Microbio Lab 10 Flashcards
metabolism
the sum total of the chemical reactions that occur in a cell
enzymes
protein molecules that catalyze individual chemical reactions that make up the metabolic pathway
most function inside the cell
catabolism
breakdown of food materials
anabolism
biosynthesis of cell constituents
exoenzymes
function outside of bacterial cell to degrade large macromolecules (because they cannot carry out phagocytosis owing to rigid cell walls)
examples…. protease, DNase, and amylase
coenzymes
transfer small molecules from one molecule to another
ex. NAD+ and FAD transfer protons and coenzyme A transfers acetate groups
most are derivatives of vitamins
only required in catalytic amounts and must converted back for process to continue
Two means by which bacteria obtain energy?
respiration and fermentation
respiration
organic molecules are completely degraded to carbon dioxide and water. ATP is generated by the energy created from a proton gradient that is established across the cell membrane when protons are transported from the cytoplasm to the outside of the cell
oxidative phosphorylation
shuttling of electrons down an electron transport chain involving cytochromes facilitates the movement of the protons to the outside of the cell. Reduced coenzyme NADH generated in metabolic reactions is converted back to NAD+ because oxygen acts as the terminal electron acceptor and is converted to water
fermentation
the partial breakdown of organic molecules to alcohols, aldehydes, acids, and gases such as carbon dioxide and hydrogen. Organic molecules in metabolic pathways are serve as terminal electron acceptors .and become the end products in a fermentation pathway
substrate level phosphorylation
metabolic intermediates in pathways directly transfer high-energy phosphates to ADP to synthesize ATP.
Why does butter become rancid?
bacteria ferment butterfat, producing volatile and odoriferous organic acids
O/F glucose test
differentiates between the two modes of metabolism
inoculate O/F tubes with unknown, a fermentative, and oxidative organism (E. coli) and an oxidative organism (P. aeruginosa)
anaerobic (yellow) and aerobic (yellow)…… oxidative and fermentative metabolism
anaerobic (green) and aerobic (yellow)….. oxidative metabolism
anaerobic (green) and aerobic (green)….. glucose not metabolized
fermentation tests
O/F glucose test
specific sugar fermentations
mixed-acid fermentation (methyl red [MR] test)
butanediol fermentation (Voges-Proskauer [VP] test)
citrate test
oxidative tests
oxidase
catalase
nitrate
specific sugar fermentations (glucose, lactose, and mannitol)
if yellow, acid has been produced. If a bubble is present in the Durham tube, gas was produced (hydrogen; carbon dioxide). If at least 10% of the liquid has been displaced, gas was formed from sugar fermentation
sugar is at concentration of 0.5%
mixed-acid fermentation (Methyl Red test)
important in differentiating some of the gram-negative intestinal bacteria
MR-VP medium is a glucose broth that is buffered with peptone and dipotassium phosphate
the pH indicator, methyl red, is added and turns red if acid is present… these bacteria are mixed-acid fermenters and generally produce gas because they produce the enzyme complex formic hydrogenlyase
formic hydrogenlyase
splits formic acid to produce CO2 and H2
2,3-Butanediol Fermentation (Voges-Proskauer test)
a more neutral end product of fermentation
an organism positive for the VP test is usually negative for the methyl red test
2,3-butanediol is converted to acetoin by oxidation of the 2,3-butanediol, which reacts with Barritts’s reagent.
If acetoin is present the tube will turn pink
citrate test
some bacteria can cleave citrate to produce oxaloacetate and pyruvate, which are fermented to produce several end products such as formate, acetate, lactate, acetoin, and CO2
medium contains ammonium salts that serve as sole nitrogen source. Must use ammonium source to degrade citrate which produces ammonia causing the medium to become alkaline and the pH indicator turns from dark green to a deep Prussian blue
oxidase test
assays for the presence of cytochrome oxidase, and enzyme in the electron transport chain.
occurs in bacteria that carry out respiration where oxygen is the terminal electron acceptor
Positive test is detected when electrons are transferred to a artificial electron acceptor, N,N,N’,N’-tetramethyl-p-phenylenediamine, and it changes from yellow to purple
catalase test
catalase is an enzyme which degrades hydrogen peroxide ( a dangerous by-product of respiration) into harmless oxygen and water.
A positive test is indicated by bubbling due to the breakdown of hydrogen peroxide and production of gas when hydrogen peroxide is placed on a sample.
nitrate reduction
assay for end products of nitrate reduction: gas or nitrite. Cultures are grown in beef extract medium containg potassium nitrate. Gases produced from nitrate reduction are captured in Durham tubes placed in the nitrate medium. Partial reduction of nitrate to nitrite is assayed for by adding sulfanilic acid followed by dimethyl-alpha-naphthylamine. If nitrite is produced by reduction, it will form a chemical complex to give a dark red color.
nitrate respiration
some facultative anaerobes can use nitrate as a terminal electron acceptor in a type of anaerobic respiration. Reduce nitrate to a gaseous end product such as N2O or N2, or they partially reduce nitrate to nitrite.