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what are the methods to classify and distinguish bacteria?
- macroscopic: colony appearance
- microscopic: gram stain, immunostaining (ELIZA)
- serologic: reactions with antibodies
- Molecular: nucleic acid or protein detection/sequence, cell surface structures
- metabolic: growth on/use of certain nutrients, by products, aerobic/anaerobic
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describe macroscopic traits
- colony morphology, solor, shape, size, patterns
- hemolysis on blood agar
- growth and color on selective and differential media
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describe this types of hemolysi
- Alpha-hemolysis
- partial lysis leaving greenish/brownish halo around colonies
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describe this type of hemolysis
- strong B-hemolysis
- total clear zone around the colonies
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describe this type of hemolysis?
- weak beta hemolysis
- lighter cloudy zone around the colonies
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describe this type of hemolysis
- non hemolytic
- no clear zone or color on blood agar
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describe types of gram stain for microscopic traits
- gram pos: stain purple, crystal violet stain gets trapped in thick peptidoglygan wall surrounding inner membrane
- gram neg: stain red, crystal violet cannot penetrate outer membrane and safranin red stain adheres to thin peptidoglycan cell wall between two cell membranes
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what are some problems that arise when using gram stain on old/weakened bacteria
may not stain properly
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gram +/- ?
Gram (+)
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gram +/-?
gram (-)
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describe the crystal violet dye, decolorizer, and safranin red steps in gram stain
- crystal violet dye: ppt'd by iodine and trapped by the thick outer cell wall of G(+) bacteria
- decolorizer (ethanol/acetone): disperses the outer membrane of G(-) bacteria and strips the crystal violet. Creates holes on outer membrane
- safranin red: a counterstain that binds to now exposed thin cell wall of G(-) bacteria
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what can you observe from gram stain morphology
- cell shape: may differ depending on cell's development, environment, nutrient availability, stress
- arrangement of cells
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what are some of the stains used to reveal bacteria? (4)
- gram stain
- silver stain
- giemsa stain
- immunostaining: fluorescent
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what is darkfield microscopy
a technique used to see very small bacterial not visible by typical staining methods
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how is serology used to ID bacteria
- "antigenic classification"
- using antibodies to detect the specific antigens on bacteria
- ex) ELIZA, fluorescence, clumping/agglutination
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what is serology useful for
- organisms that do not grow well in culture
- organisms that are too dangerous to grow in the lab
- organisms that need to be ID's quickly
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how is molecular identification used for bacterial classification
- "culture independent characterization"
- ID using highly conserved DNA which is amplified by PCR (polymerase chain reaction)
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what is molecular ID useful for
- differentiate different strains of the same bacterial species, such as pathogenic vs commensal strains
- presence/absence of genes
- comparing DNA,RNA sequences to large databases and can identify species or strains without any previous information about the organism
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what is the most common gene sequence used to determine bacteria and is found in all bacteria
- 16S rRNA
- the most highly conserved bacteria ribosomal RNA
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how is metabolic activity used to classify bacterial DNA
- certain bacteria metabolizes specific carbohydrates and different compounds using carbon, nitrogen, and water as an energy source.
- ex) catalase +/-
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how is bacteria classified by oxygen requirements
- obligate anaerobes: can only grow without oxygen. Ex) clostridium perfringens
- obligate aerobes: can only grow in oxygen. Ex) mycobacterium tuberculosis
- facultative anerobes/aerobes: can grow with/without oxygen. Are able to adapt and change their metabolism based on the oxygen environment but may grow better in one condition than the other
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what are the enzymes released by aerobic (obligate and facultative) bacteria and what is its function
- superoxide dismutase
- catalse
- f(x): detoxifies hydrogen peroxide and superoxide radicals (toxic by products)
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does fermentation or cellular respiration produce more energy
cellular respiration
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what are the fermentation products
produces organic acids that reduces the pH level
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what is the indicator that is used to ID pH and does each color change indicate?
- bromocresol purple
- acid: yellow <6.3
- basic: purple
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what are the three tests used to determine enzymes or metabolic processes
- gelatinase: hydrolyze (+), remain solid (-)
- urease: red (+), yellow (-)
- indole test: for tryptophanase pathway. Red (+), gold (-)
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