Chapter 3 - Methods of Microbiology Flashcards
What microscope type is most commonly used for observing microbes?
A compound light microscope
In the compound microscope, the objective lens forms a magnified _____ _________ inside the tube of the microscope.
Real image
What lens of the compound microscope forms a magnified real image?
The objective lens
The ____________ lens magnifies the real image produced by the objective lens.
Ocular lens
Does the ocular lens produce a image?
No, it produces the illusion of an image, which we call a virtual image
The ultimate magnification of a compound microscope is the product of the individual magnifications produced by which two lenses?
The objective and ocular lenses
Which lens of the compound microscope do you look through?
The ocular lens
Resolution of the light microscope is normally limited to about ____ µm.
0.2 µm
What is resolution?
Resolution is the ability to distinguish between two closely spaced objects; it is the smallest resolvable distance between two objects
What is the limit of resolution (d)?
The distance separating the centers of two dots that can just be barely distinguished as separate
What two characteristics relate to the limit of resolution?
- The wavelength of the light being used (λ)
- The numerical aperture of the objective lens (NA)
What equation demonstrates the relationship between the limit of resolution, the wavelength of light used, and the numerical aperture?
d = 0.5λ/NA
What is the numerical aperature defined as?
The range of angles over which the system can accept or emit light
The numerical aperture is a function of two physical properties of the objective lens: _____________ and __________ __________.
- Aperture
- Working distance
What is the working distance of an objective lens?
How close to a specimen the objective lens must come for the specimen to be in focus
How is the numerical aperture quantitatively determined?
NA = n sinα
In the following equation, what does n represent?
NA = n sinα
The refractive index of the material in between the lens and the specimen, usually air
In the following equation, what does α represent?
NA = n sinα
α is one-half the objective’s opening angle
Because the physical properties of each lens are fixed and cannot be altered, the only way to improve resolution with a given lens is to increase what?
The refractive index of the material between the lens and specimen
A shorter _______ would increase resolution but hurt the eyes.
Wavelength
How does oil help with resolution of a microscope?
Oil has a higher refractive index than air, and thus it increases the resolution of an image
____________________ _____________________ is used for observing stained cells.
Brightfield microscopy
Under compound light microscopy, why does the specimen appear darker than the surroundings?
Because the specimen has absorbed some of the light
When a specimen is very small, like a microbe, it appears nearly translucent. Why is this?
Microbes are so small that they absorb very little light
When is brightfield microscopy used today?
When used with differential staining
__________________ ______________ uses dyes that stain some microbes but not others.
Differential staining
Why is staining not commonly used with microbes?
Staining often kills microbes and prevents the ability to see living microscopes
What type of staining is most commonly used?
The Gram stain
Due to differences in the thickness of a _____________________ layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process
Peptidoglycan
Why do Gram-positive bacteria retain the crystal violet stain?
Because they have thicker peptidoglycan layers
Why do Gram-negative cells fail to retain the crystal violet stain and stain later with the safranin?
Because they have slimmer or non-existent peptidoglycan layers
What type of microscopy can resolve very small objects?
Darkfield microscopy
How does darkfield microscopy work?
A condenser is set such that it illuminates the specimen with a cone of light; the specimen scatter somes of the light, which enters the objective lens, thereby illuminating the specimen while keeping the rest of the field dark
Extremely small objects up to ____ nm can be seen utilizing darkfield microscopy.
20 nm
What component of a microscopy limits the light scattering that enters the objective?
The iris diaphragm
Because the iris diaphragm can affect light, a stained sample can be misread using darkfield microscopy. Thus, caution should be used with stained samples, using the “quick and dirty” method. What are the three steps to this method?
- Focus on the stained preparation with iris wide open
- Close the iris until the field just begins to darken
- Repeat for each objective lens
What type of microscopy is routinely used for observing live microbial cells?
Phase-contrast microscopy
Phase-constrast microscopy is achieved by modifying two microscope parts. What are they?
- Condenser
- Objective lens
How is the condenser modified in phase-contrast microscopy?
The addition of an annular diaphragm
How is the objective lens altered in phase-contrast microscopy?
Addition of a phase plate
Like darkfield microscopy, phase-contrast microscopy illuminates a specium with a cone of light because the condenser has a ____________ _____________, a transparent ring in an otherwise opaque plate.
Annular diaphragm
What is the purpose of the annular diaphragm in phase-constrast microscopy?
To bend the light into a hollow cone with the specimen at its apex
What is the purpose of the phase plate in phase-contrast microscopy?
The phase plate has a ring of tinted glass that is sized and positioned such that all of the light in the illuminating cone goes through it, dimming the light by more than 70%
Although most of the light in phase-contrast microscopy passes through the specimen, some of the light is ______________. This light is not dimmed because most misses the tinted phase ring; however, the rest of the phase plate is make of thicker glass, which shifts the phase of the deflected light by one half wavelentgh, reducing the brightness of the object and contributing to contrast.
Deflected
How does phase-contrast microscopy generally work?
The _________________ allows more light to enter an objective.
Condenser
What is the working aperture of a condenser?
The sum of aperture angles of the objective and the condenser
What is a phase shift?
When light enters a dense medium, its velocity decreases; as its velocity decreases, there is simultaneously the formation of destructive interference, decreasing the amplitude and reducing the intensity of light
_________ ___________ _________________ is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image
Phase-shift microscopy
What is the halo effect?
The halo effect describes the appearance of a bright edge for positive phase contrast or a dark edge for negative phase contrast around large objects
____________________ microscopy can locate specific molecules within cells.
Flourescence
What is the purpose of an iris diaphragm?
It limits the light scattering that enters the objective of a microscope, making it easier to see clear samples, like live cells
Since an iris diaphragm affects light, it can cause a ________________ sample to be misread; thus caution should be used with these types of samples.
Stained
What are the three steps to the quick and dirty method for looking at stained methods?
- Focus on the stained preparation with the iris wide open
- Close the iris until the field just begins to darken
- Repeat for each objective lens
What type of microscopy is routinely used for observing live microbial cells?
Phase-constrast
How is phase-contrast microscopy achieved?
Via two modifications to the condenser and the objective lens
The addition of an annular diaphragm and a phase plate in phase-contrast microscopy ___________ some of the light, resulting in light that is out of ________. This ultimately results in clearer images of the edges of objects, like the borders of cells or organelles, because the _______________ of the light changes.
Slows
Out of phase
Amplitude
In phase-contrast microscopy, the amplitude of light _______________.
Decreases
What type of microscopy is also known as Nomarski Microscopy?
Differential Interference Contrast (DIC)
What is Differential Interference Contrast microscopy useful for?
Seeing transparent samples
How does DIC microscopy work?
DIC works by separating a polarized light source into two orthogonally polarized mutually coherent parts which are spatially displaced (sheared) at the sample plane, and recombined before observation. The interference of the two parts at recombination is sensitive to their optical path difference. The contrast is proportional to the path length gradient along the shear direction, giving the appearance of a three-dimensional physical relief corresponding to the variation of optical density of the sample, emphasising lines and edges though not providing a topographically accurate image
_________________________ microscopy, also known as Nomarski microscopy, is an optical microscopy technique used to enhance the contrast in unstained, transparent samples. DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo.
DIfferential interference microscopy
What is important to remember when looking at images produced via DIC microscopy?
The three-dimensional image of a specimen may not be accurate: the enhanced areas of light and shadow might add distortion to the appearance of the image
Differential interference contrast microscopy is similar to plane-contrast microscopy in that it uses differences in _________________________; however, a DIC microscope uses __________ of light instead of ______. In addition, prisms split each light beam, adding contrasting colors to the specimen. Therefore, the resolution of a DIC microscope is higher than that of a standard phase-contrast microscopy, and the image is brightly colored and appears nearly three-dimensional.
Refractive indexes
Two beams of light
One beam of light
Which images were taken with DIC microscopy? Which were taken with phase-contrast microscopy?
The top images were taken by DIC microscopy
The bottom images were taken by phase-contrast microscopy
The halos in the bottom images indicate phase-contrast microscopy
A ____________________ is linked to the molecule to be visualized in flourescence microscopy.
Fluorophobe
What are the three basic steps to flourescence microscopy?
- A fluorophore is linked to the molecule to be visualized
- The specimen is illuminated with a beam of light with a wavelength that will be absorbed by the fluorophore
- The specimen is observed through filters that cut out most of the light except the wavelengths that are emitted y the excited fluorophore
Most fluorescence microscopes are constructed around a set of filters and a ___________________ __________________, a piece of glass that acts as a mirror for some wavelengths but is transparent to others.
Dichroic mirror
The _________________ in fluorescence microscopy can be changed to allow different fluorphores to be used.
Filters
A modification to the fluorescence microscope, termed ______________________________, removes light that is out of focus.
Confocal microscope
What is minE in E. coli?
The MinE protein is one of three proteins of the Min system encoded by the minB operon required to generate pole to pole oscillations prior to bacterial cell division as a means of specifying the midzone of the cell, as seen in E.coli.
Confocal microscopy uses a _______________ light opening and a Z axis to provide ____________.
Smaller light opening
Depth
What is the advantage of confocal microscopy?
Efficient at getting rid of fluorescent light that is out of focus
The ______________ microscope uses electromagnetic lenses to bend a beam of electrons.
Electron
What does the electron microscope use instead of a light beam?
An electron beam
What bends the beam of electrons to form an image in electron microscopy?
Electromagnetic lenses
The resolution of the electron microscope is a _________-fold better than the light microscope.
1000
Specimesn in the electron microscope need to be _______________.
Dehydrated
Why must specimens be dehydrated before using electron microscopy?
Electron microscopy uses vaccums, which distort specimens as they evaporate water and dissolved gases from cells
Specimens for use in electron microscopy are ______________ prior to dehydration using chemical agents that _______________ the macromolecules of the cell together.
Fixed
Cross-link
_____________ _________________ slices through cells and shows interior structures.
Thin sectioning
In electron microscopy, a ______________ lens is used instead of an ocular lens; this lens produces a second real image on a screen, which has a __________________ coating that glows when it is hit by the electron beam.
Projector lens
Phosphorescent coating
What is the most common form of sample preparation for electron microscopy?
Thin sectioning in which the specimen is embedded in a small block of plastic after fixation and dehydration and then cut into very thin slices
Thin sections must be _____________ before viewing because organic materials are nearly ________________ to an electron beam.
Stained
Transparent
It is wise to remember that what we see in a thin section is not the cellular material iteself but the ______ bound to it.
Stain
Two types of electron microscopes were discussed in class. What are they?
Transmission electron microscope
Scanning electron microscope
What is the difference between transmission and scanning electron microscopy?
The main difference between SEM and TEM is that SEM creates an image by detecting reflected or knocked-off electrons while TEM uses transmitted electrons (electrons which are passing through the sample) to create an image. As a result, TEM offers valuable information on the inner structure of the sample, such as crystal structure, morphology and stress state information, while SEM provides information on the sample’s surface and its composition.
What is the magnification range of TEM?
10,000X - 1,000,000X
What is the maximum magnification of SEM?
10,000X
What is the resolution of TEM and SEM respectively?
0.2 nm
5 nm
___________________________________- is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device
Transmission electron microscopy
______________________________ is a type of electron microscopy that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample.
Scanning electron microscopy
In which type of electron microscopy is the specimen coated with metal?
Scanning electron microscopy
A sample in SEM requires fixing and dehydration. The sample is coated with a very think coating of _____________ or other metal or sometimes carbon.
Gold
What is a simplified version of image formation in SEM?
- Electromagnetic lens squeeze the illuminating electrons into a beam
- The electron beam is scanned across the specimen in a pattern of closely spaced lines
- Positive charges on a collector attracts the expelled electrons
- The current that comes from the collector is proportional to the surface over which the beam is scanned
- The scanning pattern on the monitor is in exact register with the scanning patter of the electron beam; the brightness is proportional to the current coming from the collector
What is the principle behind atomic force microscopy?
_________________________ is a very-high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
Atomic force microscopy
What type of staining reveals the texture of the outside of cells and isolated subcellular organelles?
Negative staining
How does negative staining occur?
A drop of staining solution is placed on top of the specimen; the specimen is dehydrated; the stain precipitates on and around the specimen
The metal accumulation around the specimen results in electron deflective material at the surface of molecules
What process shows the surface of very small and isolated organelles?
Shadow casting
In __________________________, the fixed and wet specimen is placed in a drop of a heavy metal salt stain that precipitates on and around the specimen; it is especially useful for showing surface structures.
Negative staining
________________________________ uses the deposition of a heavy metal around a specimen to show surface structure, the fixed, dried specimen is placed in a vacuum chamber, and the metal is evaporated from an electrode.
Shadow casting
________________________ does not require fixation or dehydration; the specimen is frozen very rapidly and then fractured with a sharp, cold knife. The fractured and etched specimen is shadowed, and a layer of carbon is deposited on top of the shadow. THis _____ is then lifted off of the specimen, taking the stain with it and observed under electron microscopy.
Freeze etching
Replica
Why is freeze etching particularly useful?
Live cells are quickly frozen and thus protected from distorting effects of fixing and dehydrating; freeze etching is often used to help decide whether structures in other techniques, like thin sectioning, are real or artifacts
Freeze etching is an especially useful way to visualize what part of the cell?
Membranes
Do pure cultures consist of identical cells?
No; they normally consist of a clone of cells but could be in different stages of growth
What is a clone of cells?
A population all derived from a single ancestral cell not very far in the past
Pure culture technique consists of three interrelated techniques. What are they?
- Sterilization of growth media and glassware
- Introduction of desired cells into sterile growth media or removal of samples from pure culture without accidential introduction of contaminating microbes
- Isolation of single cells, or their progeny, to obtain pure culture
________________ means the absence of living organisms, and it is in principle an all or nothing phenomenon although it can be expressed as a probability.
Sterility
What is the most common method for sterilization?
Autoclaving
What is an autoclave?
A fancy pressure cooker that maintains a pressure of 15 lb/in2 at which pressure the boiling point of water is 121 degrees celsius; to prevent boiling, the atmosphere in an autoclave is saturated with water vapor
How long do specimens stay in an autoclave?
10 to 15 minutes
How are heat-sensitive solutions sterilized?
Via filtration
In most cases, what size pore is sufficient for sterilization via filtration?
0.45 micrometers
How is glassware commonly sterilized?
By dry heat
How is plastic ware sterilized at a factory?
Exposure to gamma irradation (cobalt-60) or the toxic gas ethylene oxide
What helps to prevent contamination during transfer into and out of containers?
Bunsen burner flames
How does a Bunsen burner flame help to prevent contamination?
- Ionization of the test tube, causing electrostatic interactions that prevent microbial entry
- Updraft generation, preventing microbes from falling into the test tube (so long as within one foot of the flame)
_____________________________ is a process that destroys or eliminates all forms of microbial life, including spores and viruses, and is carried out in healthcare facilities by physical or chemical methods. Steam under pressure, dry heat, ethylene oxide gase, hydrogen peroxide gas plasma, and liquid chemicals are the principal sterilizing agents used in healthcare facilities. It is intended to convey an absolute meaning and not generally associated with inactivating prions.
Sterilization
____________________ is a process that eliminates many or all pathogenic microorganisms except bacterial spores on inanimate objects. It can include prions.
Disinfection
How are pure cultures isolated on solid media?
Streaking
How is streaking accomplished?
A small amount of inoculum is picked up on a sterile inoculating loop made of wire; the loop is drawn repeatedly across the surface of the plate, depositing microbes as it moves; the plate is incubated and colonies are visible some time later
Normal colonies contain about 10__ to 10__ cells
7 to 8 cells
______________ media typically have a mixture of many different organic compounds, like amino acids and peptides, vitamins, fatty acids, etc.
Rich media
_______________ media contain mineral salts of major bioelements, a carbon source, and a nitrogen source.
Minimal media
What is M9 mineral media composed of?
M9 salts (Na2HPO4, KH2PO4, NaCl, NH4Cl)
Glucose
MgSO4
CaCl2
What is the difference between complex media and chemically defined media?
In complex media, we don’t know the specific ratios of organic compounds; in chemically defined media, the specific ratios are known
Which media type is more restrictive: complex/rich media or minimal media?
Minimal media since no organism with a growth factor requirement can grow unless it is specifically added
What are obligate aerobes?
Organisms that require oxygen to grow
What are obligate anaerobes?
Organisms to which oxygen is toxic
What are facultative anaerobes?
Organisms that can grow in the presence or absence of oxygen
What is a microaerophile?
An organism that needs only a little oxygen to grow
How many biosafety levels are there?
Four
Which BSL category corresponds to working with nonpathogenic microbes like nonpathogenic E. coli?
BSL1
What BSL category corresponds to pathogens that do not readily transmit by the aerosol route, like pathogenic S. aureus and HIV?
BSL2
What does it mean that a pathogen can readily transmit via aerosol route?
The pathogen contains smaller droplet nuclei particles with aerodynamic diameter below 5 micrometers that can remain airborne for longer periods
Larger droplets are believed to settle to ground within 1 to 2 metes are not considered _______________ transmission
Aerosol
What BSL category corresponds to pathogens that transfer readily by the aerosol route and pose a serious health threat, like the West Nile Virus, Mycobacterium tuberculosis, Yersinia pestis?
BSL3
What BSL category corresponds with pathogens that transmit readily by the aerosol route and that cause fatal diseases for which this is no cure, like Ebola?
BSL4
Be familiar with this chart.
