Ch 2 & 3: Microscopy & Laboratory culture of microorganisms Flashcards
what are microscopes are used for
magnify an image and increase resolution
What is resolution
ability to distinguish 2 adjacent objects as distinct + separate
Whats the benefit of better resolution
more detail
What is the resolution of the human eye
~100 um
what lense does compound light microscope have
2 lenses - objective + ocular
in compound light microscope, where is the light source focus
focused on the specimen by a condenser
what does resolution depend on
wavelength and light gathering ability of the lens
what does immersion oil do
increase light that enters the lens
how do you find total magnification
objective magnification x ocular magnification
what is a bright field microscope
range of techniques used for illumination of samples in light microscopes
Why are staining dyes used
to increase contrast for bright-field microscope
what is the charge of most dyes
positive charges - theyre basic in nature
how do staining dyes work
dyes are positively charged, they bind to negatively charged cell components (nucliec acids, proteins etc)
what is a differential stain
stain different kinds of cells different colors
most common differential stain
gram stain
gram stain divided bacteria into what groups
gram-positive (purple)
Gram - negative (pink)
what do gram stains happen
due to differences in cell wall structures
where are acid fast stains used
used to identify acid fast organisms
What does mycolic acid do
attaches to cell wall surface, producing wax like hydrophobic coating
What color is the initial stain and counterstain
red and bluw
what does initial stain do
penetrates and is retained by mycolic acid (red) , the other cells destain and take up the blue counterstain
what is the best resolution with light microscope
~0.2uM
how are specimens visualized in a bright field microscope
slight differences in contrast between them and the surroundings
Can we see bacterial in a bright-field miscroscope
Typically difficult to see bacterial cells, unless the organisms are pigmented
some examples of staining dyes
methylene blue, crystal violet, safranin
What is a gram stain positive bacteria
thick cell wall
what does gram stain negative bacteria
thin cell wall
Types of light microscopy
phase contrast and dark field
In phase contrast microscope, how do different materials show light
by different amount
In phase contrast microscope, what is used to increase contrast between background and specimen
phase ring amplifier
In phase contrast microscope, what is the contrast like
dark cells on light background
in dark field microscope, how does light reach specimen
central portion of light beam is blocked, light only reaches from the sides
in dark field microscope, what is the contrast like
light cells on a dark background
in dark field microscope, what light reaches the lens
only light scattered by the specimen
what is fluorescence
emission of light of one colour after absorbing light of another colour
why does fluorescence occur
Occurs due to excitation of electrons to a high-energy state
when will cells fluorescence
due to naturally fluorescent substances
examples naturally fluorescent substances
Chlorophyll emits red fluorescence
if cells don’t have naturally occurring fluorescent dye, how can they get dyed
by getting stained with a fluorescent dye. Egm DAPI binds with DNA and emits blue light
How does a differential interference contrast or DIC
uses a polarizer and prism to generate 2 distinct beams of light that pass through the specimen then are recombines
How does differential interface contrast (DIC) make cells appear
3d appearemce
How does confocal scanning laser micrscopy work
using a computerized microscope coupled with a laser source to generate a 3D image
How does the computer in confocal scanning laser micrscope focus on specimen
can focus on single layers
how can different layers be viewed in confocal scanning laser micrscopy
they can be compiled to construct to 3D image
in confocal scanning laser micrscopy, how can you observe cells in different layers
in bacterial biolfilm
in confocal scanning laser microscopy, what is the resolution improved to
0.1 micrometeres
How does the electron microscope work
uses electrons instead of visible light (photons) to image cells and cell structures
what has shorter wavelengths, electrons or photons
electrons
what is the signficance of the length of wavelength and image resolution
shorter wavelength = better resolution
Can electrons penetrate
yes, but they are poor at it
How can you use an electron microscope to view the internal cell structures
you would need thin sections of the cell because a single cell is too thick
what are the 2 types of electron microscopes
- Transmission
- Scanning
How does the transmission electron microscope work
electron beam passes through ultrathin tissues sections of cell or very small specimens
In transmission electron microscope , what are samples first stained with
high atomic weight stains
Why is high atomic weight stain used in transmission electron microscope
allows electrons to pass more freely in areas that bind less. they absorb energy from the electron beam
what is the resolution of transmission electron microscope
0.2 nm
what is the fold improvement of transmission electron microscope
1000
How does the scanning electron microscope work
electron beam scans back and forth and the scattered electrons are collected and projected onto a monitor to produce an imag
What color are the images in electron microscope
black/white but fake colour can be added
how do you prep a specimen for electron microscope
coat it with thin film of heavy metal
what can you view of the object with electron microscope
the surface
what is required to culture microbes in lab
all nutrients required for growth
what are nutrienta
compounds of chem elements that can be used by the cell to support growth and metabolism
In what amounts are macronutrients needed
large
in what amounts are micronutrients needed
small
what are the essential elements
CHONPSSe
Carbon, hydrogen, oxygen, nitrogen, phosphate, sulfur selenium
What is culture media
nutrient solution used to grow microorgs in labs
what is a defined media
contains precise amounts of pure chemicals in distilled water where the exact composition is known
What are complex media made from
digests of microbial, animals or plants
in complex media, is the nutrient composition known
yes but not precisely
what are some examples of digests of microbes/animals/planst
casein - milk protein, beef extract, tryptic soy broth, yeast extract etc
what is selective media
contains compounds that inhibit the growth of some microorgs but not others
what is differential media
contains compounds or additives that allow microorgs to be distinguised by the appearance of colony or surrounding media
what are enriched media used for
nutritionally demanding microbes
what do enriched media contain
complex media plus additives like serums or blood
examples of enriched media
chocolate agar and blood agar
what does the blood agar distinguish between
hemolytic and non hemolytic bacteria
how do you prepare a culture media before use
by sterilizing it. by heating in an autoclave
how are liquid culture media solidified
by adding 1-2% agar and poured into sterile petri plates
what does a solid media do to the cells
immobilizes cells, allowing them to grow and form isolated masses called coloniess
what are colonies
isolated masses
