MGY277H1 Lecture Notes - Lecture 2: Glycan, Flagellin, Enzyme

126 views11 pages

For unlimited access to Class Notes, a Class+ subscription is required.

Unit 2 - Bacteria
Microscopy
Light Microscope Electron Microscope (1931)
Use visible light Use Electrons
Glass Lenses Electromagenitc lenses
Bare eyes Florescent screen electron micrograph
Magnify objects ~1000x Magnify in excess of 100,000x (0.3nm)
Easy to observe cell size, shape and
motility (alive)
Wavelength 1000x shorter than light M,
1000x more resolution Reveal fine
details of cell structure
Limit - to visible spectrum (400-
750nm),can have contract problem
Limit – Must be in vacuum (air molecule
would interfere with e-), B&W image,
cell must be dead, toxic stain
Cheap and easy to operate Expensive, complicated, large, high
maintenance
I.e. Bright field and Dark field I.e. Scanning EM and Transmission EM
Light Microscopy
Use lenses to bend light for focusing and enlarging a small object
Light is either directed onto OR passed through the sample been viewed
Refraction
Physical phenomenon when light rays change direction due to change in the
medium through which they travel
Lenses use refraction to focus light, and light bend only at the boundry of the
medium
Refraction Index measure of relative speed of light as it passes through a
medium
Refraction is the bases for light microscope, the bend is sharper when the
light hits at an angle
3 Basic parameter of all microscope: MRC
Magnification  The increase in the apparent size of the object compare to the size
of the actual object
Resolution  The ability to see objects or points as distinct instead of a blur that
combines them
Defined as the minimum distance at which 2 point can be distinguished as
individuals
Bigger is not always better, the resolution has to be good
Contrast the ability to see objects against the background
find more resources at oneclass.com
find more resources at oneclass.com
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 11 pages and 3 million more documents.

Already have an account? Log in
How to improve contrast?
Changing the microscope optic (Dark-field, Phase contrast *, Differential
interference *)
Staining the sample
oPositive staining (cells are darker)
oNegative staining (backgrounds darker)
oFluorescent stains
Bright-field microscope
Bright Field Microscope Most basic and common used
Ocular = 10x
Objective = 4x, 10x, 40x, 100x
Magnification
Condenser focuses light from the lamp but DOES NOT magnify object
Light passes through specimen from below into the condenser lenses
(provides a perfect white background)light pass through the objective
lenses gets magnified, and then gets further magnified through the ocular
lens
Resolution
0.2 micrometer max resolution
Physical limitation of visible light wavelength
Can see the shape of bacteria but not detail, Can NOT see viruses
The 100X lens requires oil to displace air and prevent light from missing
objective lens (Oil has the same refraction index as glass)
Light Microscope
Bright Field Dark Field
Light project towards specimen from
below and pass through lenses directly
Light project towards specimen at an
angle, then only light catched by
specimen enter object
Even illuminate the entire field of view Cell stand out from dark background
Wet mount procedure (slide +drop of liquid+ cover slide)
Electron Microscope
Transmission EM Scanning EM
Observe fine detail of cell structure Observe surface detail
(coat by metal stain)
Directing electron either pass through or
scatter
Electron is scanned over surface of
specimen and reflect back to viewing
chamber
Thin-sectioning allows observation of
internal details, but it will distort cells
Relative large specimens can be views
3D effects
Dark areas = dense portion
find more resources at oneclass.com
find more resources at oneclass.com
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 11 pages and 3 million more documents.

Already have an account? Log in
***Choice of microscopy techniques must balance cost and effort Vs. Potential
benefit and the type of data needed by the researcher
Stains
Steps to Staining
1. Fixation  a process of treating/killing cells to preserves cell structure,
because many cells fall apart during staining procedures
a. Done by denature protein to form chemical bond and make cells stuck
in place
b. Killing cell can allow them to better accept stains
2. Permeabilizaiton disrupt the cell membranes to allow entry of the stain
into the cell (NOT necessary if the stain is meant to bind the outside cell
surface)
a. Often use a detergent or organic solvent
b. Act as a double safety if fixation didn’t work well
3. Mounting step of attaching the sample to the slide surface (or cell can grow
on the slide directly)
4. Staining  after fixation and mounting, immerse the compound into
coloured dye
5. Addition of mordant  a chemical that combines with the primary stain to
make it insole so it is less likely to wash away (forms insoluble colour dye)
6. Washing wash away the excessive dye
7. Counterstaining (Optional)
Simple Staining involved one dye
Differential staining distinguish different types of bacteria
Heat-smear  a method to fix specimen by passing it over the flame before staining
it which gives better contrast to BF microscopy allow
Dyes and Stain
Basic Dyes Acidic Dyes
Most commonly used, stain the cell Stain the background NOT cell (cells are
clear and white)
Positive Charge Negative Charge (won’t stick to cell)
Attracted to negatively charged cellular
components (bacteria surface)
Not attached to cellular components
Use the Heat-Smear method Avoid Heat-smear = Avoid cell distortion
Methlene blue / crystal violet Can be done as wet mount
Gram Stain (stain inside the cell, NOT cell wall)
Developed by Dr. Hans Christian Gram
Most commonly used stain for bacteria
Distinguished Gram+ve from Gram-ve bacteria (fundamental difference in
cell wall composition)
find more resources at oneclass.com
find more resources at oneclass.com
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 11 pages and 3 million more documents.

Already have an account? Log in

Get access

Grade+
$10 USD/m
Billed $120 USD annually
Homework Help
Class Notes
Textbook Notes
40 Verified Answers
Study Guides
1 Booster Class
Class+
$8 USD/m
Billed $96 USD annually
Homework Help
Class Notes
Textbook Notes
30 Verified Answers
Study Guides
1 Booster Class