Microbiology- Microscopy & Specimen Preparation
Acidic dyes -what? -examples
-Dyes w/ NEGATIVE charge that bind to positively charged structures such as... -cytoplasm -RBCs & -collagen -EX: Acid fuchsin, Eosin, rose bengal
Basic dyes -what? -examples
-Dyes w/ POSITIVE charge that bind to negatively charged structures such as... -proteins -nucleic acids & -surface of bacterial cells -EX: methylene blue, crystal violet, safranin
Scanning Electron Microscope (SEM) - what does it do? how does it do it?
-Electrons *reflect* from the surface of a specimen to create a 3D image of a specimen's *SURFACE FEATURES*
Transmission Electron Microscope (TEM)
-Electrons *scatter* when they pass through thin sections of a specimen -Denser regions = more e' scattering & appear darker -Good for looking at *INTERNAL STRUCTURES*
Electron Microscopy -what is it? -good for? -2 types?
-Electrons replace light as the illuminating beam -wavelength of e' beam is shorter than light (higher resolution!) -Mag. 100,000 -Good for fine details & viewing *VIRUSES* TYPES: Transmission & Scanning
Heat Fixation - What is it? what's preserved & what's destroyed?
-Fixates bacteria & archaea to a slide through the use of heat -Preserves *overall shape of an organism*, but internal structures are destroyed
Chemical Fixation
-Fixates larger, more delicate organisms to a slide through the use of chemicals -Preserves *internal structures* -used on delicate organisms (tissues) -more gentle method of fixation than heat fixation
Flagella Staining
-Flagella are too thin to be seen on microscope so they're coated with mordant so dye will stick to it & make it look thicker
Purpose of Dyes & Simple staining
-Make internal & external structures of a cell more visible by *increasing contrast w/ background*
Gram Staining -what is it? -what does it tell us?
-Most widely used *differential staining procedure* -Divides BACTERIA into 2 groups based on *differences in cell wall structure* (1) Gram positive (2) Gram Negative -Good for checking purity of a culture
Capsule Staining
-Negative stain - stain fluid around objects, but not the objects themselves -capsules end up colorless against a stained/dark background
Fixation -what is it? -2 types
-Preserves internal & external structures & fixes/secures them in place on a microscope slide -Organisms are usually killed,but you can see how they look (1) Heat fixation (2) Chemical fixation
Acid-Fast Staining -what is it? -what does it work with? -why?
-Specific type of differential staining useful for staining members of the genus *Mycobacterium* -Have a high lipid content in cell walls called mycolic acid -Are Hydrophobic which prevents most dyes from getting into cells -Have developed antibiotic resisitance
Differential Stains -what? -examples
-Use *more than one dye* to stain *features* -used to detect presence of absence of structures -*differentiates types of cells* -EXs: Gram Staining; Acid Fasting
Simple Stains - what is it? what's it good for? how do you make one?
-Use a *single dye* -Good for seeing cell size 1. make smear; air dry 2. fix it; stain it; remove excess 3. Look under microscope
Bright-Field Microscope
-a dark image against a brighter background -field is bright, image is dark
Resolution (& what's a major factor?)
-ability of a lens to separate/distinguish small objects that are close together (clarity) -wavelength of light is a major factor
Dark-Field Microscope -what is it? *What's it used for?* how does it work?
-field is dark, image is bright -image is formed by *light reflected or refracted by a specimen* -used to observe *LIVING, UNSTAINED* preparations (b/c they usually die when you stain them) -*BACTERIA* & internal structures in eukaryotes
Endospore staining (and how to do it)
-heated, double staining technique (stain w/ heat; counterstain) -bacterial endospore ends up 1 color, & the vegetative/dead cell ends up a different color
endospore
-stress/heat resistant organisms that form inside bacterial cells -resist boiling, UV radiation, & disinfectants -ex: Bacillus
numerical aperture (NA)
-the measure of light gathering ability -higher numerical aperture = better resolution
upper limit of resolution
0.2x
2 common features of dyes
1. Ability to bind to cells 2. All have a *chromophore group* - chemical group that makes the color
Why do microbiologists stain specimens on slides? (3 reasons)
1. Increases visibility 2. Clarifies fine features (ex: flagella hard to see unstained) 3. For preservation - stained slides keep longer -Unstained specimens are really hard to see on a bright background
4 steps to preparing a stained slide
1. Spread culture in thin film over slide 2. Air dry 3. Pass slide through flame to fix 4. Flood slide w/ stain; Rinse & dry
Steps in the gram staining process
1. Stain w/ Crystal violet - Primary Stain (everything will be purple now) 2. Add Iodine (a mordant) - (everything still purple) 3. Wash w/ Ethanol & Acetic Acid (G+ has peptidoglycan that holds on to crystal violet dye; G- does not, so dye washes off & they appear clear) 4. Secondary stain w/ Safranin (pink) - stains anything not stained by Crys. V. (So G- is stained pink)
Steps in the Acid-Fast Staining process
1. Use Primary Dye - Fuchsin (plus heat & phenol) - now everything appears pink 2. Wash w/ acidified ethanol 3. Counterstain w/ methylene blue
upper limit of magnification
1500x
Function of Mordants
To increase the binding of dyes to cells
refractive index
a measure of how greatly a substance slows the velocity/speed of light
higher numerical aperture =
better resolution
shorter wavelength =
better resolution (smaller number)
smaller # of resolution means....
better resolution/clearer view of object
resolution equation
d=(0.5y)/[nSin(theta)] d=resolution y=wavelength of light denominator = numerical aperture
shorter focal length =
greater magnification 4x has a LONGER focal length & WORSE magnification 100x has a SHORTER focal length & BETTER magn.
compound microscope
has 2 sets of lenses light microscopes
Increasing refractive index will do what to resolution and numerical aperture (NA)?
increase resolution & NA
increasing theta will do what ?
increase the numerical aperture
total magnification calculation
mag. of ocular lens x mag. of objective lens
parfocal microscope
one where an object in focus at 1 objective lens should be in focus at all other objectives without adjustment
parcentric microscope
one where an object stays centered from objective to objective without adjustment
changing the refractive index can change what?
resolution
focal length
the distance btwn center of lens and focal point
working distance
the distance from the lens to the surface of an object
focal point
where light rays are focused