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