Microbiology Chap 3 Flashcards
Total Magnification=
objective lens+ ocular lens
ocular lens
10x
objective lens
10x, 40x, 100x
Resolution
ability of lens to distinguish between two points
refractive index
measure of light bending ability of medium
brightfield illumination
dark objects are visible against bright objects
darkfield illumination
light objects are visible against dark objects
Phase-contract microscopy
permits detailed examination of living specimens
Differential Interference Contrast (DIC) Microscopy
Accentuates diffraction of light that passes through a specimen
-Uses 2 beams of light; prisms split light beam adding color
Fluorescence Microscopy
- Uses UV light (short wavelength)
- Fluorescent substances absorb UV light & emit visible light
- Cells may be stained with fluorescent dyes (fluorochromes)
- Uses fluorescent antibodies to identify unknown microbes
Scanning Acoustic Microscopy (SAM)
Measures sound waves that are reflected back from an object
Electron Microscopy
Uses electrons instead of light; have electromagnetic lens to focus beams of electrons
-Shorter wavelength of electrons gives greater resolution
Transmission Electron Microscopy
-Beam of electrons passes through specimen, then electromagnetic lens,
then focused on a projector lens
-Specimens may be stained with heavy metal salts
-Magnifies objects 10,000 to 100,000; resolution of 10 pm
Scanning Electron Microscopy
- Electron gun produces beam of electrons that scans surface of whole specimen
- Secondary electrons emitted from specimen produce 3-D image
Scanned-Probe Microscopy
Scanning tunneling microscopy (STM) uses metal probe to scan specimen
Staining
coloring microbe with dye that emphasizes certain structures
Smear
thin film of solution of microbes on slide
Smear is fixed by ….
Smear is fixed (attached) to slide, usually through heat or methyl alcohol stain is applied rinsed with water blotted with absorbent paper
Stains
consist of a positive and negative ion, one of which is colored (chromophore)
Stains in different dyes
In basic dye, chromophore is cation
In acidic dye, chromophore is anion
Negative staining
Staining background instead of cell
Simple stain
use of single basic dye
-Highlights the entire microorganism to visualize cell shapes and structures
Mordant
(chemical that intensifies stain) may be used to hold stain or coat specimen to enlarge it
Differential Stains
Used to distinguish between bacteria
Gram stain
Acid-fast stain
Special Stains
Used to distinguish parts of cells
Capsule stain
Endospore stain
Flagella stain
Gram-positive bacteria
tend to be killed by penicillin & detergents
Gram-negative bacteria
are more resistant to antibiotics
Acid-Fast Stain
Stained waxy cell wall is not decolorized by acid-alcohol
Negative Staining for Capsules
- Cells stained using simple stain
- Negative stain using India Ink or nigrosin
- Capsules are not stained but appear as halos around cells
Capsules:
gelatinous covering of microbe; can determine virulence
Endospore Staining
- Primary stain: Malachite green, usually with heat
- Decolorize cells: Water
- Counterstain: Safranin
- Endospores appear green in red or pink cell
Endospore
special resistant, dormant structure formed within cell to protect it from adverse environmental conditions
Flagella Staining
- Flagella are used for locomotion & are too small to be seen with light microscope without stain
- Mordant & simple stain carbolfuchsin build up flagella until it is visible under light microscope
