Microscopy and Specimen Preparation Flashcards
What is light microscopy?
compound light microscopes use visible light to illuminate specimens
What are the types of light microscopy?
- bright field
- dark field
- phase contrast
- fluorescence
What is the refractive index?
- describes how much light is refracted (bent) when passing from one medium to another
- light rays bend towards the normal once they pass through the lens
What focal length (f) gives you greater magnification?
shorter focal length (distance between the normal point and the focal point (F))
How are specimens visualized in Bright-Field microscopy?
due to differences in contrast (density) between specimen and surroundings
How is total magnification calculated?
magnification of ocular lens x magnification of objective lens
upper limit for Bright-Field= 1000x
What is resolution (d)?
the ability to distinguish between 2 objects as distinct and separate when viewed under a microscope
d= 0.5(λ)/n(sinθ) d= 0.5(λ)/NA
upper limit for Bright-Field= 0.2 micrometers
What are ways to achieve a better resolution?
- use a shorter wavelength
- decrease the working distance (bring objective lens closer to specimen)
- increase refractive index (n)
What is the numerical aperture (NA)?
- a measure of light gathering ability
- larger=higher resolution
What does fixation do?
- preserves specimens and fixes them in position
- organisms are usually killed and firmly attached to the microscope slide
What are the two types of fixation?
- heat fixation: routinely used for bacteria and archaeons
- chemical fixation: used with larger, more delicate organisms
What does staining do?
makes internal and external structures of a cell more visible my increasing contrast with the background
What are the two common features of stains?
- chromophore groups: chemical groups with conjugated double bonds, gives color
- the ability to bind cells
What are basic stains and what do they bind to?
- dyes with positive charges
- bind to negatively charged structures: nucleic acids, proteins, surfaces of bacteria and archaea
What are acidic stains and what do they bind to?
- dyes with negative charges
- bind to positively charged structures
What is simple staining?
- the use of a single stain
- for visualization of morphological shape and arrangement
What is differential staining?
- the use of two contrasting strains separated by a decolorizing agent
- can be used for identification: gram stain, acid fast stain
- -presence or absence of structures
- can be used for visualization of structure: spore stain, capsule stain (can be simple or differential)
What are the steps of the Gram stain?
- Flood the heat-fixed smear with crystal violet for 1 min
- Add iodine solution for 1 min
- Decolorize with alcohol for 20 sec
- Counterstain with safranin for 1-2 min
What color are gram-negative and gram-positive cells after flooding with crystal violet?
both purple
What color are gram-negative and gram-positive cells after being treated with iodine?
both purple
What color are gram-negative and gram-positive cells after decolorizing with alcohol?
- gram-negative is colorless
- gram-positive is purple
What color are gram-negative and gram-positive cells after counterstaining with safranin?
- gram-negative is pink
- gram-positive is purple
What does the Gram stain divide bacteria based on?
- differences in cell wall structure
- gram-positive cells have a thick layer of peptidoglycan and no outer membrane
- gram-negative cells have a thin layer of peptidoglycan with an outer membrane
What is the acid-fast stain used for?
- useful for staining members of the genus Mycobacterium that don’t stain well with the Gram stain
- acid-fast cells have high lipid (mycolic acid) content in their cells walls which is responsible for their staining characteristics
- wax-like, impermeable cell walls
What do the colors represent in acid-fast staining?
- pink=acid-fast cells
- blue=non acid-fast cells
What is endospore staining?
- heated, double staining technique
- bacterial endospores are stained green (malachite green) and vegetative cells are stained pink (safranin)
- the heat is used as the mordant
- bacteria with endospores are harder to stain because of their layers of protein
What is capsule staining?
- uses a negative stain (colors the background)
- the capsules are colorless against a stained background (look like a white halo)
- capsules are made out of sugar and therefore do not pick up the stain
What is flagella staining?
- mordant is applied to increase the thickness of the flagella so that it can be viewed under the microscope
- flagella are fragile and difficult to stain
- uses basic stain
How are specimens prepared and stained?
- Spread culture in thin film over slide
- Dry in air
- Pass slide through flame to heat fix
- Flood slide with stain; rinse and dry
- Place drop of oil on slide and examine with 100x objective lens
