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Topic : Types Microscopy
Define Resolving power
ability to completely separate to two objects in a microscopic field
d = λ /NA
- -[d = resolution, λ = wavelength of light, NA = numerical aperture of lens]
- -NA is a mathematical expression that describes how the condenser lens concentrates and focuses the light rays from the light source; its value is maximized when the light rays are focused into a cone of light that passes through the specimen and into the objective lens; NA is diminished by refraction [diaphragm, condenser, immersion oil]
immersion oil has what?
the same refractive index as glass.
What can be can more easily observed using darkfield microscopy?
Delicate transparent living organisms
A microscope that is able to differentiate transparent protoplasmic structures and enhance the contrast between living cells and their surroundings, without the necessity of staining, is what microscope?
The phase-contrast microscope.
Hoffman modulation contrast is another technique which does what?
Increases the contrast in specimens (by changing the amplitude of light rather than the wavelength as in phase)
What are the 6 different shapes?
- spherical (cocci)
- cylindrical (rods/bacilli)
- helical (spirilla)
- square (some Archaea)
- multicellular (filamentous, mycelial)
What are the 2 diff. types of Arrangement?
- -cocci (single, diplococci, streptococci, staphylococci, tetrads)
- -bacilli (single, streptobacilli, palisades)
involves preparing a thin smear of cells, adhering cells to the microscope slide to prevent them from washing off, and limiting shrinkage of the cells. (Heat-fix vs. chemically fixed with methanol) and involves chemicals that possess color-bearing ions called chromophores. Basic dyes have a cationic (positively charged) chromophore. Acidic dyes have an anionic (negatively charged) chromophore. Bacterial cells possess a slightly negative charge.
Simple staining (direct stain)
involves the use of a single stain to color a bacterial cell.
cause the background area around a cell to be opaque or dark; useful for viewing shape, morphology, capsules
Differential staining techniques
utilize more than one chemical stain in order to differentiate between different microorganisms or structures/cellular components of a single organism.
Gram staining define - full process
- differentiates bacterial cells with thick peptidoglycan walls (gram-positive) from those without (gram-negative).
- -Apply primary stain (crystal violet)
- -Apply mordant (Gram’s iodine) to form CV-I complex [large molecules that cannot be washed out of gram+ cells]
- -Apply decolorizing agent (EtOH or EtOH-acetone) [dissolves LPS and CV-I washes out through thin peptidoglycan layer]
- -Apply counterstain (Safranin)
- *PROBLEMS: old cell walls are degraded and do not retain primary stain (false negative); over-, under-decolorizing
Acid-fast staining define - full process
- Acid-fast staining differentiates bacterial cells with mycolic acid in their cell walls (Mycobacterium, Nocardia) from those without.
- -Apply primary stain (carbolfuchsin with 5% phenol) either with heat (Ziehl-Neelsen method) or high concentration (Kinyoun)
- -Apply decolorizing agent (acid-alcohol)
- -Apply counterstain (methylene blue)
utilizes special stains to penetrate the endospore. (Schaeffer-Fulton, Dorner)
utilizes direct and negative stains to differentiate cell, capsule, background
Topic: Microbiological medium
What are the Nutritional requirements? (4)
- -carbon/energy source (absent from media for autotrophic growth)
- -nitrogen source (absent from media of nitrogen fixers)
- -growth factors (numerous for fastidious organisms, minimal for prototrophs)
Media composition, define the 2 types
- -complex medium (exact composition unknown)
- -defined or synthetic medium (exact composition known); minimal medium is a type of defined medium which meets minimal nutritional requirements for a specific strain
Media for Isolation name and define the 3 types.
-Selective media – contains compounds to inhibit the growth of certain bacteria (antibiotics, dyes, salt, etc.)
-Differential media – contains chemicals that act as indicators of certain physiological characteristics
-Enrichment media – contains chemicals that preferentially promote the growth of certain bacteria
Culture techniques name one the one.
What does Aseptic technique do?
- -insures that an aseptic technique is maintained when handling organisms. Its two major principles involve:
- -No contaminating microorganisms are introduced into culture or culture materials.
- -The microbiologist is not contaminated by cultures that are being manipulated.
Proper aseptic technique involves:
- -Work area disinfection
- -Flaming of loops/needles
- -Culture tube flaming/inoculation
Pure cultures enable microbiologists to do what?
Study the cultural, morphological, and physiological characteristics of an individual species. Two techniques for obtaining pure cultures are the streak plate and the pour plate
Both streak plate and pour plate do what? (3)
- -Both involve dilution of bacteria to obtain pure colonies (assumed to be identical progeny of an individual cell but actually probably arise from a group of similar bacteria; thus officially known as a colony-forming unit or CFU)
- -Growth in liquid cultures does not produce colonies so isolation of pure cultures is impossible; growth in liquid culture is referred to as turbidity.
- -Pure colonies can be subcultured to produce pure cultures.
Media Preparation for broths, slants, deeps, plates involve what?
- -Solid media contain agar, complex polysaccharide from seaweed; properties of agar include (1) melting temperature of 100oC, (2) solidifying temperature of 45oC, (3) does not serve as a nutrient for most bacteria (except for a few marine organisms)
- -Sterilization with moist heat by autoclaving involves heating materials to 121oC for at least 15 minutes at 15 psi (material must be in contact with steam to be sterilized); filter sterilization involves passing solutions through bacteriological filter (pore size, 0.45 um)
reparation of stock cultures is necessary to do what?
preserve and maintain pure cultures (working culture vs. reserve culture)
ways of preparation
- -Freezing (often with glycerol to prevent formation of ice crystals; -196oC [liquid N2] better than conventional freezer [-20oC])
- -Storage under mineral oil
Populations or communities of microorganisms that attach and grow on a solid surface that has been exposed to water (i.e. pellicle)
ability of bacteria to communicate and coordinate behavior (signaling chemical called an inducer)
Cultivation of anaerobes
- -fluid thioglycollate medium (sodium thioglycollate [reducing agent], resazurin [O2 indicator], low level of agar)
- -GasPak anaerobic jar (H2, CO2 generated, Pd catalyzes 2H2 + O2 Â–> 2H2O, methylene blue indicator strip)
Organisms that require oxygen to live.
an organism, especially an aerobic bacterium, that lives and thrives in environments low in oxygen
Organisms that can live in the presence or absence of oxygen. fermentation or respiration
Aerotolerant anaerobes do not utilize oxygen, and is not affected by it.
Organisms for which the presence of molecular oxygen is toxic.