Observing the Microbial Cell Flashcards
What is light microscopy?
LM resolves images of individual bacteria by their absorption of light. Light microscopy is limited by the wavelength of light.
What are the types of light microscopy?
(1) Bright field
(2) Dark field
(3) Fluorescence
(4) Phase contrast
What is electron microscopy?
EM uses beams of electrons to resolve details several orders of magnitudes smaller than those seen under light microscopy
What are the types of electron microscopy?
(1) Scanning electron microscopy
(2) Transmission electron microscopy
(3) Cryo-EM
With is atomic force microscopy?
AFM uses intermolecular forces between a probe and an object to map the 3D topography of a cell, or cell parts.
How are bacterial cells perceived in bright field microscopy?
As dark silhouettes blocking the passage of light. Hence the speciment is viewed as as a dark object against a light filled field or background.
What is the maximum resolution of bright field microscopy?
200 nm or 0.2 micrometers.
What is the limit magnification of bright field microscopy?
x1000
So in the case of bright field microscopy, if we have a magnification of more than 1000, we won’t see more details because of the physical limitation of our eye
What does bright field microscopy require?
Stains
Single lens vs Compound lens
The advantage of compound lens microscopes over single lens is that much higher magnifications can be achieved with two lenses. Also, lenses often have aberrations within the glass, so having multiple lenses can correct aberrations. As you increase the glass curvature, the affects of aberrations increases faster than magnification. Having multiple lenses can multiply their magnification with minimal aberrations (as magnification is multiplicative whilst aberrations are additive).
What are the two basic types of preparation used to view specimens with a light microscope?
Wet mounts and fixation
Explain the wet mount preparation method
The specimen is placed on the slide in a drop
of liquid. Sometimes the liquid used is simply water, but oftenstains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope.
Explain the fixation preparation method
The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.
How do you heat-fix a sample?
A thin layer of the specimen is spread on the slide (called a smear) and the slide is then briefly heated over a heat source.
Are heat or chemical fixatives preferred for tissue speciments?
Chemical
What is simple staining?
A simple stain adds a dark color specifically to cells but not to the external medium or surrounding tissue. It does not differentiate between different cell types.
What is differential staining?
A differential stain stains one kind of cell but not another.
What is an important feature of methylene blue (simple) and gram (differential) staining?
These stains are positively charged, allowing them to bind to the negatively charged surface of bacterial cells.
What is the difference between gram negative and gram positive bacteria?
Gram positive bacteria have a thicker cell wall whilst gram negative bacteria have thinner cell walls
How many layers do gram negative bacteria have and what are they?
3 Layers:
(1) Cytoplasmic/inner/plasma membrane
(2) Cell wall
(3) Outer membrane
Hoe many layers do gram positive bacteria have and what are they?
2 Layers:
(1) cytoplasmic/inner/plasma membrane
(2) The cell wall
Why do gram positive bacteria need a thicker more protective cell wall than gram negative bacteria?
Because they have no outer membrane
What is the gram staining procedure?
(1) Crystal violet is added to the bacterial cells
(2) Iodine solution is added
(3) A decolorizer such as alcohol is added
(4) A weaker stain such as water-soluble safranin is added to stain the sample red
(5) Bright field microscopy is used
Why do we add crystal violet in the gram staining procedure?
It stains the peptidoglycan layer
Why do we add iodine solution in the gram staining procedure?
It will form a complex with the crystal violet and this complex is a larger molecule than the original crystal violet stain and is insoluble in water
Why do we add a decolorizer in the gram staining procedure?
It will release the loosely bound stain (i.e. the crystal violet/iodine complex bound to the peptidoglycan)
What does the weaker stain do in the gram staining procedure?
Because it is lighter than crystal violet, it will not disrupt the purple coloration of gram positive cells but will stain gram negative cells red
What are other kinds of differential stains besides gram staining?
- Acid fast
- Spore stain
- Negative stain
What is acid fast stain used for?
To identify mycobacterium tuberculosis
Why do we need acid fast staining to detect mycobacterium tuberculosis?
Mycobacterium contain large amounts of lipid substances within their cell wall called mycolic acid which resist staining by ordinary methods like gram staining.
What is the acid fast staining proecure?
(1) Carbol fuchsin is added and, since it has an affinity for mycolic acid, it will bind to the tuberculosis bacterium and stain it pink
(2) With alcohol and heat, carbol fuchsin is removed from cells that are unprotected by a waxy-lipid layer (i.e., non M. tuberculosis cells)
(3) Methylene blue is added to stain the other cells and the excess stain is rinsed off
so M.tuberculosis is stained pink whilst the other cells are stained blue
Why do we need spore staining to detect spores?
Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures
What is the spore staining procedure?
(1) Staining is done with malachite green
(2) Heat is also involved. When samples are boiled with malachite green, the stain binds specifically to the endospore coat.
(3) Spores are then visualized using bright field microscopy
What do we use negative stains for?
Negative staining is mostly used to stain cells thart are too delicate to be heat fixed.
How can negative stains be visualized?
With either light microscopy to electron microscopy
What is the negative staining procedure for LM?
(1) Nigrosine is used which is a black inked fluid. Nigrosine will readily give up hydrogen ions and become negatively charged.
(2) Since the surface of most bacterial cells are negatively charged, the cell surface will repel the stain
(3) Therefore, the glass of the slide will stain but not the cell
(4) The stain darkens the surrounding medium and reveals transparent components such as the outer capsule of a pathogen
How do negative stains appear with bright field microscopy?
The bacteria show up as clear spots against a dark background
Why is dark field and phase contrast microscopy useful?
It enables us to visualize structures that are too difficult or impossible to detect under a bright field microscope
What is the ideal case in which dark field microscopy should be used?
In the case where samples cannot be stained (i.e. they are too sensitive or too small). It is also useful for observing live samples, as well as motile samples. Even viruses can be detected.
What is the main limitation of dark field microscopy?
The low light levels seen in the images. This means that the sample must be very strongly illumintaed to see details, but this can damage the speciment. Any dust can also scatter light so you need a medium that is extremely clear.
How do speciment appear in final images that use dark field microscopy?
Microbes are visualized as halos of bright light against a dark background.
What is the main property dark field microscopy relies on?
The scattering of light. It uses scattered light to detect objects too small to be resolve dby any light rays. Extremely small microbes and thin structures can be visualized. The shapes of the objects are not resolved.
What does detection of scattered light require?
A modified condenser arrangement that excludes all light that is transmitted directly. The spider light stop allows only a hallow cone of light to focus on the object. The incident light converges at the object and then generates an inverted hollow cone radiating outward
How does phase contrast microscopy work?
Phase contrast microscopy exploits differences in refractive index between the cytoplasm and the surrounding medium or between different organelles. The waves that pass through the object will be out of phase compared to the waves that do not. The waves that pass through the obejct will also get bent
What does phase contrast optics depend on?
The principle of interference between two waves in a pattern of alternative zones of constructive and destructive interference. The slight differences in the refractive index of the various cell components are transformed into difference in the intensity of transmitted light
What can be seen with phase contrast microscopy
Live cells with transparent cytoplasm, the organelles of eukaryotes can be obserrved with high contrast. The specimen appears darker than the background.
What is fluorescence?
The emission of light by a substance that has absorbed light or other electromagnetic radiation.
How does fluorescence microscopy work?
The speciment is illuminated with light of a specific wavelength which is absorbed by the fluorophores, causing them to emit light of longer wavelengths/lower energy. So, the light we see comes from the sample itself. The specimen absorbs light of a defined wavelength and then emits light of higher wavelengths.
What happens to the excited electrons?
The electronsof the atoms of the sample get excited to a higher-energy orbital and then, when they come back to the ground state, they release a photon.
Which has a longer wavelength: the emitted light or the absorbed light?
The emitted light. The longer wavelength means it has a lower energy as well
What are fluorescence molecules typically used for in microscopy?
To reveal specific cells or cell parts. Fluorescence can be observed in live organisms, but you can’t resolve the detailed shape of a protein. So you can detect the presence of a specific protein, but you cannot resolve its detailed shape.
What is the difference between light microscopy and fluorescence microscopy?
In fluorescence microscopy, there is a filter after the original light source that absorbs all colors except for one, depending on the microscopy. So the light coming to the sample is monochromatic
What kind of cells does DAPI detect?
Only dead cells whose DNA is intact
What is a dichroic mirror?
A material that reflects light below a certain wavelength but transmits light above that wavelength
How do fluorescence dyes differ from bright-field stains?
Bright field stains will bind to the specimen and stain the entire specimen (ex: acid fast staining, gram staining, spore staining) or will be repelled by the specimen and will stain the glass slide (ex: negative staining). On the other hand, a fluorescent dye will
selectively bind to a particular molecule for which it has a high affinity (ex: FITC conjugated to an Ab that will
bind a protein specific to that Ab) and, after having absorbed light at a certain wavelength, will emit light at a longer wavelength. Moreover, the fluorescent dyes will allow the detection of specific molecules, such as
proteins, due to higher resolution, whereas brightfield
stains only allow visualization of entire cells, not
subcellular structures and proteins.
What are the common fluorophores?
(1) FITC
(2) AO
(3) DAPI
Fluoresceinisothiocyanate (FITC)
FITC binds to amino acids in protiens. They can be conjugated to antibodies and the antibodies are used as probes
Acridine-orange (AO)
AO stains double stranded nucleic acids (DNA) and single stranded nucleic acids (RNA) in different colors
DAPI
The aromatic rings of DAPI mimics a base pair, enabling intercalation between DNA base pairs. It binds strongly to the A-T rich regions in DNA. It gives a blue stain when it absorbs UV waves.
When do we use dyes with chemical affinities?
To label DNA (like with DAPI) and membranes
What are dye-labelled antibodies?
Dyes conjugated to antibodies. Antibodies that specifically bind a cell component and are chemically
linked to a fluorophore molecule. The use of antibodies linked to fluorophores is known an
immunofluorescence.
What are dye-labelled DNA probes for hybridization?
Dyes conjugated to DNA to localize specific DNA sequences.
What is gene fusion?
The expression of a protein fused with a fluorescent protein (reporter protein like GFP)
What is one problem associated with GFP?
Proteins fused to GFP may behave differently to the original, non-fused protein.
What is super resolution imaging?
Super resolution imaging can define the position of a fluorescent protein with a tenfold better precision than the resolution limit of light magnification. It uses peak intensity in order to generate the microscopy.
What does confocal mean?
Confocal = excitation + emitted light are focused together
What kind of sample can laser confocal microscopy be used on?
Thick tissue samples
How does laser confocal microscopy work?
Reflected emission rays are focused and pass through a pinhole which eliminates unfocused light. This technique allows for an increased optical resolution and contrast by focusing together both the excitation light (from the fluorescence) and the emitted light. It is an advanced form of fluorescence microscopy that uses laser beams to resolve subcellular details, and even to visualize cells in 3D.
What does a microscopic laser light source do?
It scans across the specimen, focusing the light in order to remove background noise.
When do we use LCM?
We use LCM to eliminate or reduce background noise and if the specimen is lightsensitive. In fact, too much light can be detrimental to the sample. Therefore, laser confocal microscopy is advantageous, since only what we are looking at will be exposed by light, conserving other parts of the sample
What is intra-vital microscopy?
A type of microscopy that allows observing biological processes in live animals at a high resolution and makes distinguishing between individual cells of a tissue possible. This variation of LCM allows us to focus on what we want to see. A type of stain is applied on living cells allowing us to visualize the movement of specific molecules (e.g. proteins)
