Chapter 3 Tools of the Laboratory: The Methods for Studying Microorganisms Flashcards
ability to enlarge objects extent of enlargement is the
Magnification
ability to show detail
Resolving power
The objective lens forms the magnified ___________
real image
The real image is projected to the ocular where it is magnified again to form the ____________
virtual image
______________ of the final image is a product of the separate magnifying powers of the two lenses
Total Magnification
The capacity to distinguish or separate two adjacent objects and depends on
The wavelength of light that forms the image along with characteristics of the objectives
Resolution
of lens ranges from 0.1 to 1.25
Shorter wavelength and larger numerical aperture will provide better resolution
Numerical apertur
most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens
Bright-fiel
brightly illuminated specimens surrounded by dark field; used for live and unstained specimens
Dark-field
transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures
Phase-contrast
Modified microscope with an ultraviolet radiation source and filter.
Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence
Useful in diagnosing infections
Fluorescence Microscope
Uses a laser beam of light to scan the specimen.
Integrates images to allow focus on multiple depths or planes.
Scanning Confocal Microscope
Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds
Electron waves are 100,000 times shorter than the waves of visible light
Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength
Magnification between 5,000X and 1,000,000X
Electron Microscopy
transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.
Transmission electron microscopes
provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.
Scanning electron microscopes
allow examination of characteristics of live cells: size, motility, shape, and arrangement
Wet mounts and hanging drop mounts –
are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.
Fixed mounts
Dyes are used to create contrast by imparting color
Staining
cationic, positively charged chromophore
Basic dyes
surfaces of microbes are negatively charged and attract basic dyes
Positive staining
anionic, negatively charged chromophore
Acidic dyes
microbe repels dye, the dye stains the background
Negative stainin
one dye is used; reveals shape, size, and arrangemen
Simple stains
use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)
Differential stain
reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains
Structural stains
If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells— a colony. A colony consists of one species.
Isolation
If a single species is growing in the container, you have a pure culture but if there are multiple species than you have a mixed culture.
Check for contaminants (unknown or unwanted microbes) in the culture.
Inspection
Cell and colony morphology or staining characteristics
DNA sequence
Biochemical tests to determine an organism’s chemical and metabolic characteristics
Immunological tests
Ways to Identify a Microbe:
liquid, semisolid, and solid
Physical state
synthetic (chemically defined) and complex
Chemical composition
general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration
Functional type
broth; does not solidify
Liquid
contains solidifying agent
Semisolid
firm surface for colony formation
Contains solidifying agent
Liquefiable and nonliquefiable
Solid
The most commonly used solidifying agent
Solid at room temperature, liquefies at boiling (100oC), does not re-solidify until it cools to 42oC
Provides framework to hold moisture and nutrients
Not digestible for most microbes
Agar
liquid medium containing beef extract and peptone
Nutrient broth
solid media containing beef extract, peptone, and agar
Nutrient agar
contains pure organic and inorganic compounds in an exact chemical formula
Synthetic
contains at least one ingredient that is not chemically definable
Complex or nonsynthetic
grows a broad range of microbes, usually nonsynthetic
General purpose media
contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes
Enriched media
contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes
Selective media
allows growth of several types of microbes and displays visible differences among those microbes
Differential media:
contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria
Reducing medium
– contains sugars that can be fermented, converted to acids, and a pH indicator to show this reaction
Carbohydrate fermentation medium
