Microbio Microscopy

What does microscopes enable us to see?

Images that are too small to see with the naked eye

What tools or methods are used to achieve microscopy?

Using light, electron, or a scanning probe

What happens to light when it passes from one medium to another?

Light is refracted (bent) when passing through one medium to another

What is the refractive index?

A measure of how much a substance slows the velocity of light

What determines the direction and magnitude of bending?

The refractive indices of the two media forming the interface

What is the focal point?

(F) the specific point where light rays are focused

what is the focal length?

(f) the distance between the center of the lens and the focal point

what is the strength of a lens related to?

the focal length

What three things do microscopes use to get a clear image of a specimen/

Magnification, resolution, and contrast

what is the difference between objective lens and ocular lens/

Objective lens: found closest to the specimen

Ocular lens: used to view the specimen

what does the term parfocal mean on a microscope?

the focus remains when changing the objective lens

How do you calculate the maginifcation?

objective magnification x ocular magnification

What is the total magnification when using a 10x low power objective, a 40x high dry objective, or a 100x oil immersion objective with a standard 10x ocular lens?

Low power (10x objective): 100x total magnification.

High dry (40x objective): 400x total magnification.

Oil immersion (100x objective): 1,000x total magnification.

just multiply.....

what is resolution in microscopy?

the ability of a lens to distinguish small objects that are close

how can resolution be increased?

-resolution can be increased with immersion oil

what makes immersion oil effective?

-immersion oil has a RI similar to glass which is better than air

what types of microscopes are there?

-bright field microscope

-dark field microscope

-phase contrast microscope

-Fluorescence microscope

what kind of image does a bright field microscope produce and can samples be stained?

-produces a dark image against a bright background

-samples can be stained or unstained

what kind of image does dark field microscopes produce, how is the image formed and what is it used for?

-produces a bright image against a dark background

-image is formed by light being refracted or reflected by the specimens

-it is used to observe living unstained samples

how does phase contrast microscope turn light into a image? what components are used?

-phase shifts in light (deviated or undeviated) are converted into an image with contrast in amplitude/density

-a phase plate and condenser annulus are used to produce a cone/ring of light passing through the specimen and refocusing light.

what types of specimens are best viewed using a phase contrast microscope?

-living and unstained specimens

what kind of image does fluorescence microscope produce, and what are specimens stained with?

-bright image (dark background) of an object resulting from fluorescent light emitted by the specimens

-specimens are stained with fluorochrome, which differs in color and usage. Each tag/dye represents a specific wavelength. It can also examine natural fluorescing specimens (ex. P. aeruginosa)

What are the uses for Acridine orange, DAPI, FITC, and TRITC/rhodamine?

Acridine orange: Stains DNA.

Diamidino-2-phenylindole (DAPI): Stains DNA.

Fluorescein isothiocyanate (FITC): Often attached to DNA probes or to antibodies that bind specific cellular components.

Tetramethyl rhodamine isothiocyanate (TRITC or rhodamine): Often attached to antibodies that bind specific cellular components.

What are the general purposes of fixing and staining specimens?

-Helps visualize & identify specimen.

-Accentuates specific morphological features.

-Preserves specimens.

What is the difference between heat fixation and chemical fixation (including examples)?

-Heat fixation: Routinely used with microbes; preserves overall morphology but not internal structures and can distort the image.

-Chemical fixation: Used with larger, more delicate organisms (ex. protists); protects subcellular morphology.

-Examples: Ethanol, gluteraldehyde, formaldehyde.

What is the main purpose of using dyes, and what two components make up a dye?

-Purpose: They make internal and external cellular structures more visible and increase contrast with the background.

-Components: A chromophore group (the color component) and binding properties (such as ionic, covalent, or hydrophobic).

What is the difference in charge and targets between basic dyes and acidic dyes?

-Basic dyes: Have positively charged groups and bind to negatively charged molecules like nucleic acids, many proteins, and bacterial/archaeal cell surfaces (examples include methylene blue and crystal violet).

-Acidic dyes: In their ionized form, have a negative charge and bind to positively charged cell structures (examples include eosin and acid fuchsin).

What are the key features and uses of simple stains?

-Uses a single dye in a fast procedure.

-Used to determine the size, shape, and arrangement of bacteria.

What is the purpose of differential stains?

-Divides microorganisms into groups based on staining properties.

-Used to detect the presence or absence of structures (such as endospores, flagella, and capsules).

What is the most widely used differential stain, what groups does it divide bacteria into, and what are the four main reagents used?

-It is the most widely used differential stain.

-It divides bacteria into two groups: Gram-positive and Gram-negative.

The four reagents used are Crystal violet, iodine (mordant), alcohol (decolorizer), and safranin

What occurs during each of the four steps of the Gram stain procedure?

-Step 1 (Crystal violet): Applied for 1 minute; all cells stain purple because the dye is taken up equally well.

-Step 2 (Iodine): Applied for 1 minute as a mordant to enhance dye binding; cells remain purple.

-Step 3 (Alcohol): Used as a decolorizer for 10-30 seconds; Gram-positive cells remain purple, while thin-walled Gram-negative cells become colorless.

-Step 4 (Safranin): Applied as a counterstain for 30-60 seconds; Gram-negative cells appear pink or red, while crystal violet in Gram-positive cells repels the counterstain so they stay purple.

What is the purpose of an acid-fast stain, what does it target chemically, and what genus is it famously used for?

-Purpose/Target: Uses acidified alcohol for bacteria with cell walls rich in lipids composed of mycolic acids.

-Genus: Used for staining members of the genus Mycobacterium (such as M. tuberculosis and M. leprae).

what are the defining features of endospore stain?

Uses heat to force dye into the spore; used to identify spore-forming bacteria like Bacillus and Clostridium.

what are the defining features of capsule stain?

Used because not all dyes can penetrate the capsule (which is a network of polysaccharides).

what are the defining features of Flagella stain?

Used to determine motility; flagella are too thin to be seen with a bright-field microscope unless stained.

What replaces light as the "illuminating" beam in electron microscopy, and what effect does its wavelength have on the image?

-Illuminating beam: Electrons replace light.

-Wavelength and resolution: The wavelength of the electron beam is much shorter than light, which results in a much higher resolution.

What is the main benefit or application of using electron microscopy for studying microorganisms?

It allows for the study of microbial morphology in much greater detail.

How is the electron beam focused in a Transmission Electron Microscope (TEM), and what type of lenses are used?

-A beam of electrons is focused on the specimen by a condenser.

-Magnetic lenses are used instead of glass lenses.

How is the image formed in a TEM, and why do denser regions appear darker?

-Image formation: Some electrons scatter, but most travel through the specimen to form an image on a screen.

-Density effect: Denser regions in the specimen scatter more electrons, causing those areas to appear darker.

What condition must the transmitted electrons be under, and how must specimens be prepared for a TEM?

-Vacuum: Transmitted electrons are under a vacuum.

-Preparation: Specimens must be cut very thin after embedding in a matrix (such as wax, epoxy, or resin).

Can living specimens be viewed using a Transmission Electron Microscope (TEM)?

No, living specimens cannot be viewed using a TEM.

How does a Scanning Electron Microscope (SEM) create an image, and what kind of view does it provide?

-Mechanism: Uses electrons excited from the surface of a specimen (unlike TEM, which goes through it).

-Image type: Produces a realistic 3D image that allows for greater surface details.

What are the main limitations of using a Scanning Electron Microscope (SEM)?

-No living samples can be used.

-The sample must have a thin metal coating.

What can scanning probe microscopy visualize?

It can visualize molecules and atoms.

What is the magnification of a Scanning Tunneling Microscope, and how does it create an image?

-Magnification: 100 million X.

-Mechanism: It can view atoms on the surface of a solid and uses the up/down movement of a probe while maintaining a current to create a surface image (such as a DS DNA helix).

When was the Atomic Force Microscope (AFM) developed, and what was its main purpose?

-Developed in 1985.

-Created to improve on the limitations of the Scanning Tunneling Microscope (STM).

What are the key operational features of an Atomic Force Microscope regarding conductivity and probe movement?

-Conductivity: Works well on specimens that do not conduct electricity well.

-Probe mechanism: A sharp probe moves over the specimen surface, and the distance between the probe and the surface is kept constant.

What size ranges are covered by the light microscope, electron microscope, and scanning tunneling microscope on the scale?

-Light microscope range: Covers from about 100 µm down to 1 µm.

-Electron microscope range: Covers a much broader range from 100 µm down below 1 nm.

-Scanning tunneling microscope: Focuses on the smallest scale, down to 0.1 nm (individual atoms like a hydrogen atom).

What are some examples of biological structures found at the 10 µm, 1 µm, and 100 nm marks?

-10 µm: Red blood cells and white blood cells.

-1 µm: Rickettsia bacteria, E. coli,

Staphylococcus, Mycoplasma, and Poxvirus.

-100 nm: AIDS virus and Poliovirus.