Microbio 2 - Microscopy

Define magnification

The extent to which the image of an object is larger than itself

Define resolution or resolving power

The degree to which details are retained in a magnified image; the ability to distinguish between two points

Resolution = R = 0.5 λ/ NA

λ = wavelength of light

NA= numerical aperture (The light gathering ability of the lens)

What type of microscope is most common in general microbiology labs

bright field microscope

how is light utilized with a bright field microscope?

light is transmitted through the specimen

How is the contrast between the background and the cells generated with birght field microscopy?

absorption or scattering of light

Only cells with what are easily seen with a bright field microscope

natural pigments

is staining needed with bright field microscopy?

Staining of the cells is generally required

What type of bacteria are simple staines used for?

Generally used for all bacteria

T or F: Stains are generally uncharged molecules

False

Are basic dyes cationic or anionic

cationin (positively charged)

What do basic dyes bond to?

negatively charged cell components

What are examples of negatively charged cell components?

• polysaccharides

• Nucleic acids

• Phospholipids

Acidic dyes are positively or negatively charged?

anionic ( negatively charged)

What do acidic dyes bind to?

Bind to positively charged cell components

What are examples of positively charged cell components?

• proteins

• amino groups

Are basic or acidic stains more commonly used and why?

Bacterial cell surface carries a negative charge, therefore basic stains are most commonly used for the most simple staining methods

What is the purpose of a differential stain?

Divides a mixture of bacteria into two groups based on a unique property

A Gram stain is an example of a _____ stain

differential

What is the difference in the cell wall between a gram-positive and gram-negative cell?

The cell wall is thick in a gram-positive cell while it is thin in a gram-negative cell

What is the difference with the surface area hit by light in a bright field vs dark field microscope?

• In the bright field microscope the whole area receives light

• In the dark field microscope light is only on the edges

The source light reaches the eye in _____ field microscope

bright field

In dark field microscopy the _____ light reaches the eye

scattered

In dark field microscopy the only light which reaches the specimen comes from the ____

side

In dark field microscopy how does the light reach the objective lense?

The only light which enters the objective lens is that which is scattered by the specimen

How do the cells appear against the background in dark field microscopy?

cells are bright against a dark background

Do you need to stain cells for fark field microscopy?

No

Why do you not need to stain cells for dark field microscopy?

cant see colors/stains

Does bright field or dark field microscopy provide better resolution?

dark field

(provides better resolution so we can see smaller cells or structures)

Does phase contrast microscopy require a stain?

no stain required

What does phase contrast microscopy allow you to visualize?

it allows for visualization of live cells

How does a fluorescence microscope work?

3. Front: How are fluorescent dyes and tagged proteins used in fluorescence microscopy?
Back: Fluorescent dyes stain specific cell structures, while fluorescently tagged proteins allow specific proteins to be localized within cells.

Light of one wavelength excites the specimen, causing it to fluoresce and emit light at a different wavelength.

What is autofluorescence?

When cells or cellular components naturally fluoresce without fluorescent staining.

How are fluorescent dyes and tagged proteins used in fluorescence microscopy?

Fluorescent dyes stain specific cell structures, while fluorescently tagged proteins allow specific proteins to be localized within cells.

How is the image created by transmission electron microscope?

The electrons that pass through the sample are collected to make the image

How does a scanning electron microscope (SEM) produce an image?

It collects electrons scattered back from the sample’s surface to produce an image.

How does atomic force microscopy work?

Tiny stylus is scanned across and just above the surface of the sample

How does an atomic force microscope (AFM) produce an image?

Repulsive forces between the stylus and sample cause the stylus to deflect up and down; these deflections are recorded to create a surface contour map.

What are the advantages and limitations of atomic force microscopy?

Provides extremely fine surface detail, but can be difficult to use with live, sticky, or flexible samples.