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.
zinc test
To the negative culture, add a pinch of zinc powder and shake the tube vigorously. if a red color develops in the tube, nitrate was reduced by zinc metal, confirming the negative test for nitrate reduction. If no color develops, a nongaseous end product may have been formed, which means you unknown reduced nitrate
starch hydrolysis test
starch hydrolysis is detected by adding Gram’s iodine to starch medium. iodine complexes with the starch macromolecule and causes the medium to turn blue. if starch has been degraded, the medium adjacent to the bacterial growth will be clear after the addition of the iodine
casein hydrolysis test
casein gives milk its characteristic white color
the protein that degrades casein is lysyl endopeptidase that cleaves at lysine residues
test done on skim milk agar and clear zone around bacterium shows casein hydrolysis
fat hydrolysis test
liipases cleave fatty acids from glycerol
spirit blue agar contains peptone as source of carbon, nitrogen, and vitamins. it also contains tributyrin, a natural animal triglyceride that serves as a substrate for lipases. release of fatty acids from tributyrin via lipase activity results in the lowerying of the pH of the agar to produce a dark blue precipitate. if the tributyrin is not hydrolyzed all the way, the pH is not sufficiently lowered and all there may be is the depletion of fat or oil droplets in the agar to indicate lipase activity.
tryptophan degradation test
amino acid, tryptophan, is degraded to produce indole, ammonia, and pyruvic acid by tryptophanase
degradation can be detected with Kovac’s reagent, which forms a deep red color if indole is present
Tryptone broth is used
Urea hydrolysis test
urease is responsible for urea hydrolysis, and splits it into carbon dioxide and ammonia
urease is produced by some gram-negative enteric bacteria.
urea medium contains yeast extract, urea, a buffer, and the pH indicator phenol red. Urea is added after autoclaving
when urease is produced by organism, the resulting ammonia causes the pH to increase and the phenol red changes from yellow to a bright pink
phenylalanine deamination test
gram-negative bacteria can oxidatively deaminate the amino acid phenylalanine to produce phenylpyruvic acid and ammonia. Catalyzed by the enzyme phenylalanine deaminase, a flavoprotein oxidase.
enzyme can be detected by the addition of 10% ferric chloride, which forms a green-colored complex with a-keto acids such as phenylpyruvic acid
Kligler’s Iron Agar
multiple test medium that will detect the fermentation of glucose and lactose and the production of hydrogen sulfide resulting from the breakdown of the amino acid cysteine
contains 0.1% glucose, 1% lactose, peptone, ferrous salts, and phenol red as a pH indicator
prepared as a slant and is inoculated by streaking the slant and stabbing the butt of the tube.
useful in the differentiateion of the gram-negative enteric bacteria
Possible Results:::
alkaline (red) slant/acid (yellow) butt……… means only glucose was utilized….. the organism utilized the low concentration of glucose initially and then degraded the peptone in the medium…….the slant is alkaline (red) because glucose wad degraded aerobically, and the ammonia released form peptone utilization casued the aerobic slant to become alkaline…. the butt is yellow (acid) because glucose was fermented anaerobically to produce enough acids to cause the acidic reaction in the but…. if gas is produced, it will be evident by the splitting of the medium and the formation of gas bubbles in the agar slant
acid (yellow) slant/acid (yellow) butt….. the organisms has fermented both glucose and lactose, producing acids that cause the pH indicator to turn yellow….. lactose is present in 10 times the concentration of glucose and sufficient acid is produced to cause both the slant and butt to be acidic (tubes must be read at 24 hours because they can revert to alkaline in 48 hours if the lactose becomes depleted and peptones are utilized, producing ammonia)
alkaline (red) slant/alkaline (red) butt; alkaline (red) slant/no change butt…… no fermentation of either sugar has occurred…. some enteric bacteria can use the peptones both aerobically and anaerobically, causing both the slant and butt to become alkaline….. others can only use the peptone aerobically, producing an alkaline slant but no change ***……
Hydrogen sulfide:::::: a sulfide is removed from cysteine (cysteine desulfurase) to produce pyruvic acid, ammonia, and hydrogen sulfide. Kligler’s iron agar contains ferrous salts that will react with the hydrogen sulfide liberated by cysteine desulfurase to produce an insoluble black precipitate, ferrous sulfide.
SIM Medium Tests
multiple test medium that detects the production of hydrogen sulfide and indole and determines if an organism is motile or not.
medium contains hydrolyzed casein, ferrous salts, and agar (0.7%), which makes the medium semisolid (inoculated by stabbing)
breakdown of tryptophan in the medium will produce indole, which can be detected by adding Kovac’s reagent after incubation (will turn red)….. if cysteine is degraded, hydrogen sulfide will be released, which will combine with the ferrous salts to produce a black precipitate in the tube
because the medium is semisolid, motile bacteria can be detected by growth out from the line of inoculation
IMViC tests
I: indole test
M: methyl red test
V: Voges-Proskauer test
C: citrate test
E. coli and E. aerogenes produce opposite reactions for these test:::::
E. coli ( positive indole and methyl red; negative Voges-Proskauer test and citrate)
E. aerogenes (positive Voges-Proskauer and citrate; negative indole and methyl red)
API 20E System
miniaturized version of conventional tests that is used for the identification of members of the family Enterobacteriaceae and other gram-negative bacteria.
utilizes plastic strip with 20 separate compartments that contain a specific dehydrated medium…. allow user to determine 23 different biochemical characteristics of the inoculated bacterium
to provide anaerobic conditions for some of the compartments, it is necessary to add sterile mineral oil to them
incubated for 18-24 hours, test reactions are recorded and test reagents are added to some
a code number is tabulated that corresponds fto the Analytical Profile Index
EnteroPluri-Test system
used for rapid identification of Enterobacteriaceae
incorporates 12 different conventional media and 15 biochemical tests into a single ready-to-use tube that can be simultaneously inoculated with minimal equipment
each of the 12 compartments of an EnteroPluri tube contains a different agar-based medium
a code number is tabulated that corresponds to the EnteroPluri-Test codebook.
Enterobacteriaceae
large family of diverse gram-negative rods
ubiquitous and can be found soil, water, vegetation, and the intestinal tracts of most organisms
all are facultative anaerobes
divided into potential human pathogens and true intestinal pathogens
E. coli
opportunistic pathogen
found in large numbers as resident of colon
can cause urinary tract infections, sepsis, wound infections, and meningitis
can acquire virulence factors encoded on plasmids or in bacteriophage DNA causing some strains to have enhanced virulence
E. coli 0157:H7 produces a toxin that damages blood vessels and causes very severe diarrhea
characteristics used in identification:::::: motility, its ability to produce indole, and its inability to use citrate as a carbon source
Proteus
opportunistic pathogens that are normally found in the intestinal tract and considered harmless
can cause urinary tract infections, wound infections, and septicemia
motile and produce many different types of fimbriae, which account for their ability to adhere to the epithelium of the urinary tract and cause urinary tract infections.
most species of proteus produce large quantities of urease, which then raises the urine pH and can cause formation of crystals.
unable to ferment lactose but can ferment glucose (not considered coliforms)
true intestinal pathogens
Salmonella, Shigella, Yersinia
not part of normal skin microbiota and have well-developed virulence factors
Salmonella
infections involve gastroenteritis, septicemia, and typhoid fever
when grown on differential or selective media, can produce non-lactose-fermenting colones with black centers if the media contain indicators for hydrogen sulfide production.
have flagella and are negative for indole and urease
Shigella
intestinal pathogens that cause dysentery
humans are the only known reservoir for infection
do not ferment lactose or hydrolyze urea
non-motile and do not produce hydrogen sulfide
Yersinia
causes bubonic plague
non-lactose fermenters when cultured and do not produce hydrogen sulfide and are generally considered nonmotile and urease positive
