Module 3 Flashcards
(40 cards)
1
Q
Micrometer
A
- um
- Used to measure size of cells
- One-millionth of a meter, 10^-6m
2
Q
Nanometer
A
- nm
- Measures cellular organelles and viruses within a cell
- One-billionth of a meter, 10^-9
3
Q
Resolution
A
- Ability to distingush 2 points that are close together (resolving power)
- High resolution: distinguishable and clear
- Low resolution: blurry
4
Q
Constrast
A
- Difference of light absorbance btwn 2 objects or the object and its background
- Most microorganism are colorless
> staining increases contrast
5
Q
Microscopy
A
Using light or electrons to magnify objects
6
Q
Measurement
A
To measure an object, the unit must be smaller-scale than the object itself
7
Q
Magnification
A
- Increases the size of an object
- Results when radiation (light) bends as it passes through a lens
8
Q
Condenser
A
Converges light beams into a focused area on a sample
9
Q
Light Microscope
A
- Use visible light and blue wavelengths for improved resolution
- Magnification: 1x-2000x
- Resolution: 10mm-200mm
- Can see whole cells and organelles
10
Q
Types of Microscopy
A
- Light microscope
- Electron microscope
- Probe microscope
11
Q
Bright Field Microscopy
A
- Count microorganisms or cells
- View stained specimen
- View live unstained specimen
- Bright background with clear or colored specimen
(most commonly used)
12
Q
Phase Contrast Microscopy
A
- View internal structures of live specimen
- Observe motility (cilia & flagella)
- No staining needed
- Image shows light and dark areas of microbe
13
Q
Dark Field Microscopy
A
- View living, unstained specimens
- Filter inhibits light from going through organism and instead light is reflected by organism
- Dark background with bright specimen
14
Q
Types of Light Microscopy
A
- Bright Field
- Phase Contrast
- Dark Field
- Fluorescence
- Confocal
15
Q
Fluorescence Microscopy
A
- Localize specific structures or molecules
- Diagnostic tool
- Uses UV light to excite fluorophores
- Visualize whole cells, specific structures, or proteins and watch movement or interactions
- Dark background with florescent structures
16
Q
Immunofluorescence
A
Florescent dyes are linked to antibodies which find a cellular target and bind to it bringing the dye that can visualize and located the structure
17
Q
Confocal Micrscopy
A
- Highly detailed structures
- 3D renderings
- Biofilms
- Uses a laser to focus plane by plane through the specimen
- Single plain of structures stained with fluorescent dyes
18
Q
Electron Microscopy
A
Electron beams give shorter wavelengths than light to increase magnification and resolution
19
Q
Scanning Electron Microscopy (SEM)
A
- Visualize surface details
- 3D view of surface of cell or cellular structure because electron beam reflects off surface of specimen treated with gold or palladium
- 3D rendering of surface of object or shell
20
Q
Transmission Electron Microscopy (TEM)
A
- Visualize internal ultrastructural details of cells, viruses, and bacteria
- 2D view of subcellular organelles, substructures, or viral particles as electron beam passes through specimen
- 2D highly magnified picture
21
Q
Staining
A
- Using dyes to improve resolution and contrast of a sample for visualization via light microscopy
22
Q
Benefits to Staining
A
- Increase contrast
- Allows for determining cellular…
> shape
> size
> arrangement
> number
23
Q
Steps for Preparing a Slide
A
- Smear
> liquid culture
> solid culture - Fixation (adhere to slide)
> heat
> chemical
24
Q
Chemistry of Staining
A
Stains are salts, which break down into positively and negatively charged ions
25
Chromophore
Colored ion in the dye
26
Acidic Dyes (Na+ Dye-)
- Anionic (-) chromophores that stain positively charges structures in acidic conditions (low pH)
> mostly used for "negative staining"
27
Basic Dyes (Dye+ Cl-)
- Cationic (+) chromophores that stain negatively charged structures in basic conditions (high pH)
> most common
28
Simple Stain
A single basic dye with and involve soaking in the dye for 30-60 seconds and rinsing
> ex: crystal violet, safranin, fuchsin or methylene blue
29
Differential Stain
At least 2 dyes are used to show different cells, structures, or chemicals in sample and is a multistep process
> ex: gram, acid-fast, endospore, hematoxylin, and eosin staining
30
Special Stains
Simple stains that visualize specific structures
> ex: negative stains, flagellar staines and fluorescent stains
31
Gram Stain
- Takes advantage of difference in the bacterial cell wall
- Divides bacteria into 2 groups
32
Gram Positive Bacteria
Thick peptidoglycan layer
33
Gram Negative Bacteria
- Thin peptidoglycan layer
- Outer membrane
34
Step 1 - Gram Stain
- Slide is flooded with crystal violet for 1 min, then rinsed with water
- Result: all cells are stained purple
35
Step 2 - Gram Stain
- Slide is flooded with iodine for 1 min, then rinsed with water
- Result: iodine acts as a mordant, all cells remain purple
36
Step 3 - Gram Stain
- Slide is rinsed with solution of ethanol and acetone for 10-30 sec, then rinsed with water
- Result: smear is decolorized; gram-positive cells remain purple, but gram-negative cells are now colorless
37
Step 4 - Gram Stain
- Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry
- Result: gram-positive cells remain purple, gram-negative cells are pink
38
Negative Staining
- Acidic, negatively charged
- Cells remains colorless but background is stained to give contrast
- Creates "capsule" effect
39
Acid-Fast Staining
- Stains mycobacterium and nocardia
> cells that resist decolorization
- Primary dye: carbofuchsin (red)
- Counterstain: methylene blue
- Cell remains colorless but background is stained to give a contrast
- Tuberculosis, leprosy, lung and skin infections
40
Giemsa
- Detects blood parasites
- Mix of multiple dyes that gives it neutral charge
- Mix of methylene blue, eosin, and azurer
- Stains nuclei of WBCs bluish/purple and cytoplasm pink
- Diagnosis of malaria and blood protozoa, Chlamydia trachomatis, Borrelia, Histoplasma, Pneumocystis jiroveci cysts
