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What is the relationship between the millimeter, micrometer, and nanometer?
1 milimeter (mm) is the size of a lower case "o"
1 mm= 1000 Mm(micrometer)
1Mm= 1000 nm (nanometers
How does wavelength relate to the visible color spectrum (roygbv)?
Light travels in waves.
- Red:780-622 nm
- Orange: 621-597 nm
- Yellow:596-577 nm
- Green: 576-492 nm
- Blue: 491-455 nm
- Violet:454-390 nm
Visible light waves are between 780-400nm
Define Magnification and Resolution. Whats the difference?
Magnification: degree of which the scope enlarges the specimen's image
Resolution: a measure of clarity of the image
You can magnify something as big as you want, but if you do not have resolving power then it will be blurry and pointless
What is the resolution limit? What is the smallest thing that we can then see with a light microscope?
The resolution limit is ~1/2 wavelength in nm
The resolution limit for a light microscope with the eye is 200 nm the smallest visible wavelength is violet at 400nm
other things that can limit resolution: quality and cleanliness of the lens
What makes a specimen visible under a light microscope?
In order for a specimen to be visible under a microscope, it must distort the light waves passing though it
Why is immersion oil used in light microscopy?
immersion oil is used in order to bend the light waves directly into the objective lens and it get a better picture of the MO. Otherwise, the light waves would miss the objective lens. The oil acts as an extension of the glass lens directly onto the slide
Describe the total magnification of a light microscope with the ocular lens and the objective lens
Ocular lens is 10x magnification. Multiply this by whatever the objective lens is:
- 4x times 10x=40 total mag
- 10x times 10x = 100 total mag
- 40x times 10x =400 total mag
- 100 times 10x =1000 total mag
What is the approximate size of a eukaryotic cell?
What is the approximate size of a bacterium?
What is the approximate size of a virus?
What is the approximate size of a protein molecule?
What is a light microscope and how does it work? What are three types?
- Most commonly used is the brightfield microscope. It is best for stained cells, as live cells are transparent. It's application is that its used to view stained cells
What is the application of a darkfield microscope and how does it work?
- Only the light that travels through the MO is seen. That means that the MO does not to be stained and it can be viewed live. It's application is to check motility and to look for cells in a sample
what is the application of doing florescence and how does it work?
Flourencent dye molecules (flourochromes) are added to the MO and a UV lamp is used (light waves too short for human eye to see). The flourochromes make the light waves longer so that we can see them, typically a green color.
- how does it work? Its a key and lock system for antibodies that attach to the MO. Specific flourochromes are added to specific antibodies that attach to the MO and create an immunofluoresence
- *a very good way to detect specific MO in a sample
How does a TEM Microscope work and what is it used for?
- Transmission Electron microscope
- shoots electron beams of a much shorter wavelength. This allows us to view much smaller MO. There is a vacuum in the TEM because e- beams are scattered by air and dust
-application is to view high resolution pics of SLICES of cell interiors (harsh prep and cells are dead)
How does a scanning microscope work and what is it used for?
The microscope actually scans the exterior of the cell and therefore the application us ti see high resolution pics of the cells surface (also dead)
Why do the short wavelengths of electron beams allow for higher resolution images?
because the resolution limit is 1/2 the wavelength, shorter wavelengths let us view smaller objects
What is the magnification and resolution of the unaided eye?
- magnification: 1x
- resolution: 100Mm (size of the smallest visible object)
What is the magnification and resolution of the light microscope?
- magnification: 1000x
- resolution: .2Mn (200nm)
What is the magnification and resolution of the Scanning EM?
- Magnification: 1000-10,000 x
- Resolution: 10nm
What is the magnification and resolution of the Transmission EM?
- Magnification: 10,000-100,000x
- Resolution: 2.5 nm
How do you prepare a bacterial smear for staining?
- Use the aseptic technique to put the smear on the slide
- Air dry and then heat fix
What is the difference between a simple and a negative stain? What are their applications?
Simple stains are one step. The bacteria is usually (-) charged and the dye is (+) charged such as crystal violet, safranin, and methalyne blue
Used to simply view bacteria, not to ID them necessarily
Negative stains are a (-) bacteria and a (-) stain. The bacteria are not distorted as much.
Why are gram stain and acid-fast called "differential stains"?
They use several steps and different stains such as a counterstain in order to stain the MO for viewing
Define: primary stain, decolorizer, and counterstain
Primary stain will color the MO you are looking for. The decolorizer is normally some kind of acid which removes the stain from some MO (such as gram -). The counterstain stains those MO which were decolorized in order to provide a method of ID as well as contrast
Who invented the Gram stain?
H.C Gram (Danish) in 1884 developed the staining technique we call the Gram stain
What is the procedure for a gram stain? What will gram + and Gram - look like after proper staining?
- 1. Apply crystal violet to heat fixed slide for 1 minute
- 2. Rise off Cv with water
- 3. Apply iodine as a mordant for 1 minute
- 4. Decolorize with 95% ETOH
- 5. Counterstain with safranin
Gram + will be dark purple from the cv, while the gram - will be pink from the safranin. The gram + have thick walls which hold in the cv, while the thin walls of the gram - are easily decolorized
What is the procedure for Acid-fast stain? What genera of bacterium are acid-fast and why?
- 1. Apply Carbohlfucshin to the heat-fixed smear
- 2. gently heat this slide over steam for 5 min
- 3. Decolorize with 95%ETOH/3%HCL
- 4. Counter stain with methalyne blue
Mycobacterium have thick waxy exteriors that make them hard to dye, but once they are, they will stay pink from the carbohlfucshin. Non-acid-fast will appear blue from the counterstain.
What is the endospore staining procedure? What color will the endospores be? Name 2 bacterial genera that form spores
- 1. Apply malachite green to heat fixed smear
- 2. Heat slide over steam for 5 min
- 3. Rinse heavily with water
- 4. Counterstain with safranin
Also very difficult to stain, but one they are, they will hold onto the malachite green. Dead and vegetative cells will be pink from the counterstain.
Endospores are dormant and durable forms of the cells.
Bacillus and Clostridium are two genera that form spores
What is the main purpose for differential staining?
To ID the MO.
Gram stains are done first to help ID. There are no known acid fast or endosore that are gram -.