Microbiology CH. 3 Observing Microorganisms Flashcards
Microorganism
too small to be seen with unaided eye
microscope
derived from “micro” — small
greek word “skopos”: to look at it
antoni van Leeuwenhoek
invented microscope
when was the first clear mention of microorganisms and their discovery
1676
describe Leeuwenhoek microscope
self-made
hand-held
single lens
magnified 100X
drawings indicative of species are still known to be present in materials he examined
3 types of microscopes
light; electron; scanned probe (diamond/metals)
what does light microscopy refer to ?
any microscope that uses visible light
what are the several types of light microscopy
compound light
darkfield
phase-contrast
differential interface contrast (DIC)
Fluoresce
confocal
how do you find out the total magnification of a compound light microscope
total magnification is calculated by multiplying the magnification of objective lens by the magnification of ocular lens
ex. objective lens 40x
ocular lens 10x
40x10=400x
what are some components used with the compound light microscope
resolution, refractive index, and immersion oil
resolution def?
resolving power —- lenses ability to distinguish fine details of structure
refractive index ?
measure of light-bending ability of a medium
immersion oil def?
reduce light loss between slide and the lenses
darkfield microscopy
uses special condenser with opaque discs that block light from entering objective lens directly
what does darkfield microscopy show
light silhouette of organism against dark background
what is darkfield microscopy most useful for
it is most useful for detecting presence of extremely small organisms
what does darkfield microscopy examine
unstained microorganisms suspended in media
ex. paramecium
what is phase-contrast microscopy
brings direct reflected/diffracted light rays together (in phase) to form image of specimen on ocular lens
what does phase-contrast microscopy allow
detailed observation of living organisms internal structures
what does phase-contrast microscopy not require
staining
how does differential interface contrast microscopy (DIC) work
uses 2 beams of light separated by a prism
what kind of image does DIC microscopy provide
a colored 3 dimensional image of object being observed
what does DIC microscopy allow
detailed observations of living cells
what does DIC microscopy not require
staining
what is Fluorescence Microscopy FM
specimens are stained with fluorochromes and viewed through compound microscope using ultraviolet light source
how do microorganisms appear on FM microscope
bright objects against a dark background
ex. T. pallidum
what is FM microscopy primarily used in
diagnostic procedure called fluorescent-antibody (FAb) technique
or
immunofluorescence to detect and identify microbes in tissues or clinical specimens
what is confocal microscopy
uses laser light to illuminate one plane of specimen at a time
what type of image is produced in confocal microscopy
2 and 3 dimensional images of cells for biomedical applications
what is electron microscopy EM
uses beam of electrons instead of light
what is used instead of glass in EM microscopy to focus, illuminate, and magnify
electromagnets
what processes in EM microscopy does electromagnets use to control focus, illuminate, and magnify
transmission/scanning EM
what does Transmission EM examine
viruses or internal ultra structure in thin sections of cells
how does transmission EM work
electrons pass through the specimen
what is the magnification range on transmission EM
10,000X - 100,000X
what type of image is produced in transmission EM
2-dimensional image
what is scanning EM
e-beam (electron beam) is directed at intact specimen from the top rather than passing through sections
electrons leaving the surface of specimen (2ry e-) are viewed on television like screen
what is scanning EM used to study
surface features of cells and viruses
what is the range of magnification of Scanning EM
1000x-10,000x
what are the 2 processes used in scanned-probe microscopy
scanning tunneling and atomic force
what is scanning tunneling in scanned-probe microscopy
uses thin metal probe that scans specimen and produces an image revealing bumps and depressions of the atomic on the surface of the specimen
what is atomic force in scanned-probe microscopy
uses a metal and diamond probe gently forced down along the surface of the specimen
what type of image does atomic force in scanned probe microscopy produce
3-dimensinal image
what are the 3 different staining techniques
simple, differential, and special
what are stains
salts composed of a positive (NA+) and a negative ion (Cl-)
sodium chloride
Chromophore
part of molecule responsible for stain color
bacteria (neg charged) and pos ion of chromophore (basic dye ) will stain it
What the common basic dyes with positive charges
crystal violet
methylene blue
safranin
malachite green
what are the common acidic dyes with negative charges
eosin
carbolfuchsin
nigrosin
what are simple stains
aqueous or alcohol solution of basic dyes methylene blue, crystal violet, and safranin
what is the purpose of simple staining
visualize shapes and arrangements of cells
what is mordant used for
improve bonding between the stain and the specimen
what are differential stains
react differently with types of bacteria
what are the 2 most common differential stains
gram stain and acid-fast stain
when was gram stain used and who invented it
in 1884 by Hans Christian Gram
what is the most important stain used in bacteriology
gram stain
what does gram stain do?
divides bacteria into 2 groups
Gram (+) and Gram(-)
how does gram stain and acid fast stain differentiate bacteria
differentiate according to bacteria reactions to the stain
what does gram stain procedure use
purple stain (crystal violet)
iodine (mordant)
alcohol decolorizer
red counterstain(safranin)
what is the application process of gram stain
application of crystal violet (purple dye)
application of iodine(mordant)
alcohol wash (decolorizer) 95% alcohol or acetone
application of safranin (counterstain)
what does application of safranin (counterstain) provide in gram stain
provides contrast to the 1ry stain
what is the gram stain mechanism
crystal violet-iodine crystals form in cell
gram positive def?
alcohol dehydrates peptidoglycan
CV- 1 crystals do not leave
gram negative def?
alcohol dissolves outer membrane and leaves holes in peptidoglycan
CV-1 ashes out
What do the results look like in gram stain
Gram (+) purple
Gram (-) red
what determines the difference of bacteria in gram stain
structure of cell wall
gram(+) thick cell wall
gram (-) thin cell wall
what are acid-fast microbes members of
genera
what does mycobacterium and nocardia do in acid-fast stain
retain carbolfuchsin after acid alcohol decolorization and appear red
non-acid fast microbes take up which counterstain and appear what color
take up methylene blue counterstain and appear blue
what are special stains used for
used to color and isolate specific parts of microorganisms
which microorganisms are special stains used to color and isolate
endospores
flagella
capsules
what is the process of negative staining for capsules
microbiologist mix bacteria in solution containing fine colloidal suspension of colored particles (india ink or nigrosin )
and then stained with simple stains of safranin
what do capsules not accept and how do they appear
don’t accept most stains and appear as unstained halos around bacterial cells and stand out against a dark background
ex. K pneumoniae
what is the process of endospore staining(specials staining)
Schaeffer Fulton endospore stain uses malachite green (1ry stain)
heat/wash
add safranin (counterstain) to stain portions of the cells other than endospores
ex. bacillus cereus
what is the process of flagella staining (special staining )
used a mordant and the stain carbolfuchsin to build up the diameters of flagella until they become visible under the light microscope
the mordant intensifies the stain or wats a structure to make it thicker and easier to see after it is stained
ex. salmonella typhosa
what can a mordant do for flagella
because flagella can’t be normally seen the mordant can be used to increase the diameter of the flagella before it is stained
