Week 1 - History, Microscope Types, Stains Flashcards
Eukaryotes (uni or multi, DNA storage)
- Unicellular AND multicellular
- Linear DNA
- Has a nucleus
Prokaryotes (uni or multi, DNA storage)
- Unicellular
- Circular DNA
- No nucleus
Viruses (uni or multi, DNA storage)
- Acellular (not made of cells)
- BOTH linear and circular DNA
- No nucleus
Names of Light microscopes
- Brightfield
- Darkfield
- Phase contrast
- Differential Interference Contrast (DIC)
- Fluorescence
- Confocal
- Two-photon
Light microscopes (magnification and what is used to view image)
- Magnification: up to 1000x
- Use visible or ultraviolet light to produce an image
Electron microscope (magnification and what is used to view image)
- Magnification: 20 - 100,000x or more
- Use electron beams focused with magnets to produce an image
Names of electron microscopes
- Transmission (TEM)
- Scanning (SEM)
Scanning probe microscopes (magnification and what is used to view image)
- Magnification: 100 - 100,000,000x or more
- Use very short probes that are passed over the surface of the specimen and interact with it directly
Scanning probe microscope names
- Scanning tunneling (STM)
- Atomic force (AFM)
What is a Brightfield microscope?
- A compound microscope with two or more lenses
- Most common type of microscope
- Produces a DARK IMAGE on a BRIGHT BACKGROUND
- Light is transmitted, absorbed, reflected, or refracted by different structures (chromophores or stains)
- Ocular lens is typically 10x
- Oil immersion
- Can see objects as small as bacteria (visible at 400x), but not smaller objects like viruses
- Can’t be used to view live cells (instead used to view external structures)
What are chromophores?
- Pigment that absorb/reflect particular wavelengths of light
- Affects how colors behave as they interact
- Added using stains —> increases contrast/resolution
What are immersion oils?
- Increases the maximum angle at which light can strike the lenses
- Different oils for different lights
- Only use immersion oil with a specialized oil objective (usually 100x)
What are Darkfield microscopes?
- Similar to brightfield
- Has an opaque light stop between the illuminator and condenser that blocks light, producing a hollow cone of light that’s focused on the specimen
- Done without stains —> Used on living specimens
- Bright on dark background
- Good for small organisms
What are Phase Contrast microscopes?
- Use refraction and interference caused by structures in a specimen to create high contrast/ resolution images
- Annular stop in the condenser produces a cone of light focused on specimen —> specimen refracts/reflects the light —> light directly from condenser and light from the specimen are out of phase when they pass through the objective and phase plates —> wavelengths in phase add up and wavelengths out of phase cancel out (destructive interference)
- Done without stain —> Used for live specimens
- Good to view organelles in eukaryotic cells and endosperm’s in prokaryotic cells
What are Differential Interference Contrast (DIC) microscopes?
- AKA, Nomarski optics
- Two beams of light are created in which the direction of wave movement (polarization) differs
- Similar to phase contrast —> Use interference patterns
- Beams pass through the specimen or free space and are recombined —> causes differences in the interference patterns generated —> 3D appearance
- Can be done without staining —> Good for live specimens
- Images can reveal structures within cells
What are Fluorescence microscopes?
- Used for immunofluorescence
- Uses fluorescent chromophores (called fluorochromes) that can absorb energy from a light source, then emit it as visible light
- Fluorochromes include natural substances (ex: chlorophylls)
- Microscope transmits and excitation light (form of EMR with short wavelength like UV light) towards the specimen —> chromophores absorb the excitation light and emits visible light with longer wavelengths
- Bright colors against dark background
- Useful to distinguish between living and dead cells, pathogens, or particular species
What is immunofluorescence?
- A technique used to identify certain disease-causing microbes by observing whether antibodies bind to them
What are the two approaches of immunofluorescence?
- Direct immunofluorescence assay (DFA)/ primary antibody stain —> Specific antibodies are stained with a fluorochrome
- Indirect immunofluorescence assay (IFA) —> Secondary antibodies are stained with a fluorochrome and don’t attach directly to pathogen (BIND TO PRIMARY ANTIBODIES —> many secondaries may attach)
What are confocal microscopes?
- Uses a laser to scan multiple z-planes (other light microscopy focuses at a single distance)
- Produces multiple two dimensional images at various depths —> can be constructed into 3D images
- Fluorescence stains are generally used
- Useful for examining thick specimens (ex: biofilms)
What are Two-photon microscopes?
- Uses a scanning technique, fluorochromes, and long-wavelength light (ex: infared)
- Low energy from the long wavelength light —> two photons must strike a location at the same time to excite the fluorochrome
- Low energy is less damaging to cells
- Long wavelength easily penetrates THICK specimens (ex: biofilms)
- Useful to examine living cells with intact tissues
What are Electron Microscopes (EM)?
- Uses short wavelength electron beams rather than a light to increase magnification and resolution
- Electrons produce wavelengths of 0.005 nm
- For sub cellular structures and some molecular structures
- NOT FOR LIVING MATERIAL
What are Transmission Electron Microscopes (TEM)?
- A type of EM
- Similar to brightfield
- Uses an electron beam above that’s focused using a magnetic lens (rather than a glass lens) and projected through specimens and onto a detector (captures image)
- Specimens must be VERY THIN (20-100 nm) and DEHYDRATED
- Image produced by varying opacity in various parts of specimen —> can be enhanced with staining
What are Scanning Electron Microscopes (SEM)?
- Form images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons
- Specimens are DRIED and prepared with FIXATIVES that reduce artifacts (ex: shriveling) —> coated with a thin layer of METAL
- Can be used to view the three-dimensional surface details of specimens and larger objects
Who created the Environmental Transmission Electron Microscope (ETEM)?
- Pratibha L. Gai (later created the Environmental Scanning Transmission Electron Microscope (ESTEM) and was the first person to see atoms interacting)
- Edward D. Boyers
What are Scanning Probe Microscopes?
- Use very sharp probes that are passed over the surface of the specimen and interacts with it directly
- Doesn’t use light or electrons
What are Scanning Tunneling Microscopes (STM)?
- A type of scanning probe microscope
- Uses a probe passed horizontally at a constant distance just above the specimen while the intensity of the current is measured
- Can map the structure of surfaces at the atomic level
- Works best on conducting materials but can also be used to examine organic material (ex: DNA) if fixed on a surface
What are Atomic Force Microscopes (AFM)?
- Several ways
- Using a laser focused on a cantilever to measure the bending of the tip or a probe passed above the specimen while the height needed to maintain a constant current is measured
- Useful to observe specimens at the atomic level
- Can be more easily used with nonconducting samples
What are the two basic types of preparation used to view specimens with a light microscope?
- Wet mounts
- Fixed specimens
What is the wet mount preparation method?
- The specimen is placed on the slide in a drop of liquid (specimen —> liquid)
- Water or stains can be used
What is the fixation method?
- The process of attaching cells to a slide
- Heating or chemically treating the specimen
- The process kills microorganisms
- Chemical fixatives are often preferable for tissue specimens
What are stains?
- Used your apply color to certain features of a specimen before examining
- Contains salts with a positive ion and a negative ion
- The positive or the negative ion may be the chromophores (the colored ion) —> The un colored ion is called the counterion
What are basic dyes?
- Dyes in which the chromophore is the positively charged ion
What are acidic dyes?
- Dyes in which the chromophore is the negative ion
What are positive stains?
- Dye that is absorbed by the cells/organisms
- Add color to objects of interest to make them stand out against the background
What are negative stains?
- Dyes that are absorbed by the background but not by the cells/organisms
- Produces an outline or silhouette of the organisms against a colorful background
Cell walls are typically ______ charged? How does this influence how the chromophores in dyes interact?
- Cell walls are typically NEGATIVE
- Positive chromophores (absorbed) in basic dyes (positive colored ion) tend to stick to the cell walls
- Negatively charged chromophores in acid dyes are repelled by negative charged walls
Examples of basic dyes/positive stains
- Basic Füchsin
- Crustal violet
- Malachite green
- Methylene blue
- Safranin
Examples of acid dyes/negative stains
- Acid fuchsin
- Eosin
- Rose bengal
What is simple staining?
- A single dye is used to emphasize specific structures in a specimen
- Will make all of the organisms in a sample appear to be the same color
What is differential staining?
- Distinguishes organisms based on their interactions with multiple stains
- Ex: two organisms —> two different colors
What are examples of differential staining?
- Gram staining
- Acid fast staining
- Endoscope staining
- Flagella staining
- Capsule staining
Why was Gram staining created? By who?
- Developed by Hans Christian Gram
- To distinguish between bacteria with different types of cell walls
What is the process of Gram staining and what are the results?
1.) PRIMARY STAIN (crystal violet) is applied to a heat fixed smear —> all cells are purple or blue
2.) MORDANT (Gram’s iodine) is used to set/stabilize stains and dyes —> makes the crystal violet iodine complex climb and stain contained in thick layers of peptidoglycan in the cell walls
3.) DECOLORIZING AGENT (usually ethanol or acetone/ethanol mix) —> Gram positive cells remain purple or blue (cells that have thick peptidoglycan layers in their cell walls) OR Gram negative cells become colorless (washes the dye out of cells with thinner peptidoglycan layers)
4.) SECONDARY COUNTERSTAIN (usually safranin) —> Gram positive cells remain violet or blue OR Gram negative cells become pink/red
How is Gram staining used in a clinical setting?
- Helps classify bacterial pathogens in a sample into categories associated with specific properties
- Gram negative bacteria tend to be more resistant to certain antibiotics than gram positive
What are Acid-fast stains? What are the two types?
- Stains that are able to differentiate between two types of gram positive cells (those hat have waxy mycolic acids in their cell walls and those that don’t)
- Zeihl-Neelsen technique
- Kinyoun technique
What are similarities between the two types of methods for acid-fast stains?
SIMILARITIES
- Both use carbolfuchsin as the primary stain (acid-fast cells retain the carbolfuchsin even after a decolorizing agent is applied)
- Secondary counterstain (methylene blue) renders non-acid fast cells blue
DIFFERENCE
- Ziehl-Neelsen —> Uses HEAT to infuse carbolfuchsin into acid-fast cells
Kinyoun method —> NO HEAT
How do Acid-fast bacteria (AFB) results look and why are they important?
- AFB present —> Red/pink color against blue background
- A number of specific diseases are caused by AFB
What is the capsule/ its characteristics?
- A protective outer structure
- Certain bacteria and yeast have this
- Directly related to a microbe’s virulence (ability to cause disease)
- Don’t absorb most basic dyes —> negative staining is used
What is capsule staining?
- The process of using a NEGATIVE STAINING TECHNIQUE, making HALOS appear around the boarders of the cell
- No need to be heat-fixed
- India ink/nigrosin may be used
- A COMBINATION OF POSITIVE/NEGATIVE STAINING (positive stains the color of the body, negative stains the background, leaving a halo around each cell)
What are endospores?
- Structures produced within certain bacterial cells that allow them to survive harsh conditions
- Appear Lear when gram-stained cells are viewed
What is endospore staining?
- Uses two stains to differentiate endospores from the rest of the cell
- Schaeffer-Fulton method (most commonly used) uses heat to push the PRIMARY STAIN (malachite green) into the endospore —> washing the cell decolonizes cell but endospores are still green —> COUNTERSTAIN (safranin) makes the cell pink
- Important for identifying Bacillus and Clostridium (genera of endospore-producing bacteria)
What is a flagellum?
- Flagella are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes
- Typically can’t be seen under a light microscope without special technique
- The number and location of labella can help identify/classify bacteria
What is flagella staining?
- A processes that thickens the flagella
- First, MORDANT (generally tannic acid, but may also be potassium alum) coats the flagella, then the specimen is stained with PARAROSANILINE OR BASIC FUCHSIN
How are samples prepared for a TEM?
- Must have very thin sections —> cells are embedded in plastic resin, dehydrated with a series of ethanol solutions (replaces the water in the cells and dissolves the resin, entering the cell and solidifying) —> thin sections are cut using an ultramicrotome —> samples are fixed to fine copper wire or carbon-fiber grids and stained with substances that have electron-dense heavy metal atoms
How are samples prepared for an SEM?
- Dehydrated using an ethanol series (drier than needed for a TEM) —> critical point drying with inert liquid carbon dioxide under pressure is used to displace water —> Specimen is sputter-coated with meal by knocking atoms off a palladium target with energetic particles (sputter-coating prevents specimens from becoming charged by SEM’s electron beam)
How are samples for fluorescence and confocal microscopy prepared?
- Similar to samples for light microscopy but dyes are fluorochromes
- Stains are often diluted in liquid
- some dyes attach to an antibody (immunofluorescence)
- Others may attach to DNA molecules (Fluorescence in situ hybridization (FISH)) —> help stain certain DNA sequences
How are samples prepared for two-photon microscopy?
- Similar to fluorescence microscopy, but use infrared dyes
How are samples prepared for Scanning tunneling microscopy (STM)?
- Need to be on a very clean and atomically smooth surface
- Often mica coated with Au(111) and fixed with toluene vapor
Which organisms (prokaryotes, eukaryotes, bacteria) have a cell wall?
- ALL PROKARYOTES
- Some eukaryotes (ex: plants, fungi)
- Some bacteria
In 1858, Louis Pasteur performed his famous swan neck flask experiment. What theory did this disprove?
Spontaneous Generation (the twist in the flask caught all the microbes, preventing bacteria from growing in the flask)
What is mm?
Milimeter
What is µm?
Micrometer/micron
What is nm?
Nanometer
mm = ? m
mm = 10^-3 m
µm = ? m
µm = 10^-6 m
nm = ? m
nm = 10^-9 m
What size does an organism need to be in order to be visible to the naked eye?
At least 100 µm
What is the size order of microbes?
Viruses < Prokaryotes < Eukaryotes
What is the size range for eukaryotes?
Between 10 - 100 µm
Around what size are prokaryotes?
~ 1 µm
What is the typical size of viruses?
~ less than a nanometer (nm)
Who discovered the simple microscope?
Anthony Van Leeuwenhoek
Who discovered the compound microscope?
Robert Hooke
What is numerical aperture?
The ability to gather light
Viruses are _____ outside of hosts
- Inert; (can’t replicate)
- Can multiply within
What are examples of eukaryotes?
- Animals
- Plants
- Fungi
- Protists
- Protozoa
- Fungi
Coccus
- Prokaryotic cell shape (round)
Bacillus
- Prokaryotic cell shape (rod)
Spirillum
- Prokaryotic cell shape (spiral)
Coccus (arrangement)
- Prokaryotic cell arrangement (single coccus)
Diplococcus
- Prokaryotic cell arrangement (pair of two cocci)
Tetrad
- Prokaryotic cell arrangement (Grouping of four cells arranged in a square)
Streptococcus
- Prokaryotic cell arrangement (chain of cocci)
Staphylococcus
- Prokaryotic cell arrangement (cluster of cocci)
Bacillus (arrangement)
- Prokaryotic cell arrangement (a single rod)
Streptobacillus
- Prokaryotic cell arrangement (a chain of rods)
Sarcina
- Prokaryotic cell arrangement (two layers of four cocci - total of 8 cocci)
The divide in one plane (vertical through a single cocci) can lead to…
- Chain arrangement (strepto)
- Diplo arrangement
The divide in two planes (horizontal divide through two cocci) can lead to…
- Tetrad
- Sarcina (if there is a third plane division through all 4 in the tetrad)
An irregular divide (slanted divide) can lead to…
- Cluster
Bacillus typically divide…
- Only one plane of division —> can only form chains of rods (streptobacillus)
