Microbial Microscopic Techniques Flashcards
UNITS OF MEASUREMENT
Microorganisms are measured in smaller metric units than we’re used to seeing; micrometers and nanometers.
A micrometer (μm) is equal to 0.000001 m (10-6 m). The prefix micro indicates that the unit following it should be divided by 1 million.
A nanometer, (nm) is equal 0.000000001 m (10 -9 m).
LIGHT MICROSCOPY
Refers to the use of any kind of microscope that uses visible light to view specimens
Compound Light Microscopy – A compound light microscope (CLM) has a series of lenses and uses visible light. The magnification is achieved when light passes from the illuminator through a condenser, which has lenses directing light rays through the specimen. The image is magnified again via the ocular lens (eyepiece). Most CLMs have several objective lenses including 10x, 40x, and 100x (oil immersion). Some CLMs can achieve magnification of 2000x w/ the oil immersion lens. Resolution is the ability of the lenses to distinguish fine detail and structure. Total magnification
DARKFIELD MICROSCOPY
A darkfield microscope is used for examining live microorganisms that are either invisible via light microscopy, cannot be stained by standard methods, or are so distorted from staining that their characteristics cannot be indentified. Instead of a normal condenser, a darkfield microscope uses a darkfield condenser that contains an opaque disc. The disc blocks light that would enter the lens directly. Only light that is reflected off (turned away from) the specimen enters the lens – the specimen appears light against a dark background.
PHASE-CONTRAST MICROSCOPY (PCM)
This is especially useful because it permits detailed examination of internal structures in living organisms. In addition, it’s not necessary to fix or stain the specimen – procedures that could distort or kill the microorganisms.
The principle behind PCM is based on the wave nature of light rays and the fact that light rays can be in phase – (their peaks and valley’s match) or out of phase. If the wave peak from one source coincides with the wave peak from another source, the waves interact to produce reinforcement (relative brightness). If the wave peak from one source coincides with the wave trough from another source, the rays interact to produce interference (relative darkness).
DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY
Like phase-contrast, DIC uses differences in refractive indexes to produce images. Uses two beams of light – each beam being separated or split by prisms; which does two things: 1. The specimens appear colored as a result of the prism effect and, 2. The resolution is higher than a PCM.
There is no staining required with DIC
FLUORESCENCE MICROSCOPY
Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (ultraviolet), and give off light at longer wavelengths (visible).
If the specimen doesn’t naturally fluoresce, it’s stained with one of a group of stains called fluorochromes
When microorganisms stained with a fluorochrome are viewed with an ultraviolet (or near ultraviolet) light source, they appear luminescent, bright objects against a dark background.
CONFOCAL MICROSCOPY
A fairly recent development in light microscopy. Like fluorescent microscopy, specimens are stained with fluorochromes so they will emit, or return light. In confocal microscopy one plane of a small region of a specimen is illuminated with a laser, which passes the returned light through an aperture aligned with the illuminated region. Each plane correspondes to a n image of a fineslice that has been physically cut from a specimen.
Because confocal microscopy uses a pinhole aperture, it eliminates the blurring that occurs from other microscopes resulting in an exceptionally clear two-dimensional images with improved resolution of up to 40%.
SCANNING ACOUSTIC MICROSCOPY
SAM – basically consists of interpreting the action of a sound wave sent through a specimen.
SAM is used to study living cells attached to another surface, such as cancer cells, artery plaque, and bacterial biofilms that foul experiment.
