Chapter 2 microscopes Flashcards
How do light waves react with materials
By being reflected, absorbed, or transmitted
when does reflection occur
when a wave bounces off of a material
When does absorbance occur
when a material captures the energy of a light wave
When does transmission occur
when a wave travels through a material, like light through glass
what is transmittance
the process of transmission
What is diffraction
when light waves interact with small objects or opening by bending or scattering
When is diffraction larger
when the object is smaller relative to the wavelength of the light
When does refraction occur
When light waves change direction as they enter a new medium
What is the refractive index
the extent to which a material slows transmission speed relative to empty space
(the extent in which it bends)
What happens when there are large differences in refractive indices of two materials
a large amount of refraction when light passes from one material to the other
What is the clarity of the image
resolution
what is resolving power
the distance at which it can still distinguish between two separate points that are close together
what is contrast
the difference between two or more parts of an object (light and dark areas of microbe)
what does refraction refer to
light bending as it passes through a substance such as air, water, gas and oil
What happens when light is refracted away from the lens
it is lost and the image loses resolution
when is immersion oil used
with the highest magnification of of the microscope
why does the 100X objective require immersion oil
It is so close to the side that when light passes from the slide into the air it gets refracted and is not captured by the lens
how does immersion oil help
it has a similar refractive index to glass somber light is captured.
Who first saw the cell through a microscope
Robert hooke
what kind of microscope did van Leeuwenhoek use
simple microscope (one lens)
what kind of microscope did Hooke use
compound microscope (objective and ocular lens)
Where is the ocular lens in a binocular compound microscope
Inside the eye piece
what is the interpulpillary distance
distance between pupils adjusting allows both eyes to focus together on the image
what is the objective lens
the lenses that are changed to increase magnification
what is total magnification
the sum of magnification from the objective and ocular lens
how do you calculate the total magnification
power of ocular lens is multiplied by the power of the objective lens.
ocular is usually 10X
what kind of microscope did Galileo use
compound microscope
what kind of microscope did Leeuwenhoek use
simple microscope
what did Robert Hooke observe through his microscope
he looked a sample of cork and was the first to observe cells
who invented the light microscope
Joseph Jackson lister
what are the types of light microscopes
Brightfield microscopes
dark field microscopes
phase contrast microscopes
differential interference contrast
fluorescence
confocal scanning laser
two-photon microscopes
describe the brightfieqld microscope
coumpound microscope with two or more lenses that produce a dark image on a bright background
What is the stage of a microscope
the platform where the specimen is set
What ;do they X-y mechanical stage knobs do
move the slide on the surface of the stage but does not raise or lower
what is the coarse focusing knob
knob for large scale movements
what is the fine focusing knob
used for small scale movements especially with more powerful lenses
why do images become dimmer with more magnification
there is less light per unit area of image
what provides light in a microscope
the illuminator
where does the light for the illuminator pass through
the condenser lens
what does the condenser lens do
focuses all of the light rays on the specimen to maximize illumination
What does the diaphragm do
Helps adjust the light striking the specimen
what is a rheostat
a dimmer switch that controls the intensity of the illuminator.
When is a fluorescent microscope the best choice
when observing organisms that fluoresce or can be tagged with a fluorescent antibody
how is a dark field microscope changed from a bright field
There is an opaque light stop block most light from the illuminator as it passes through the condenser producing a hollow cone o flight focused on the specimen. it shows bright images on a dark background. the light is deflected or scattered from specimen
When is a dark field microscope usefull
for creating high contrast, high resolution images without stains. great for live specimens.
how does a phase-contrast microscope work
uses refraction and interference caused by structures in a specimen to create high-contrast, high resolution images without staining. it alters wave lengths.
what are phase contrast microscopes good for looking at
Live specimens, organelles in eukaryotic cells
endospores in prokaryotic cell
What appearance is created with a differential interference contrast microscope
a high contrast image of living organisms with a three dimensional appearance
What are chlorophylls in fluorescence microscope
texas red
PITC
nucleic acid dyes
acridine orange
What are fluorescence microscopes useful for
identify pathogens
find a species within an environment
find locations of particular molecules or structures in a cell
what is immumofluorescence
a technique used to identify certain disease causing microbes by see what antibodies bind to them
How does an electron microscope increase magnification and resolution
uses short-wavelength electron beams rather than light
what are the two types of electron microscope
transmission electron microscope and the scanning electron microscope
What are stains
dyes composed of organic compounds
what is a chromophore
an ion in the dye that is colored
What happens when ions are dissolved in water
the positively and negatively charged ions will separate
what charge is bacteria
negative
What is a positive stain
a stain with a positively charged chromophore so it sticks to the negative bacteria
what is a negative stain
a negative charged chromophore that leaves the bacteria colorless
What is differential staining
process that distinguishes between two groups of bacteria
which gram bacteria are harder to treat
gram-negative
What does a gram stain do
distinguishes between the cell wall components of bacteria
what is the difference between gram negative and gram positive bacteria
gram positive bacteria have multiple layers of a wall and gram negative only have one surrounded by a membrane
what does the primary stain dye
any bacteria
what is the primary star of the gram stain
crystal violet (purple stain)
what is the second step of the gram stain
mordant
what is the mordant
iodine
why is iodine the mordant
it will bind with crystal violet forming a complex
what is the 3rd step in gram staining
decolorizing
what is added in the decolorizing step
alcohol
what does alcohol do to the stain
dehydrates the cell
what happens after alcohol is added in a gram positive stain
crystal violet/ iodine complex dehydrates and becomes bound within the layers of walls so they retain the primary stain when rinsed
what happens to gram-negative stains when they are rinsed
they are colorless
what is the counterstain in the gram stain procedure
safranin (red stain)
what does safranin do to gram negative stains
It will dye the the gram negative stain because they are colorless
what colors are at the end of gram staining
Pink (gram -negative)
purple (gram positive)
What is the primary stain in an acid-fast stain
carbolfuchsin (red dye)
what happens to to acid fast cells when washed with acid
the retain the primary stain
what is the decolorized in an acid-fast stain
acid alcohol
what is the counterstain in an acid-fast stain
methan blue
what does the counterstain in an acid-fast stain do
color non acid fast cells blue
why is acid fast staining important
it distinguishes cells that are in the genus mycobacterium
what are some diseases caused by myobacterium
TB
leprosy
what do acid fast cells contain
a waxy coat called mycotic acid
what is significant about acid-fast cells
they cannot be treated with general ABX and cannot be stained with gram staining procedure.
What does endospore staining do
differentiates between bacteria that produce endospores and those that do not
when are endospores produced
when the bacteria is about to die
what do endospores have
a thick resistant wall surrounding the cells DNA
how long can endospores remain doormant
up to 30 years
what are endospores resistant to
extreme environmental factors such as heat, chemicals and PH
what happens when endospores are activated
they become a vegetative cell that will reproduce in the environment or host
what are some disease caused by endospore forming bacteria
botulism
tetanus
anthrax
what is the primary stain in endospore staining
malachite green
what is the decolorized in endospore staining
water
what is the counter stain in endospore staining
safranin
how are endospores stained
heat drives the primary stain into the endospore and it cannot be washed out.
endospore is green and the rest of the cell is red
What is a capsule stain
differentiates between with a capsule or not
what are capsules
a coating on the outside of the wall of some bacteria
what capsules do
allow bacteria to resist the body immune defenses (phagocytosis
What is phagocytosis
the process where white blood cells engulf and destroy foreign invaders
what is virulence
a microbes ability to cause disease
how does the capsule stain differ from other differential staining procedures
it is a negative staining process, the capsule appears as a halo around the cells
What is flagella stain
a stain the differentiates between bacteria that possess flagella or not or determine the arrangement of flagella
why is it important to determine the arrangement of flagella
to determine type of motility and classification
why is flagella staining difficult
flagella are fragile and can break off during the process
why is a mordant used in flagella staining
so they appear thicker
What is fixation
attaching cells to a slide
how is fixation achieved
heat fixing or chemically treating the specimen
what does fixation do to a specimen
kills microorganisms while preserving the integrity of cellular components
how do you heat fix a sample
spread a thin layer of sample on the slide and briefly heat slide
what chemical agents can be used in chemical fixing
acetic acid
ethanol
methanol
formaldehyde
glutaraldehyde
when are chemical agents preferred for fixing
tissue specimens
What is a basic dye
When the positively charged ion is the chromophore
what is an acidic dye
when the negative ion is the chromophore
What are commonly used acidic dyes
fuchsin
eosin
rose bengal
What are the steps in gram staining
- primary stain (all cells purple)
- mordant (sets or stabilizes stain)
- decolorizing agent
- counterstain (cells are pink now if gram neg)
What considerations need to be taken into account when gram staining old cells
they may have damaged cells wall making them appear gram-negative when they are not
what things can affect gram stain results
old cells
leaving decolorizer on too long
what should you do if decolorized was left on too long and some look gram positive and others gram negative
consider them all gram positive instead of a mixed culture
what is the difference between the kinyoun technique and the ziehl-neelsen technique of acid fast staining
the ziehl nelson method uses heat to infuse the carbolfuchsin
benefits of dark field microscope
you can view shape and motility of unstained live organisms
great for syphiis
