Lecture 3 - microscopy cultivation and pathogens

Define magnification, resolution.

• ability to enlarge an image

• the ability to distinguish two adjacent objects as distinct and seperate.

  • Limit of resolution for light microscope is about 0.2 μ m.

  • magnification of 1000x needed for 0.2nm diameter resolution

Different types of several types.

bright field, phase contrast, differential inferences contrast, dark field, fluroescence

What is the general structure of a microscope?

• Compound light microscope uses visibel light to illuminate cells

• The condenser focuses the light

• Objective lense magnifies (10-100x)

• Ocular lense magnifies (10-30x)

How do we calculate total magnification?

total magnification = objective magnification x ocular magnification

Explain what bright field scope is?

• When specimens are visualized because of differences in contrast (absorbing or scattering light differently) between specimen and surrounding.

• Pigmented microbes add contrast

What is staining and how is it done?

• staining increases the contrast for bright field microscopy and uses dyes that are organic compounds that binds to speciifc cellular materials.

What are basic dyes?

• positively charged, bind strongly to negatively charged cell components

• examples are methylene blue

• simple stains use dried cells

What is a differential stain? And what is an example?

• Render different kinds of cells in different colors.

• Gram stain is an example which causes differences because of cell wall structure

• Gram positive bacteria appear purple and gram negative appear pink.

Explain the mechanism behind the Gram staining.

•Gram-positive bacteria have a thick peptidoglycan layer that retains the crystal violet-iodine complex, staining them purple.

•Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane, which does not retain the crystal violet and instead takes up the counterstain (safranin).

What is phase contrast microscopy?

• improves image contrast of unstained live cells

• the phase ring amplifies differences in the refractive index af the cell

What is dark field microscopy?

• Light reaches the specimen from the sides.

• Only light scattered by specimen reaches the lens.

• Image appears light on a dark background.

• Better resolution than light microscopy.

• Excellent for observing motility (flagella).

What is fluroescence microscopy?

• Used to visualize specimens that fluoresce (emit light)

• Cells appear to glow on black background due to filters.

• Fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye such as DAPI.

• Widely used in clinical diagnostic microbiology and microbial ecology for enumerating bacteria.

What is Differential interference contrast (DIC) microscopy?

• uses a polarizer to create two distinct beams of polarized light (light in single plane)

• gives structures such as nuclei and bacterial inclusions a three-dimensional appearance

What is Confocal scanning laser microscopy (CSLM)??

• using computerised fluorescent microscope coupled with laser source to make a 3D image

• focus on single layers of specimen

• 3D image can be made

What is electron microscopy?

• use of electrons instead of visible light to image cells and structures

• electromagnets functioning as lenses

• operation in vaccum

• types: transmission/scanning electron microscope

What is transmission electron microscopy?

• Much greater resolving power (0.2 n m) than light microscope.

• Enables visualization of structures at the molecular level.

• Specimen must be very thin (20–60 n m) and stained with high atomic weight substances that scatter electrons well and improve contrast.

• Negative staining allows direct observation of intact cells/components. (Figure 1.29b).

• Electron cryotomography (cryo E T): T E M used to obtain three-dimensional images.

What is scanning electron microscope?

• Specimen is coated with a thin film of heavy metal (e.g., gold).

• An electron beam scans the object.

• Scattered electrons are collected and projected to produce an image. (Figure 1.29c)

• Even very large specimens can be observed.

• magnification range of 15–100,000× only surface visualized

What is asepctic technique?

collection of practices that allow preparation and maintenance of sterile (no living organisms) media and solutions

What did louis pasteur discover?

• discovered that living organisms discriminate between optical isomers

• alcoholic fermentation was biological

• diaproved theory of spontaneous generation

What did robert koch discover?

• link between microbes and infectious diseases

• identifies agents of TB etc.

• developed solid media for obtaining pure cultures of microbes

• etc.

Why did bacteria become excellent models of life?

Because they were easy to grow under controlled conditions.

How was the first tree of life made?

• Ribosomal RNA present in all cells made it possible to build the first tree of life.

• Woese discovered Archaea using this rRNA

• Discovered that evolutionary relationships can be revealed by rRNA analysis

What is a phylogenetic tree?

• depicts phylogeny (evolutionary history) of all cells

• clearly shows three domains

• Root is LUCA (last universal common ancestor)

• evolution along two paths to form Bacteria and Archaea

• Archaea later diverged to distinguish Eukaryafrom Archaea