Chapter 3

Observing Microorganisms Through a Microscope

Conversions

• 1 micrometer = 10-6 meters = 10-3 millimeter

• 1 nanometer = 10-9 meters = 10-6 millimeter

• 1000 nanometer = 1 micrometer

• 0.001 micrometer = 1 nm

total magnification

objective x ocular lens

NA: numerical aperture of lens

an objective lens with a higher value of NA produces a more highly resolved image

compound light microscope: ocular lens

eyepiece → remagnifies the image formed by the objective lens

compound light microscope: body tube

transmits the image from the objective lens to the ocular lens

compound light microscope: objective lenses

primary lenses that magnify the specimen

compound light microscope: stage

holds the microscope slide in position

compound light microscope: condenser

focuses light through specimen

compound light microscope: diaphragm

controls the amount of light entering the condenser

compound light microscope: illuminator

light source

Resolution

the ability of the lenses to distinguish two points or ability to distinguish fine detail and structure

Resolution limit of a compound light

0.2 micrometers (200 nanometers)

Resolving power

wavelength / (2 x numerical aperture of lens)

shorter wavelengths provide…

better resolution

Refractive index

measure of the light bending ability of a medium

light may refract after passing through a specimen to an extent that it…

does not pass through the objective lens

Immersion oil

used to keep light from refracting

Fluorescence Microscopy

• uses UV as a light source for illumination

• absorb UV light and emit longer wavelengths of visible light

• cells may stain bright yellow, green, or orange

Fluorescent-antibody (FA) technique

• aka immunofluorescence

• antibodies w/ a specific pathogen are tagged with fluorochrome

• “fluorescent antibodies applied to a microscope slide that contains a pathogenic microbe

• if pathogen is present, it will glow because antibodies will adhere to it

• rapid and specific detection of pathogens in a patient

Electron microscopy

• uses electrons instead of light

• shorter wavelengths = greater resolution

• used for images too small to be seen with light microscopes

• use electromagnetic lenses to focus electron beams

• two types: transmission electron microscopy and scanning electron microscopy

Transmission electron microscopy

• path of electrons is from top to bottom

• beam of electrons passes through ultrathin sections of a specimen → then through an electromagnetic objective lens → then focused by a projector lens

• specimens can be stained with heavy metal salts for contrast

• magnification: 10,000 - 10,000,000x

• limit of resolution: 0.2 nm

Scanning Electron Microscopy

• an electron gun produces a beam of electrons that scans the surface of a specimen

• secondary electrons emitted → transmitted to an electron collector → produces 3D image

• magnification: 1000 - 500,000x

• limit of resolution: 0.5 nm

Staining methods

used to increase contrast and visibility → aids in classification

Simple stain

highlights the entire microorganism to visualize cell shapes and structures

Differential stains

used to distinguish between bacteria

ex: gram stain and acid-fast stain

• surface of bacteria is negatively charged → color portion of dye is positively charge → attracts

• gram positive → turns purple

• gram negative → turns pink

Special stains

used to distinguish parts of microorganisms

ex: capsule stain, endospore stain, flagella stain

Ziehl-Neelsen Staining

• ACID FAST STAINING

• used to identify mycobacterium tuberculosis → this is the causative agent of TB