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- Also can fuse DNA from fluorescent protein to a gene of interest (Protein Tagging)
- Fluorescent protein is then made any time your gene is made
- No antibodies needed
- We are able to see the proteins that are being made inside of the cell with color
- Some bacteria can be naturally fluorescent
- Version of Fluorescence Microscopy
- Cover everything with fluorescent dye
- 3D Images
- Laser scanning
- Puts image back together again
- UV light is used
- The scope analyzes the bacteria in the computer itself
- Advantages: It's done quickly, online, and it's easy to share with others
- Disadvantages: Limited power, costs a lot of money ($500,000-$1,000,000)
- No light waves, electrons, good for very small
- They are used with vaccum sealed air. When electrons are shot down, they must be vaccumed up or else they would scatter.
- Electromagnets: They focus the beams of electrons since there are NO lenses.
Scanning vs. Transmission? (Electron Microscopes)
- Transmission: The specimen is in a thin section of plastic, it's see through
- Scanning: At the bottom of the scope, cover with metal (electrons bounce off, computer records 3D images, artificially colored)
- The images are colored afterwards
Scanning Tunneling Microscopy (STM)
- You run a metal probe over a surface
- Electrons interact and create a current
- It can get down to visualize the individual atomic level
- 3D Structures
- Like reading braille
Atomic Force Microscopy (AFM)
- Visualizing atoms to 1 micrometer
- The technology is newer
- It has the same idea as STM
- 3D Structures
- Like reading braille
Wet Mounts - Regular vs. Hanging Drop?
- Regular Slide: A drop of cells is put on a flat surface, more difficult to see since the cells are spread out over such a large surface area.
- Hanging Drop: Instead of being flat, there's a divot where the drop is placed and organisms are better seen. However, more expensive.
- Must air dry before heat fixing because if not, you'll end up boiling the cells and killing them
- 3 Things of Heat Fixing: Kills bugs, denatures proteins so they adhere to the slide, makes bugs take up dye easier.
Most are basic (+) so they are attracted to bacteria cell walls (-)
Simple Stain vs. Differential Stain
- Simple Stain: Everything is the same color
- Differential Stain: For gram staining, 2 or more dyes (different cells are different colors)
Gram Stains - Which are used? Color? Order?
- Crystal Violet: Turns everything purple
- Iodine: Mordant, it locks in Gram +
- Alcohol: Turns Gram - invisible
- Safranin: Turns the invisible Gram - cells pink
- If one of the steps is missed, the cells will be misinterpretted and all will become crystal violet
Acid Fast/Negative Stain
- Sometimes bacteria have an outer capsule on top of the peptidoglycan that makes the bacteria invisible in out bodies, so then they won't stain
- This type of staining will help us see the actual cells (which are dangerous) with the clearing around them
Use a metal stain with a hanging drop to see the bacteria around the flagella
Heat is used to break down the spore wall and the inside becomes green
- Pro=Before, Karyon=Nucleus (Before Nucleus)
- For Eukaryotic: Eu=True (True Nucleus)
- Some big difference between Pro and Euk: Nucleus, Circular DNA vs. Linear, and true organelles are only in Eukaryotes
- Single: Only one
- Diplo: Two stuck together
- Strepto: Chain
- Staphylo: Circular cluster
- Tetrad: 4 circular
- Sarcinae: 8 circular
Shapes of Cells
- Bacillus: Long
- Cocci: Circular
- Vibrio: Comma-shaped
- Spirillium: Wave-like, ALWAYS have flagellum
- Spirochete: Wavier, DO NOT have flagellum
- NAG and NAM: Sugars, chain-link fence, make up peptidoglycan
- Peptide cross bridges
- Cell membrane, and then a thick wall of Peptioglycan on top.
- Contains Teichoic Acid which fixes in the gram stain (purple).
- Makes exotoxin, which is not attached.
- Cell membrane, thin layer of peptidoglycan, and outer membrane
- Periplasmic Space: Digestive enzymes and protein pumps
- Lipid contains endotoxin (LPS) which is inside the cell and permanently stuck in the outer membrane.
Acid Fast Cell Wall - what makes it different?
There is a thick lipid layer on the outer part of the cell wall (mycolic acid) that repels stains (found in TB and leprosy)
The 2 acids for Gram +?
- Teichoic Acid: Iodine sticks to it to get crystal violet stain
- Mycolic Acid: Repels stains