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metabolic processes
require enzymes to REDUCE activation energy needed for chem rxns to occur
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Endoenzymesenzymes
catalyze rxns INSIDE the cell
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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)
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Metabolic Assays
- these test for different metabolic activities and are
- indicators of the presence of specific genes
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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
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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)
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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
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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:
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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
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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
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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
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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
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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)
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Gram + BAC
- staphylococcus Circle shaped (ie: S. aurueus)
- streptococcus circle shaped (ie: S. pnemonias)
- Bacillis ROD shaped (ie: B. subtilis)
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Gram - Bac
- Eschercia ROD Shaped (ie: E. coli)
- Proteus ROD Shaped (ie: P. vulgaris)
- Pseudomonas ROD Shaped (ie: P. aeruginosa)
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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
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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.
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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
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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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