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25 Cards in this Set
- Front
- Back
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aseptic techniques
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-insures no contamination of org being introduced
-no contamination of org present -no contamination of environment/handler |
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why are plates inverted during incubation?
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-prevent contamination
-prevent condensation (from dripping onto culture) |
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goals of smear prep (3)
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-adhere cells to slide
-so cells won't shrink during subsequent staining -think smeans |
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simple staining
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dyes with color-bearing ions that are positively charged (cationic) bc bacteria are negatively charged.
METHYLENE BLUE/CRYSTAL VIOLET |
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negative staining
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negatively charged dyes repelled by negatively charged bacteria.
-india ink & nigrosin |
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capsule staining
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negative stain - outlines the capsule surrounding the cell
positive stain - stain the cell. capsule appears as a halo. india ink, gently heat fix, crystal violet. |
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gram staining
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primary stain: crystal violet, all cells
mordant: gram's iodine, complexes w/gpos cells. decolorization: ethyl alcohol, removes violet from gneg cells counterstain: safranin, stains gpos cells |
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gram positive
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retains crystal violet/iodine complex b/c of larger peptidoglycan layer
PURPLE ex s. aureus |
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gram negative
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violet removed by alcohol. stained by safranin. thinner peptidoglycan layer (has outer mem)
RED/PINK ex. e.coli |
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spore staining
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prepare smear. steam smear over boiling water. saturate with MALACHITE GREEN. rinse. counterstain with SAFRANIN.
GREEN spores w/in RED vegetative cells. positive control: B. megaterium |
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acid-fast staining
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prepare smear.
primary stain: CARBOLFUCHSIN decolorize: acid alcohol counterstain: METHYLENE BLUE |
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acid-fast
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high lipid content (mycolic acid)
RED carbolfuchsin ex. m. smegmatis |
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non acid-fast
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low lipid content
BLUE methylene blue ex. s. aureus |
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coliforms
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found in intestines of humans/warm-blooded animals. ferment lactose to produce acid and gas.
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why are standard plate counts reported as CFUs rather than cells?
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uncertainty in how many actual cells form a colony
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how does a counterstain differ from a primary stain?
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counterstain: less intense. gpos cell stained by primary stain while gneg cells stained by counterstain. provides contrast.
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what is the most critical step in the gram stain?
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decolorization (by ethyl alcohol)
if over applied, dye-mordant complex can be removed from gpos cells, converting them to gneg cells. |
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why is culture age important in gram stains
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ideally: 16-18 hours
18+ can convert to gram variable or gneg. |
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why is smear thickness important in gram stains
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thin smears allow for observation of individual cells and the arrangement.
thick smears entrap more than necessary stains/dyes |
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differentiate between complex and defined media
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complex: exact composition & amounts of aa, vitamins, growth factors, etc are NOT completely known
defined: all specific compositions/amounts known |
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6 basic nutritional requirements supplied in all cultural media
fastidious bacterial pathogens? |
carbon source, energy source, nitrogen, minerals, vitamins, growth factors & water
fastidious require blood/serum components |
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define sterilization:
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process by which all living cells, spores, viruses, and viroids are destroyed or removed.
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define selective medium:
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w/components that allow certain bacteria to grow but will inhibit growths of other org
ex. msa (mannitol salt agar) |
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define differential medium:
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contains substances that cause some bacteria to take on an appearance that distinguishes them from other org.
ex. mas (mannitol salt agar) |
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AGAR:
origin? why is it ideal? |
orign: seaweed, Robert Koch's lab
ideal: melts at 100C but doesnt solidify until its cooled to 45C. bacteria can be innoculated into agar w/o killing cells. also, not nutrient for most bacteria. semi-soft agar can be used to study motility |
