Microscopy and Staining Flashcards
(42 cards)
1
Q
Magnification
A
is when a focused image appears larger than the specimen itself and is achieved when the light rays from the illuminator passes though the condenser which has lenses that directs the light rays through the specimen. Then this light passes through the objective lens to the ocular lens
-so obviously the path of light goes from the light to the top
-Calculation of total magnification by multiplying the objective lens magnification (power) by the ocular lens magnification (power)
-Objective lenses (10x lower power) , (40x high power), (100x oil immersion). The ocular lenses magnify specimen by 10 so we
see total magnification of 100x 400x and 1000x respectively.
2
Q
Resolution
A
(resolving power) is the ability of the lenses to show the fine detail and structure.
- refers to the ability of the lenses to distinguish two points that are a specified distance apart
- so what ever is the resolving power is the distance of the two points
- ex. Resolving power .4nm the two points are .4nm away
- Shorter the wavelength of light used, the greater the resolution
- most important property of microscope
3
Q
To see specimen…
A
To see the specimen in a clear image you must have a different background or medium and to obtain this you must change the refractive index (the measure of the light-bending ability of a medium)
- to make this change with the refractive index is by STAINING them
- after they are stained they specimen and the medium have different refractive indexes
- when light rays go through the specimen and the medium (background) the rays change direction (refract) from a straight path by bending or changing angle causing it to have a contrast between the specimen and its background (medium).
4
Q
Objective lens
A
enlargement and resolution
5
Q
Occular lens
A
only enlargement
6
Q
Oil Immersion
A
- to achieve 1000x (high magnification) with good resolution the objective lens must be small
- we want the light traveling through the specimen and medium (Background) to refract differently to see contrast, we don’t want to lose light rays after they passed through the stained specimen this is why oil immersion is needed
- the oil helps capture the light by narrowing it out into the objective lense. Without it the light is refracted and lost
7
Q
Dark Field Microscope
A
- is used to examine live microorangisms that either r are invisible in the ordinary light microscope, cannot be stained by standard methods, or are fucked up by staining that their characteristics are obscured.
- uses a darkfield condenser that solid disk and this disk blocks light that would enter the objective lens directly
- only light that is reflected off the specimen enters the objective lens
- because there is no direct background light, the specimen appears light against the darkfield
- used to look at unstained MOs in liquid, thing spirochete like Treponema pallidum the causative agent of syphilis.
8
Q
Why use D.F microscope?
A
- allows you to view living cells to observe activities such as motility
- allows you to view delicate cells
- fixation and no staining is required
9
Q
Phase Constrast Microscope
A
-useful because the internal structures of a cell become more defined and allows for detailed examination of the LIVING MOs.
-don’t have to fix the microbes to the slide or stain the specimen that may distort or kill the microorganisms.
-one set of light comes directly from the light source and the other comes from the light reflected or diffraction from a structure in the specimen
-diffraction is the scattering of light rays as they touch a
specimens edge.
-When the two set of different rays are brought together, they form an image of the specimen on the ocular lens, containing areas that are light, gray, or black
10
Q
Differential Interference Contrast Microscopy
A
- similar in phase contrast because it uses differences in refractive indexes.
- the difference is the fact that it uses two different beams of light instead of one
- the prisms split each light beam, adding contrasting colors to the specimen so that shit has higher resolution than the phase contrast scope.
- image is brightly colored and appears three dimensional.
11
Q
Fluorescence Microscopy
A
- takes advantage of fluorescence (the ability of substances to absorb short wavelengths of light i.e ultraviolet and give off light at a longer wavelength i.e visible)
- organisms naturally fluoresce under ultraviolet light if not it is stained with dyes called fluorochromes
- when they are stained with this under a fluorescence microcope with an ultraviolet light or close to one light source they appear bright against a dark background
- Fluorochromes have special attractions for different MOs
12
Q
Fluorochrome auramine
A
O glows yellow when exposed to ultraviolet light and is absorbed by Mycobaterium tuberculosis a bacterium that causes tuberculosis.
- when this dye is applied to a sample that people may think contains that bacterium it could be detected if they could find bright yellow organisms against the dark background.
- Same thing with anthrax but it appears to green and the fluorochrome is fluorescein isothiocyanate.
13
Q
THE PRINICIPAL USE OF FLUORESCENCE MICROSCOPY
A
- the principal use of it is a diagnostic technique called fluorescent-antibody technique or immunofluorescence
- Antibodies are a natural defense molecules that are produced by humans and many animals in reaction to a foreign substance or antigen
14
Q
Steps of immunofluorescence Microscope
A
- Flourescent antibodies for a particular antigen are obtained when an animal is injected with a specific antigen such as a bacterium then the animal begins to produce antibodies against the antigen. After some time the antibodies are removed from the serum of the animal.
- next a fluorochrome is chemically combined with the antibodies then these fluorescent antibodies are added to a microscope slide containing an unknown bacterium and if this unknown bacterium is the same one that was injected to the animal the fluorescent antibodies bind to the antigens on the surface of the bacterium causing it to fluoresce.
- this detects bacteria or other pathogenic microorganisms, within cells, tissues, or other clinical specimens.
15
Q
Why is the Immunofluorescence Microscope important?
A
- Can tell who’s sickness is related to each other
- can find a correlation for people getting sick
- ITS IMPORTANT BECAUSE it can identify a microbe in minutes. It is also useful in disgnosing syphilis and rabies.
- can detect very unique microorganisms
- helps detect ecoli
16
Q
Confocal Microscopy
A
- is used to reconstruct 3D images
- like fluorescent microscopy, the specimens are stained with fluorochromes so they will emit or return light.
17
Q
Two Photon Microscopy
A
- Specimens are stained with fluorochrome
- uses long wavelength (red light) so two photons are needed to excite the fluorochrome to emit light cuz its long.
- allows deep imaging of tissues in living cells
- the longer wave length is less likely to generate singlet oxygen that damages cells
- It can also track the activity of cells in real time
- cells of the immune system have been observed responding to an antigen
18
Q
Scanning Acoustic Microscopy
A
- understanding the action of a sound wave sent through a specimen
- a sound wave of a specific frequency travels through the specimen, and a piece of it is reflected back every time its hits an interface within the material.
- used to study living cells attacted to another surface such as cancer cells, artery plaque, and bacterial biofilms that foul equipment.
19
Q
Electron Microscopy
A
- Small objects than about .2um like viruses or the internal structures of cells have to be examined by an electron microscope.
- A beam of electrons are used instead of light
- has the strongest resolving power because of the short wavelength of elections that’s why they use it to examine the smallest structures
- images are always black and white
- uses an electromagnetic lenses instead of a glass one to focus a beam of electrons on a specimen
- TWO types transmission electron and scanning electron
20
Q
Transmission Electron Microscope
A
a finely focused beam of electrons from an electron gun passes through a specially prepared, ultrathin section of specimen.
- The beam is focused on a small area of the specimen by an electromagnetic condenser lens directing the beam of electrons in a straight line to light up the specimen that is placed on a copper mesh grid instead of glass slide. The beam passes through the specimen and then through an electromagnetic objective lens that magnifies the image. Instead of ocular lens the electrons are focused by an electromagnetic projector lens onto a fluorescen screen.
- the product of all of these steps is called a transmission electron micrograph
- objects are magnified 10,000 to 10,000,000x
21
Q
Why they use staining for Transmission Electron Microscopes?
A
- because the specimen are so thin and the contrast bewtween their ultrastructures and the background is weak they could be enhanced by using a dye.
- dye absorbs electrons and produces a darker image in the stained region.
- the stains could be salts of different heavy metals like lead, osmium, tungeten and uranium
- These metals can fixed onto the specimen (POSITIVE STAINING) or used to increase the electron opacity of the surrounding field (NEGATIVE STAINING)
- Negative staining is good for the study of very small specimens such as virus particles, bacterial flagella, and protein molecules.
22
Q
Shadow Casting
A
- a heavy metal such as platinum or gold is sprayed at an 45 degree angle so that it strikes the microbe from only one side
- the metal piles up on side of the specimen and the uncoated area on the opposite side leaves a clear area behind its shadow -gives a three dimensional effect to the specimen and provides a good idea of the size and shape of the specimen.
23
Q
Disadvantages of Transmission Electron Microscope
A
- electrons have limited penetrating power so only a very thin section of the specimen can be studied effectively
- no 3d aspect
- has to fixed, dehydrated, and viewed under a high vacuum to prevent electron scattering and this causes shrinkage an distortion that makes it seem like there is additional strutures
- structures that appear as a result of the preparation method are called artifacts
24
Q
Scanning Electron Microscope
A
- overcomes the problem that the transmission electron microscope has with only being able to examine one section
- provides striking 3D views of a specimen
25
Steps of Scanning Electron Microscope
- AN electron gun produces a finely focused beam of electrons called PRIMARY ELECTRON BEAM
- electrons pass through electromagnetic lenses and are directed over the surface of the specimen
- the PEB knocks electrons out of the surface of the specimen and the secondary electrons are sent to an electron collector amplified and used to produce an image on a viewing screen called scanning electron micrograph
- Useful in studying the surface structures of intact cells and viruses
26
Staining
is coloring the MO with a dye that emphasizes certain structure
- before they could be stained they must be Fixed (attached) to the slide.
- fixing kills the MO while its being attached to the slide
- preserves various parts of microbes in their natural state with minimal distortion
- a thin film of material containing the MO is spread over the surface of the slide…this film is called smear and is allowed to air dry
- Slide is then fixed by passing it through a flame, stain is apllied and washed off with water, then the slide is blotted with aborsbent paper.
- without fixing the stain might wash the microbes off the slide
27
Stains
- are salts composed of a positive and a negative ion, one of which is colored and is known as the chromophore
- basic dyes is the cation (positive) . Acidic dyes is the anion (negative)
- to do apply basic or acidic dyes we have to use three kinds of staining techniques :simple, differential, and special
28
Basic Dyes
crystal violet, methylene blue, malachite, green, and safranin
-are more commonly used than acidic dyes.
29
Acidic Dyes
eosin, acid fuchsin, nigrosin
- are not attracted to most types of bacteria because the dyes negative ions are repelled by the negatively charged bacterial surface, so it stains the colored background instead
- Preparing colorless bacteria against a colored background is called negative staining
- valuable for observing overall cell shapes, sizes, and capsules because they are really easy to see against a contrasting dark background
- Distortions of cell size and shape are minimized because fixing isn’t necessary and the cells don’t pick up the stain
30
Positive Staining
- most common
- relies on basic dyes such as crystal violet, methylene blue or safranin
- gives colors to the cells on clear background
- simple and gram stain
31
Negative Staining
- uses acidic dye like nigrosin, eosin, or congo red
- see clear cells on colored background
- no heat fixation just fixation in drying
- used to see natural sizes and shapes of organisms and to see delicate structures such as capsules
32
Simple Stain
- an aqueous or alchohol solution of a single basic dye
- primary purpose of a simple stain is to highlight the entire MO so that cellular shapes and basic structures are visible
- applied to the fixed smear for a certain length of time and then washed off. The slide is dried and examined.
- Sometimes a chemical is added to the slide to intensify the stain i.e mordant
- Mordant increases the affinity of a stain for a biological specimen and to coat a structure to make it thicker and easier to see after it is stained.
- some simple stains are methylene blue, carbolfuchin, crystal violet, and safranin
33
Differential Stain
- unlike simple stains these stains react differently with different kinds of bacteria and thus can be used to distinguish them
- most frequently used ones are the gram stain and the acid-fast stain
34
Gram Stain
was developed by the Danish bacteriologist Hans Christian Gram
-one of the most useful staining procedures becaue it classifies bacteria into two large groups : Gram Positive and Gram Negative
35
Procedure Gram Stain
- a heat fix smear is covered with a basic purple dye usually crystal violet..this stain all of the cells so its called the Primary stain
- after a short time the purple dye is washed off then the smear is covered with iodine a mordant. When its washed off borth gram positive and gram negative bacteria appear dark violet or positive
- the slide is then washed with alchohol Decolorizing agent which removes the purple from the cells of some species but not from others
- The alcohol is rinsed off, and the slide is then stained with safrannin, a basic red dye. The Smear is washed again and blotted dry to examine.
- The purple dye and the iodine combine in the cytoplasm of each bactierum and color it dark violet or puple.
- If the bacteria retains this color after the alcohol has attempted decolorization means it is classified as gram-positive
- Bacteria that loses the dark violet or purple color after decolorization are gram-negative
- Because gram negative bacteria are colorless after the alchohol wash you cannot see them. So this is why the basic dye safranin is applied because it turns the gram negative bacteria pink.
- safranin has a contrasting color to the primary stain called Counterstains
- because gram positive bacteria retain the purple stain they are not affected by the safranin
36
acid fast stain
- differential stain which binds strongly only to bacteria that have a waxy material in their cell walls
- Use this stain to identify all bacteria in the genus Myobacterium tuberculousos (Mycobacterium tuberculosis and mycobacterium leprae)
- causative agent of tuberculosis and leprosy respectively
37
acid fast procedure
- the red dye carbolfuschin is applied to a fixed smear, and the slide is gently heated for several minutes. The heat enchances penetration and retention of the dye. Then the slide is cooled and washed with water. The smear is than treated with acid-alcohol a decolorizer which removes the red stain from bacteria that are not acid fast.
- Acid fast MOs keep the pink because the carbolfuchin is more soluble in the cell wall lips than in the acid alchohol…non acid fast Mos lack the lip components so the carbolfuchin is rapidly removed from the alchohol
- the non acid fast MOs are now colorless so a counterstain is needed in methylene blue causing the non acid fast MOs to appear blue.
38
ACID FAST AND GRAM POS
- acid fast has cell walls built like gram pos, but not gram stainable because have waxes so makes them resistant to staining, decolorization, defective solutions, and antibiotics
- M. Tuberculosis and M. Leprae
39
Special Stains
used to color parts of MOs such as eendospores, flagella, or capsules
40
Capsule Stain
uses negative stain principles to reveal delicate extracellular slime coat
demonstrating the presence of a capsule is a means of determining the organisms virulence (the degree to wich a pathogen can cause a disease)
- it is harder than the other staining procedures because the capsule materials are water soluble and may be removed during rigorous washing
- don’t accept most biological dyes because of their chemical composition
- You can mix the bacteria in a solution containing a fine colloidal suspension of colored particle to provide contrasting background and then stain the bacteria with a simple stain
41
Spore Stain
uses heat to drive primary stain to thick spore walls
- endospore or spore is a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions
- are relatively uncommon
- cant be stained by normal methods because the dye cannot penetrate the endospores wall
42
Flagella
uses mordant to enhance color intensity on this flagella
- are structures of locomotion too small to be seen with a light microscope without staining
- an annoying and delicate staining procedure uses a mordant and the stain carbolfuchsin to build up the diamaters of the flagella until it could be seen
