Metabolic Assays.txt


?metabolic processes


require enzymes to REDUCE activation energy needed for chem rxns to occur


Endoenzymesenzymes


catalyze rxns INSIDE the cell


Exoenzymes


enzymes released from the bacterial cell that carry out rxns in the environmentCan be used to INCREASE food availability (breakdown proteins to aas)Can be used to INCREASE pathogenicity (destroy tissue to allow invasion into organism)


Metabolic Assays


these test for different metabolic activities and are indicators of the presence of specific genes


Fermentation tubes: carbohydrate fermentation


a. fermentation produces acid by-products (lactic acid, pyruvic acid) and release into environment, LOWERING pHb. Phenol red: red @ alkaline/neutral, yellow @ ACIDIC pHc. Durham tube: collects gas, which can be a PRODUCT of FERMENTATION in some pathwaysd. the media in the tube contains peptone and 1% of single carbohydratee. Inoculate different sugars with diff cultures. Positive test is yellow


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) - DENITRIFICATIONE. coli: POSITIVE for DENITRIFICATION (NON-fermenter)P. aeroginosa: NEGATIVE for gas, but POSITIVE for color change to pink, which indicates the enzyme (fermenter)


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 nitrogenIf 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: NEGATIVEE. aerogenes: POSITIVE


Starch Metabolism


Amylase is an EXOenzyme that breaks down starch into GLUCOSE monomersStarch is too large to pass thru bac cell membrane so needs to be broken down 1st in order to be metabolizedThis 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/brownB. subtilis: POSITIVE for amylaseS. aureus:


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


Casein Hydrolysis Test


indicates the presence of Casease, which is an EXOENZYME that hydrolyzes milk protein CASEINPositive test is indicated by a clearing (clear color) near the bacteria growthB. subtilis: POSITIVE for CaseaseE. coli: NEGATIVE for Casease


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 presentS. aureus: POSITIVEE. coli, P. vulgaris, B. subtilis: NEGATIVE


SIM Medium Test


Tests for 3 bacterial activities1. 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 = POSITIVEP. vulgaris: Reduction to H2S = POSITIVE. Indole Production = POSITIVE. Motility = POSITIVEE. aerogenes: Reduction to H2S = NEGATIVE. Indole Production = NEGATIVE. Motility = POSITIVE


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)


Gram + BAC


staphylococcus Circle shaped (ie: S. aurueus)streptococcus circle shaped (ie: S. pnemonias)Bacillis ROD shaped (ie: B. subtilis)


Gram - Bac


Eschercia ROD Shaped (ie: E. coli)Proteus ROD Shaped (ie: P. vulgaris)Pseudomonas ROD Shaped (ie: P. aeruginosa)


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


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.


Gram -


Negatively charged, shown through a BASIC stain (+ chromogen safranin)Very thin peptidoglycan layeroutside layer - lipopolysaccharides: additional barrierHAS Porin Proteins (allow passage of stuff thru outer membrane) HAS Periplasm


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 moleculesSENSITIVE to Penicillin (binds proteins in cell wall synth)SENSITIVE to Lysozymes (breaks bonds forming the glycan chain)