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Pure culture
contain only one type of organism in any type of media
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colony
genetically identical group of organisms, arise from a single cell
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criteria for isolated colonies
distance from other colonies
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Not flaming btw Area 3 & 4
few organisms remain btw area 3 and on loop, if u flame, no bacterial growth will occur on area 4
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why can't u cross track
when u cross track, pick up more microbes and make dilution process more difficult
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loop should not go near area 1
- area 1 is confluent, diluted micrboes from area 3will be concentrated again
- no isolated colonies will appear in area 4
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Cultural characteristics
- Form
- texture
- optical characteristics
- color (pigmentation)
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Starch hydrolysis
- Enzyme1: Amylase,
- Product: Maltose
- Enzyme2: Maltase
- Product: glycosis (energy)
- Media: Starch Agar plate
- (-) amylase & maltase- blue black precipitation around growth
- (+) amylase & maltase - no starch left, no blue black precipitation
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Caesin Hydrolysis
- Enzyme: protease, caseinase
- Product: amino acids
- Media: milk agar plate
- No zone of clearing around growth: (-) proteolysis, (-) caseinase
- Zone of clearing around growth: (+) proteolysis, (+) caseinase
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Gelatin hydrolysis
- Enzyme: gelatinase
- Product: amino acid
- Media: Nutrient gelatin deep tube
- ** Need to refrigerate for 30 mins after incubation to read the result
liquid after 48hrs incubation and 30mins refrigeration: rapid gelatin hydrolysis, (+) gelatinase
Not liquid after all incubations and refrigerations: (-) gelatinase
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Why u shouldn't shake gelatin tube after incubation
Mixed hydrolyzed and unhydrolyzed gelatin = false negative
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why carbs fermentation tube results should be read in 48 hrs
after 48 hrs peptone may be broken down and alkaline products produced
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Results
- Yellow - (+) fermentation
- Original red - (-) fermentation
- deep purple or hot pink - (+) alkaline, (-) fermentation
- Gas in tube - (+) gas production
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why is it important to check bacterial growth if the tube is red?
- in order to make sure fermentation has occured,
- if no bacteria, no fermentation
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Hydrogen Sulfide test
Why use peptone iron deep
- 1. to enhance anaerobic respiration
- 2. to allow observation of motility
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H2S result
- Black precipitate along the stab = (+); H2S production
- No black precipitate along the stab = (-) H2S production
- Growth not restricted to stab line = (+) motility
- Growth restricted to stab line = (-) motility
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Indole production test
- Enzyme: tryptophanase
- Media: tryptone broth
- Reagent: Kovac's reagent
- (+) : red layer on top of media
- (-): no red layer formed
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Methyl red test
- To differentiate E.coli and Enterobacter
- Media: MRVP broth
- Reagent: methylred reagent
- (+) : red= (+) for acid end product
- (-): colorless (yellow) = (-) for acid end product
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VP test
- Media: MRVP broth
- Reagent: Barritt's Reagent
- (+) = rose color within 15 mins, (+) for non acidic and neutral end products
- (-) = no color develops within 15 mins = (-) for nonacidic or neutral end products
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Citrate Utilization test
- Enzyme: citrease permease ; citrease
- Media: Simmon's citrate agar slant
- (+) = growth of bacteria, blue coloration = (+) citrease, (+) citrease permease
- (-) = no growth on slant, remains green = (-) citrease, (-) citrease permease
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Urease test
- to identify Proteus Vulgaris
- Urease: hydrolyze urea and form alkaline end product
- Media: Urea Broth (contain phenol ph indicator)
- (+) : deep pink = (+) urease (alkaline)
- (-) : no color change = (-) urease
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