Lab Exam 2: Ex 5-4; 5-5; 5-7; 5-8; 5-9; 5-11; 5-12; 6-1; 9-5 Flashcards
What does the catalase test detect?
an organismal ability to produce catalase - an enzyme that detoxifies the cell by converting hydrogen peroxide produced in the ETC to H2O and O2 (shows bubbles).
What does the catalase test prove?
Organisms thriving in an oxygen rich environment have evolved different mechanisms to deal with oxygen radicals.
flavoprotein
an ETC carrier molecule that can bypass the next carrier in the chain and transfer electrons directly to oxygen, which produces hydrogen peroxide.
hydrogen peroxide
a highly potent cellular toxin
Why is hydrogen peroxide toxic?
it oxidizes biochemicals and makes them nonfunctional
How do organisms counteract the hydrogen peroxide they produce?
they also produce enzymes capable of breaking hydrogen peroxide down.
catalase
an enzyme that converts hydrogen peroxide into water and gaseous oxygen.
How are bacteria that produce catalase detected?
Bacteria that produce catalase are easily detected using typical store grade hydrogen peroxide. When it is added to a catalase-positive culture, oxygen gas bubbles form immediately. (if no bubbles appear then the organism is catalase-negative)
What microbes were used in the catalase test lab and what were the results?
Lactococcus lactis - no bubbles, catalase negative
Staphylococcus epidermidis - bubbles, catalase positive
What is the oxidase test designed for?
to identify microorganisms that contain a carrier molecule called cytochrome c oxidase which is used by cells to transfer electrons to oxygen (ETC)
How can microorganisms containing cytochrome c oxidase be identified?
they can be identified by using chromogenic reducing agents which change color upon becoming oxidized.
What is the name of the chromogenic reducing agent?
tetramethyl-p-phenylenediamine
How does the oxidase test work?
The reducing reagent is added directly to bacterial growth on solid media. a color change occurs within seconds if the reducing agent becomes oxidized, thus indicating that cytochrome c oxidase is present.
What color result is seen if the oxidase test is positive?
dark blue/purple
What microbes did we use in the oxidase test and what were the results?
Pseudomonas aeruginosa - purple, oxidase positive
Escherichia coli - no color change, oxidase negative
What is the Citrate Utilization Test designed to do?
It is designed to differentiate members of the Enterobacteriaceae, all of which are facultative anaerobes.
What does it mean if a bacteria is facultative anaerobic?
they have the ability to ferment carbohydrates and also the ability to aerobically respire, which means they have a functional citric acid cycle.
What does the Citrate Utilization Test tell us about an organism?
the ability of organisms to use citrate as their sole carbon source and perform citrate fermentation.
What is citrate?
an intermediate product of the Krebs cycle
What kind of medium is Simmons citrate agar?
a defined medium - the amount and source of all ingredients are carefully controlled.
What is the carbon source in Simmons citrate agar? What does it do?
sodium citrate - provides the means for bacterial species that possess the enzyme citrate permease to transport citrate into the cell and perform citrate fermentation. (cells convert citrate into pyruvate which can then be converted into a variety of products.)
What dye is used in the Citrate Utilization test and what does it do?
Bromothymol blue - pH indicator which can change from green to blue as the pH becomes more alkaline due to metabolic product production (ammonia, ammonium hydroxide, etc.)
What microbes did we use in the Citrate Utilization Test and what were the results?
Enterobacter aerogenes - blue growth, citrate positive
Escherichia coli - no growth (green agar), citrate negative
What is another name for the Decarboxylase Test?
Amino Acid Decarboxylation
What was the decarboxylation test designed for?
to differentiate Enterobacteria from other Gram negative rods
What do many organisms contain that aid cells in the breakdown of amino acids?
enzymes
What does decarboxylation medium contain?
peptone, glucose, pyridoxal phosphate and the pH indicator bromocresol blue
What is decarboxylation?
a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain.
What color will the medium turn if the sample is positive in a decarboxylation test?
purple - decarboxylation of the amino acid results in accumulation of amines, which are alkaline and turn the medium purple.
What does a yellow result in a carboxylation test mean?
the organism is a glucose fermenter but does not produce the appropriate decarboxylase.
What does a green (no color change) result in a carboxylation test mean?
the organism does not ferment glucose
What organisms did we test using the decarboxylation test and what were their results?
Escherichia coli - purple, positive result
Providencia stuartii - yellow, glucose fermenter but carboxylation negative result
What does the Phenylalanine Deaminase medium test for?
the ability of an organism to produce the enzyme deaminase.
What does the enzyme deaminase do?
it removes the amine group (NH2) from the amino acid phenylalamine and releases the amine group as free ammonia.
What is produced when the amine group is released from the amino acid phenylalanine as free ammonia?
As a result of this reaction, phenylpyruvic acid is also produced.
What is Phenylalanine agar used for?
to differentiate members of the genera Proteus, Morganella, and Providencia from other enterobacteriaceae.
What doe Phenylalanine agar contain?
nutrients and DL-phenylalanine
What is added to Phenylalanine agar after incubation? What does it do?
A reagent, 10% ferric chloride, is added to the media.
If phenylpyruvic acid was produced, it will react with the ferric chloride and turn dark green.
In a Phenylalanine Deaminase test what does a yellow test result indicate?
the organism is negative for phenylalanine deaminase production.
What microbes did we test with the Phenylalanine Deaminase test and what were the results?
Escherichia coli - green, positive
Providendcia stuartii - yellow, negative
What is starch made up of?
alpha-D glucose subunits
What organisms can hydrolyze starch?
Organisms that can secrete alpha amylases and oligo 1-6-glucosidase (i.e. Corynebacterium, Clostridium, and Bacillus)
What is in starch agar?
it is a simple plated medium of beef extract, soluble starch, and agar.
In the starch hydrolysis test, what is added after incubation and why?
Grams iodine - used to detect the presence or absence of starch in the vicinity around the bacterial growth. It reacts with starch and produces a blue or dark brown color; therefore any microbial starch hydrolysis will be revealed as a clear zone surrounding the growth.
What does it mean if a bacteria amylases?
it excretes exoenzymes
What is a simple overview of the procedure for the starch hydrolysis test?
Plate out/streak organisms onto LB starch media, incubate and then stain for the presence of starch by adding iodine.
What microbes did we test in the starch hydrolysis test and what were the results?
Escherichia coli - iodine stained the entire area around the organism, negative result
Bacillus cereus - there was a space left around the organism that did not stain with the iodine, positive result
What does the enzyme urease do?
converts urea into ammonia and carbon dioxide (NH3 and CO2)
What is urea?
a product of the decarboxylation of certain amino acids.
What is the Urease test also known as?
Urea Hydrolysis
What is the Urease test used for?
to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease (i.e. helps identify pathogens from the genus Proteus and H. pylori)
What are some examples of “rapid urease-positive bacteria”?
Proteus, Morganella morganii, and some Providencia stuartii strains
What are the components of the urea broth?
Nutrient source: urea and a trace (0.0001%) of yeast extract
Buffers
Phenol Red
What role does phenol red play in the urease test?
it is a pH indicator - yellow or orange below pH 8.4, red or pink above pH 8.4
What does a pink result indicate in a urease test in less than 24 hours?
positive result - rapid urea hydrolysis; strong urease production
What does a orange or yellow result in a urease test indicate in less than 24 hours?
negative result
What microbes did we test using the urease test and what were the results?
Proteus vulgaris - pink, positive
Escherichia coli - yellow, negative
What is the standard plate count?
a procedure that allows microbiologists to estimate the population density in a liquid sample by plating a very dilute portion of that sample and counting the number of colonies it produces. (a way to estimate the number of living bacteria in a sample)
What is the EnteroPluri-test?
a tube, enterotube II, containing 12 individual chambers with the capability of performing 15 tests.
What is the EnteroPluri-test used for?
rapid identification of bacteria from the family enterobacteriaceae and other Gram-negative, oxidase-negative bacteria.
