Metabolic Assays.txt

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Metabolic Assays.txt
2011-03-01 13:25:07
Metabolic Assay gram info

Metabolic assay & gram info
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  1. metabolic processes
    require enzymes to REDUCE activation energy needed for chem rxns to occur
  2. Endoenzymesenzymes
    catalyze rxns INSIDE the cell
  3. Exoenzymes
    • enzymes released from the bacterial cell that carry out rxns in the environment
    • Can be used to INCREASE food availability (breakdown proteins to aas)
    • Can be used to INCREASE pathogenicity (destroy tissue to allow invasion into organism)
  4. Metabolic Assays
    • these test for different metabolic activities and are
    • indicators of the presence of specific genes
  5. Fermentation tubes: carbohydrate fermentation
    • a. fermentation produces acid by-products (lactic acid, pyruvic acid) and release into environment, LOWERING pH
    • b. Phenol red: red @ alkaline/neutral, yellow @ ACIDIC pH
    • c. Durham tube: collects gas, which can be a PRODUCT of FERMENTATION in some pathways
    • d. the media in the tube contains peptone and 1% of single carbohydrate
    • e. Inoculate different sugars with diff cultures. Positive test is yellow
  6. Nitrate reduction
    • Gram - bac contain NITRATE REDUCTASE (reduces Nitrate (NO3) to nitrite (NO2))
    • Other back carryout a multistep process reducing nitrate to nitrogen gas (N2) - DENITRIFICATION
    • E. coli: POSITIVE for DENITRIFICATION (NON-fermenter)
    • P. aeroginosa: NEGATIVE for gas, but POSITIVE for color change to pink, which indicates the enzyme (fermenter)
  7. Citrate Test
    • determines if back are members of the family Enterobacteriaceae by placing in a medium with sodium citrate as bac's only carbon source and ammonium phosphate as only source of nitrogen
    • If positive, alkalinization will occur with the media turning blue/green. This shows that citrate permeases is the enzyme used to metabolize citrate as the sole source of carbon.
    • E. coli: NEGATIVE
    • E. aerogenes: POSITIVE
  8. Starch Metabolism
    • Amylase is an EXOenzyme that breaks down starch into GLUCOSE monomers
    • Starch is too large to pass thru bac cell membrane so needs to be broken down 1st in order to be metabolized
    • This is shown on a plate by the indication of hydrolyzed starch into glucose monomers surrounding the bacterial growth.
    • Iodine dye is used to stain the starch blue/brown
    • B. subtilis: POSITIVE for amylase
    • S. aureus:
  9. Urea Hydrolysis
    • To determine which bacteria contain the enzyme urease, which can hydrolyze urea to ammonia and CO2.
    • P. vulgaris: POSITIVE test for urease.. turned pink (INCREASE in pH) giving
    • E. coli: NEGATIVE for urease
  10. Casein Hydrolysis Test
    • indicates the presence of Casease, which is an EXOENZYME that hydrolyzes milk protein CASEIN
    • Positive test is indicated by a clearing (clear color) near the bacteria growth
    • B. subtilis: POSITIVE for Casease
    • E. coli: NEGATIVE for Casease
  11. Gelatin Hydrolysis Test
    • To determine which bacteria produced the extracellular enzyme gelatinase, which hydrolyzes gelatin.
    • This can be shown by stab inoculating a tube of nutrient Gelatin - if the medium become liquified, gelatinase is present
    • S. aureus: POSITIVE
    • E. coli, P. vulgaris, B. subtilis: NEGATIVE
  12. SIM Medium Test
    • Tests for 3 bacterial activities
    • 1. SULFUR REDUCTION to H2S - which is the product of CYSTEIN DESULFURASE. H2S is shown as a BLACK precipitate (FeS) when combined with FeSO4 in the medium.
    • 2. INDOLE production from try metabolism by TRYPTOPHANASE. Tryptophan hydrolysis is detected by adding Kovacs’ reagant, which will produce a red layer giving a positive test.
    • 3. Motility - by stab culture and the medium which is a reduced agar concentration.
    • E. coli: Reduction to H2S = NEGATIVE. Indole Production = POSITIVE. Motility = POSITIVE
    • P. vulgaris: Reduction to H2S = POSITIVE. Indole Production = POSITIVE. Motility = POSITIVE
    • E. aerogenes: Reduction to H2S = NEGATIVE. Indole Production = NEGATIVE. Motility = POSITIVE
  13. Catalase Test
    • To determine if certain organisms produce the enzyme CATALASE, which converts hydrogen peroxide (H2O2) into water (H20) and oxygen gas (O2).
    • S. aureus: POSITIVE (formed gas)
    • S. faecalis: POSITIVE (formed gas)
  14. Gram + BAC
    • staphylococcus Circle shaped (ie: S. aurueus)
    • streptococcus circle shaped (ie: S. pnemonias)
    • Bacillis ROD shaped (ie: B. subtilis)
  15. Gram - Bac
    • Eschercia ROD Shaped (ie: E. coli)
    • Proteus ROD Shaped (ie: P. vulgaris)
    • Pseudomonas ROD Shaped (ie: P. aeruginosa)
  16. E. coli summary
    • Gram - ROD
    • + for denitrification (non-fermenter)
    • - for citrate permease
    • - for urease
    • - for casease
    • - for gelatinase
    • - for reduction to H2S (no black color in SIMS media)
    • + for indole production (red color after Kovacs' reagant)
    • + for motility
  17. Distinguishing between Gram - & +
    • The cell wall distinguishes between the two. The cell wall is made up of a peptidoglycan layer, giving it rigidity.
    • If there is no cell wall, the cell membrane becomes stronger and more impermeable to outside substances.
  18. Gram -
    • Negatively charged, shown through a BASIC stain (+ chromogen safranin)
    • Very thin peptidoglycan layer
    • outside layer - lipopolysaccharides: additional barrier
    • HAS Porin Proteins (allow passage of stuff thru outer membrane)
    • HAS Periplasm
  19. Gram +
    • Negatively charged, shown through a BASIC stain (+ crystal violet plus iodine to form complex)
    • Thick peptidoglycan layer, this layer is permeable to sugar & other large molecules
    • SENSITIVE to Penicillin (binds proteins in cell wall synth)
    • SENSITIVE to Lysozymes (breaks bonds forming the glycan chain)

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