chapter 2 observing the microbial cell

resolution

smallest distance by which teo objects can be seperate and still be distinguished

deflection

ability to determine the presence of an object

magnification

increase in the apparent size of an image to resolve smaller seperations between objects

wavelength of visible light

400-750

conditions for electromagnetic radiation to resolve an object

1. contrast between object and medium

2. wavelength smaller than the object

3. magnification

absoption

object blocks part of the light

reflection

wavelength of the light bounces off the surface

refraction

light bends when it enters a substance that changes its speed

scattering

small fraction of the incident light is scattered in all directions

magnification

requires the bending of light rays

empty magnification

magnification without increasing detail

immersion oil

increases the resolving power of the microscope by replacing the air gap between the immersion objective lens and cover glass with a high refractive index medium and reducing light refraction

total magnification =

magnifications of the ocular multiplied by that of the objective

wet mount advantages

observation of cells in natural state

wet mount disadvantages

little contrast between cell and background, sample may dry out quickly

fixation

cells are made to adhere to a slide in a fixed position

staining

cells are given a distinct color, increases the contrast

differential stain

stains one kind of cell but not another

gram positive

bacteria retain the crystal violet stain because of thick cell wall [purple]

gram negative

pink because of thin cell wall

first step of stainijg

crystal violet - primary stain

second step of staining

mordant: iodine

third step of staining

decolorizer alcohol

fourth step of staining

counterstain safranin

fluorescence microscopy

specimen absorbs light of a defined wavelength and then emits light of lower energy

fluorophore

flurescent chemical compound

trasmission electron microscopy TEM

electrons pass through specimen, reveal internal structure of cell and microbe

scanning electron microscopy

Electrons scan the specimen surface, revealing external features of cells and microbes

tomography

acquisition of projected images from different angles of a transparent specimen avoids need to physically slice the sample

four interactions of light with matter

absorption, scattering, refraction, reflection

increase resolution of bright field microscopy

use shorter wavelenth light, immersion oil, wide lens, higher numerical aperature

prinicples and uses of bright-field microscopy

cellular structure, stained specimen, live cells. generate a dark image of an object over a light background

basic staining

stain negatively charged molecules and structures, such as nucleic acids and proteins

acidic staining

stain postively charged molecules and structures, such as proteins

negative stains

stains background, not specimen

capsule stain

used to distinguish cells with capsuels from those without

flagella stain

used to view and study flagella in bacteria that have them

endospore stain

used to distinguish organisms with endospores from those without; used to study the endospore

acid-fast stain

used to distinguish acid-fast bacteria such as tuberculosis form non-acid fast cells.

how can flurophore cell be determined

chemical affinity, labeled antibodies, DNA hybridization, gene fusion reporter

fluorescence microscopy

• Principle: Fluorophores absorb light & emit longer wavelength.

• Uses: Detect proteins, DNA, microbes (FISH), gene expression (GFP).

confocal laser scanning microscopy

• Principle: Laser scans specimen → sharp 3D images.

• Uses: Thick samples (tissues, biofilms), subcellular detail

chemical imaging microscopy

• Principle: Mass spectrometry maps chemical distribution.

• Uses: Study metabolites, microbiomes, isotope-labeled molecules

dark-field microscopy

• Principle: Oblique light → scattered rays form bright image on dark background.

• Uses: Live/unstained cells, thin bacteria (Treponema), flagella

phase contrast microscopy

• Principle: Exploits refractive index differences → interference patterns.

• Uses: Live, unstained cells & organelles.

cryo-EM

• Principle: Flash-freeze sample, no staining, electron imaging.

• Uses: High-res protein/virus/organelles structures, tomography 3D.

Atomic force microscope

• Principle: Sharp tip scans surface → measures atomic forces.

• Uses: 3D cell surface, elasticity, molecular interactions.

x-ray crystallography

• Principle: X-rays diffract through crystal → atom positions mapped.

• Uses: 3D atomic structure of proteins, DNA, biomolecules.

EM electron microscopy

• Principle: Electron beams (short λ) give nm resolution; heavy-metal staining.

• Uses: Cell ultrastructure, viruses, morphology.

TEM

Electrons pass through → internal details. / Embed, slice thin (microtome), heavy-metal stain, copper grid.

SEM

Electrons scan surface → 3D surface images. / Dehydrate, coat with heavy metal, no slicing.