall structure and composition
ex. the gram stain and the acid fast stain
what are the types of differential stains
The gram stain and the acid fast stain
The gram stain
A type of differential stain
- determines whether a bacterium is gram + or gram - (can differentiate it)
1) application of crystal violet (purple dye)
2) Application of iodine (mordant)
3) Alcohol wash (decolorization)
4) Application of safranin (counterstain)
you want to identify all bacteria that falls within the genus, Mycobacterium such as Mycobacterium tuberculosis and Mycobacterium leprae, what staining technique do you use?
A type of differential stain called the Acid fast stain (carbon fushcin and methylene blue)
Explain the Acid fast stain and what it is used to idenitfy
A type of differential stain
- binds strongly to bacteria that contain a waxy cell wall component - Mycolic acid
- The stain us used to identify all bacteria that fall within the genus Myobacterium such as Mycobacterim tuberculosis and Mycobacterium leprae (all Gram + have another layer = (waxy) Mycolic acid (= mycobacterium), which is hydrophobic - so you cant so a gram stain b/c it doesnt stick so you do an acid fast stain)
- The waxy cell wall of the mycobacterium retains the carbol fuschin (gram - stain) die that is added
- A counterstain with methylene blue leaves tissues and non-acid fast bacteria blue
Carbol Fuschin
Is the first dye that is added, which sticks to the waxy cell wall of the Mycobacterium in an acid fast stain
Methlyene blue
A counterstain that leaves the tissues and non-acid fast bacteria blue
any cell non acid fast = blue
Mycolic acid is
a waxy cell wall component on bacteria - Acid fast stain is used to identify bacteria (specifically,Mycobacterium) that contain mycolic acid
What are the types of special stains
Capsule stain, Endospore stain and flagella stain
Capsule Stain
A type of special stain
Reveals the presence of a thick polysaccaride layer outside of the bacterial cell (a capsule stain is a type of glycocalyx)
- capsule presence indicates a bacterium w/ increased virulence or increased ability to cause disease in comparison to the same bacterium that lacks a capsule
- The background is colored w/ a negative stain such as nigrosin which makes it black
- The bacterial cell is stained w/ a positive stain such as safranin making it colored pink (when safranin is used)
- the capsule does not take up dye and remain colorless appearing as a halo around the cell
Nigrosin
( used in a capsule stain) - blue-ish black
= negative stain that makes the background black, the bacteria repels the negative stain
Safranin in the capsule stain
(capsule stain)
= positive stain that makes the bacterial cell pink
What color does the capsule appear
Capsule stain
= colorless b/c it doesnt retain the dye = halo around the cell
explain why an Endospore stain is used and provide an example
A type of special stain
Recall: endospores are intracellular structures that make bacteria resistant to adverse conditions
- ordinary stains cannot penetrate the bacterial cell wall so special stains must be used to detect their presence
A primary stain w/ malachite green colors the endospore green
A counter stain with safranin colors the rest of the cell pink
an example of an endospore forming bacteria is Bacillus anthracis (gram + cell)
Why can’t we use ordinary stains on the bacterial cell wall
Ordinary stains cannot penetrate the bacterial cell wall so special stains must be used to detect their presence
Primary stain in an endospore stain
Malachite green colors the endospore green
Counterstain in an endospore stain
Safranin colors the rest of the cell pink
What type of staining technique can be used on Bacillus anthracis
Endospore stain can be used because Bacillus anthracis is an example of an endospore forming bacteria (and also a capsule stain if needed b/c they also have capsules)
Flagella Stain and what type of microscope
Recall: Flagella are extracellular bacterial structure used for motility
They too small to be seen under the light microscope w/o a stain
A mordant and stain together work to increase the thickness of the flagella (b/c they’re small and thin) so that they can be observed under the light microscope
