Review2

Review – Lab Practical #2 This exam will be 30 multiple-choice choices (2pts each) with 10 stations (4pts each) that will require short answers. **** Be familiar with reagents, colors, and reactions involved in all the biochemical tests. Did I add any reagents to help tell me, what colors were shown, and what did they mean about bacteria? 30 multiple questions, 10 station questions. IMViC SIM – tests for three things, going to see the station. 1) Hydrogen S ulfide H 2 S -- ( black precipitate ) 2) I ndole (Kovacs) – Add Kovacs reagent to determine Indole production. ~ Bacteria that hydrolyze tryptophan to produce Indole. 3) M otility (stab line) – Growth away from the stab line. Methyl Red Test MR- MRVP; Methyl Red (pH indicator) = mixed acid fermenters The color of the medium is Red at acidic and Yellow at neutral pH . Voges-Proskauer Test VP = Detect the presence of acetoin, a precursor of 2,3 butanediol . (+) will turn brownish red to pink (-) brownish green to yellow. SCA – expect Simmons Citrate Agar station. Media started army green  If turned Royal blue (+) It has the enzyme citrate permease and can use citrate as a sole carbon source.

Oxygen and Growth of Bacteria - be familiar with how it works Gas Pak- Hydrogen & CO2 was released . Both bounded (hydrogen & oxygen) to create anaerobic environment . - if it grows in Gas Pak, what does this mean? It is going into oxygen groups ( five of them ) If you can grow in the Gas Pak , you could be facultative anaerobe, obligate anaerobe, aerotolerant anaerobe. Apply oxygen groups in what is going on in the Gas Pak. Obligate aerobe and Microaerophile (-) it needs oxygen . Obligate anaerobe (+) its best with no oxygen . Aerotolerant anaerobe (+) will grow and survive , won’t grow with oxygen. Facultative anaerobe (- or +) may grow but slow . - three enzymes (catalase, peroxidase, SOD) Reactions: what is being broken down and breaking it down into Toxic oxygen products: Superoxide and Hydrogen peroxide. O2 -------------------------------------> O2 + H 2 O 2 Superoxide dismutase (SOD) SOD eliminates superoxide anions by combining them with hydrogen cations to produce oxygen and Hydrogen Peroxide . H 2 O 2 ----------------> H 2 O +O 2 Catalase H 2 O 2 is destroyed by catalase to produce water and oxygen . H 2 O 2 ----------------> H 2 O Peroxidase Peroxidase destroys hydrogen peroxide to release water only . - Major oxygen groups Obligate anaerobe - Cannot tolerate oxygen and will be killed in its presence . It lacks the enzymes necessary to eliminate toxic oxygen products. Obligate aerobe - Requires atmospheric levels of oxygen for normal growth. Facultative anaerobe - grows with or without oxygen . Usually grows faster in its presence . (more atp) Aerotolerant anaerobe - Will survive BUT will not grow in the presence of oxygen. Microaerophile - Requires reduced levels of oxygen . Ex) A candle jar test lowers oxygen. Perfect for microaerophiles.

Protein Catabolism (Gelatin/Casein hydrolysis) - expect station. Casein – large protein that gives milk its white opaque color. lost opaqueness of milk = + clearing of agar. Gelatin had to incubate for a week .  Then leave it in the fridge for 15-30 minutes. - Short incubation can give false negative. (+) Gelatin is liquid. (-) Gelatin, if it is solid (chunk)  gelatin still intact . Gelatin ----------------------------------> soluble amino acids/peptides (Solid) Gelatinase (Liquid even after refrigeration) Respiration (Nitrate and Oxidase Activities) - electron acceptors involved. OIL RIG Aerobic Respiration = final electron acceptor is oxygen . Anaerobic Respiration = no oxygen is present , other molecules must be the acceptors  Inorganic molecules- sulfate, NITRATE , carbonate. - reaction/reagent What is determined? (+) Nitrate reduction  Nitrite – capable of anaerobic respiration 5 drops each of Nitrate reagents A and B  Red (+) ~ No red could mean (-) or it is further reduced forming nitrogen gas & ammonia. ADD ZINC No red (+) Ammonia or nitrogen gas are present. Red (-) - What type of bacteria do this? Oxygen groups Facultative can do aerobic and anaerobic. Obligate aerobe , microaerophile- aerobe aerotolerant, and obligate anaerobe- anaerobe Oxidase Activity (+) capable of aerobic respiration Cytochrome c -------------------e----------------------> Oxygen =========>H20 Cytochrome oxidase

Effect of UV light station - – observe plates & questions associated What section of the plate is being used in UV light? If exposed to UV light , should have LESS growth . When two Thymine’s right next to one another, it won’t covalently bond forming- Define: Thymine Dimer is the damage due to UV light tinkering with DNA ~ Formed in order to connect covalent bonds when thymine nucleotides aren’t able to in the groups hit by the 260 nm wavelength. T.D.  Mutation, leads to a mistake in DNA. Describe two forms of repair. 1) Light repair - pyrimidine dimerase- (enzyme) Goes in and removes covalent bond and restores the strand back to normal. 2) Dark repair - DNA polymerase - (enzyme) Clip out WHOLE damaged thymine nucleotides and replace them with normal thymine nucleotides. Cut out whole damaged portion and make it a new. Antibiotic Sensitivity methodology used. - - Kirby Bauer Method with a Mueller Hinton Agar plate Plate with six disks ( measure the plate ) zone of inhibition in mm. Zone of inhibition (clearing zone) - station – be able to make measurements ( mm ) and use a table. ( 1 cm = 10 mm ) - TABLE  Resistant, Intermediate, Susceptible rating Want the bacteria to be susceptible to the antibiotic and die so the body feels better. More growth in zone of inhibition means its more susceptible. Bacteria is dying. Resistant -> do NOT recommend to someone, bacteria don’t die. No growth in zone.

It selects for or only allows Gram negative organisms to grow, and you will be able to differentiate among species based on color.