Microscopy

Produce a magnified image of the specimen

Separate details in the image

render the details visible to the human eye or camera

Magnification- size of image or point

Resolution/ Resolving power- ability to distinguish two adjacent point

highest resolution in compound light microscope
- 0.2µm

produce a dark image against a brighter background

use in lab, requires stain

total magnification = product of the magnifications of the ocular lenses and the objectives lenses

dark-filed microscope

image is formed by light reflected or refracted by specimen

Produces a bright image of the object against a dark background

Used to observe living, unstained preparations

internal structures in eukaryotic microorganisms

identify bacteria such as Treponema pallidum

phase-contrast microscope

converts difference in refractive index/ cell density into detected variations in light intensity

some light rays from hollow cone of light passing through unstained cell slowed/ out of phase ( dark against bright background )

excellent way to observe living cells

stain is not necessary, view internal structure s of living organisms

fluorescence microscope

exposes specimen to ultraviolet, violet or blue light

Ultraviolet

permits greater resolution than conventional light microscope

has a shorter wavelength( 180-400nm)

Ultraviolet radiations are invisible

image are made visible by recording on a photographic emulsion, or by display on a television screen after pickup by an ultraviolet- sensitive television camera tube

specimens usually stained with flurochromes

shows a bright image of the object resulting from the fluorescent light emitted by the specimen

fluorescence

essential tool in microbiology

spme chemical substances absorbs the energy of ultraviolet waves and emit it as visible waves of greater length- fluorescent

cell contains natural fluorescent substances (eg. chorophyll ) or has been treated with fluorescent dye( eg. auramineO)

fluorochrome- labeled probes, such as antibodies, or fluorochrome dyes tag specific cell constituents for identification of unknown pathogens

microorganism appear as bright object against dark background

has applications in medical microbiology and microbial ecology studies

confocal microscope

confocal scanning laser microscopy ( CLSM) creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface

numerous applications including study of biofilms

compound microscope

image formed by action of ≥2 lenses

uses visible light as source of illumination
( 400nm to 700nm)

illuminator - source of light

condenser- to direct the light through the specimen

objective lenses- magnifies the specimens

ocular lenses- magnified the specimens

objective lenses magnification
-10X
-40X
-100X
-oil-immersion lens

ocular lenses magnification
-10X

Total magnification = multiply the objective lens magnification by the ocular lens magnification

metric system

micrometers, nanometers and angstroms

1 meter = 10^6 micrometer

1 meter = 10^9 nanometer

Cells are microscopic

Microscope is an instrument to observe microorganisms

Light microscopes

Electron microscope

wet mount

demonstrating motility or structure od microorganisms

smears

Preparing smears

thin suspension of cells placed on glass, no cover slip needed

air dry completely

fixation

fixation

preserved internal and external structures and fixed them in position

microorganism is killed and firmly attached to microscope slide

heat fixing

chemical fixing

staining

simple staining

single staining agent is used

revels basic cell shaped and arrangements

acidic dyes frequently used

basic dyes frequently used

differential staining

gram staining

gram positive

gram negative

acid-fast staining

very intensive decolourizer

useful for genus Mycobacterium

special staining

negative staining

preparing colourless bacteria against coloured background

visualize capsules surrounding bacteria

endospore staining

used for identifiying bacteria that can produce tough, dormant spores

flagella staining

Mordant and Carbolfuchsin applied to increase thickness of flagella

employs a beam of electron in place of light wave to produce the magnified image

wavelength= 0.01A

resolving power 100 times than light microscope

Scanning electron microscope (SEM)

strcutures smaller than 0.2µm

produces a realistic 3D image of specimen's surface features

magnification - 100X to 10 000X

resolving power- 20 nm

principle used: to study the surface features of cells and viruses

Transmission electron microscope (TEM)

structures smaller than 0.2µm

image produce- two dimensional

magnnification- 10 000X to 100 000X

resolving power- 2.5 nm

principal uses: to examine viruses or then internal ultrastructure in thin sections of cell

Electron cryotomography

rapid freezing technique provides way to preserve native state of structures examined in vacuum

images are recorded from many different directions to create 3D structure

provides extremely high resolution images of
-cytoskeletal elemetns
-magnetosomes
-inclusion bodies
-flagellar motors
-viral structures