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the ph indicator in carbohydrate broth
phenol red
In the carbohydrate broths, deamination of amino acids supplied by peptone results in an alkaline reaction from the ___ that is produced and turns the medium ___ (color).
ammonia, pink/red
Acid production from fermentation of the carbohydrate in the carbohydrate broths lowers the pH below the neutral range of the indicator and turns the medium ___
yellow
Gas production from the fermentation of a sugar in the carbohydrate broths, is indicated by a bubble, or pocket, in the inverted tube in the broth, also known as a ___ tube.
durham
When inoculating microorganisms onto a mannitol salt agar plate, the selective component of the medium is ___.
salt
When inoculating microorganisms onto a mannitol salt agar plate, the differential components of the medium are the pH indicator ___ and the sugar ___.
phenol red, mannitol
EMB agar is a complex ___ and ___ medium.
differential, selective
The two functions of eosin Y and methylene blue in EMB agar are to inhibit the growth of ___ bacteria and to serve as indicators of vigorous ___ fermentation.
gram positive, lactose
MacConkey agar is a commonly used selective and differental medium. ___ and ___ inhibit the growth of Gram (+) bacteria.
bile salts, crystal violet
The differential agents found in the MacConkey agar plate include the sugar ___ and the pH indicator ___.
lactose, neutral red
Vigorous lactose fermenters growing on the EMB plate produce ___ colonies while non-lactose fermenters produce ___ colonies.
black, colorless
Vigorous lactose fermenters growing on the MacConkey agar plate produce ___ colonies while non-lactose fermenters produce ___ colonies.
red, colorless
the enzyme catalase breaks down ___ into ___ and ___
hydrogen peroxide, oxygen, water
When hydrogen peroxide is added to a catalase-positive culture, oxygen gas ___ form immediately.
bubbles
Nitrate reduction is an example of ___, reduction of (transfer of electrons to) an inorganic molecule other than oxygen.
anaerobic respiration
Nitrate broth is an undefined medium of beef extract, peptone, and ___
potassium nitrate
Many Gram-negative bacteria (including most Enterobacteriaceae) contain the enzyme ___ and perform a single-step reduction of nitrate to nitrite.
nitrate reductase
Before a broth can be tested for single-step nitrate reduction activity (nitrate reduction to nitrite), it must be examined for evidence of ___. This is simply a visual inspection for the presence of gas in the ___tube.
denitrification, durham
denitrification reaction:
___ > ___ > ___ > ___ > ___
NO3 > NO2 > NO > N2O > N2
If there is no visual evidence of denitrification upon visual inspection, nitrate reagent A ___ and nitrate reagent B ___ are added to the medium to test for nitrate reduction to nitrite.
sulfanilic acid, naphthylamine
Most of the time, we do not use CHO broths to differentiate among organisms, but ___ is an important sugar in differentiating among Gram (-) bacteria, specifically Gram (-) bacteria short rods.
lactose
Bacteria ferment sugars to produce ___ and/or gas. If bacteria do not ferment the sugar in a phenol red carbohydrate broth, it will then use the protein in the media, produce ___ and increase the pH of the solution.
acid, ammonia
Members of the Enterobacteriaceae all ferment ___ but not all of them ferment lactose. They tend to produce catalase and nitrate reductase, classifying them as ___ (oxygen preference).
glucose, facultative anaerobe
___ and ___ do not ferment any sugars because both bacteria are ___ (oxygen preference).
micrococcus, pseudomonas, obligate aerobes
When oxygen is excited to a higher state, reactive oxygen species are produced, such as ___, ___, and ___.
hydroxyl radical, peroxide anion, superoxides
___ and ___ convert peroxide to water and/or oxygen gas.
catalase, peroxidase
All of the bacteria sampled in lab tested positive for the catalase test except for ___
strep lactis
To an inoculated nitrate broth growing for over 48 hours, if the broth does not turn red after you add nitrate solutions A and B, the microbe either does not have the enzyme ___ or does have the enzyme and is a ___.
nitrate reductase, denitrifier
To confirm a negative nitrate reductase test, we add ___ powder, which is a ___, that will force nitrate to nitrite.
zinc, chemical catalyst
Most of the bacteria sampled in class are single-step nitrate reducers. Only two bacteria, ___ and ___ do not reduce nitrate, and only ___ is a denitrifier.
micrococcus, streptococcus, pseudomonas
Selective agents in MacConkey's Agar?
Bile Salts, crystal violet
What does MacConkey Agar inhibit growth of
Gram positive bacteria
What are the differential components in MacConkey's agar?
neutral red, lactose
What does EMB stand for?
Eosin Methylene Blue
Functions of E/MB in EMB agar?
dyes inhibit growth of gram positive bacteria and indicators of fermentation of lactose
pH indicator in carb broths?
Phenol red
Differential components in MSA plate
Phenol red, mannitol
Selective agent of MSA plate?
Salt. Encourages growth of halophiles
Nitrate reduction reaction and enzyme?
Nitrate (NO3) converted to NO2 by nitrate reductase
Catalase reaction and enzyme?
H2O2 converted to H2O + O2 by catalase
What is the key step of gram staining?
Alcohol (decolorizer)
The SIM medium is used for determination of three bacterial activities: (answers in S, I, M order please)
Sulfur reduction, indole production, motility
Sulfur reduction to H2S can be accomplished by bacteria in two different ways, depending on the enzymes present: thiosulfate reductase and
cysteine desulfurase
Bacteria possessing the enzyme _______ can hydrolyze tryptophan to ____________ and __________ (these 2 answers in alphabetical order), with the waste product ammonia.
Tryptophanase, indole, pyruvic acid
Urea can be hydrolyzed to _______ and _______ (these 2 answers in alphabetical order) by bacteria possessing the enzyme __________ .
Ammonia, carbon dioxide, urease
The pH indicator in the urea broth is ________ , which turns the media hot pink when the pH rises due to the increased levels of __________.
Phenol red, ammonia
Organisms that produce the enzyme _________ can be identified by their ability to deaminate the amino acid ___________ .
Phenylalanine deaminase, phenylalanine
Starch is a complex polysaccharide compound composed of _________ and ________ (answers in alphabetical order). There are three enzymes involved in starch digestion: an alpha-1,6-glucosidase and an alpha and beta-___________ .
Amylose, amylopectin, amylase
Gelatin is a protein derived from collagen and can be hydrolyzed by the enzyme _________ . Because the enzymes that hydrolyze both starch and gelatin are secreted extracellularly, they are referred to as __________ .
Gelatinase, exoenzymes
The purpose of the IMViC battery of tests is to differentiate among Gram-________ short rod ___________ __________.
Negative, lactose, fermenters
What is Gram's Crystal Violet in a gram stain?
Primary stain
What is Gram's Iodine in a gram stain?
Mordant
What is Safranin in a gram stain?
Counterstain
What is a SIM stab composed of?
0.75% agar, iron salts (protein)
Sulfur reduction reaction
Cytesine ——→ (Cytesine desulfurase) pyruvic acid + NH3 + H2S
Indole production reaction
Tryptophan + water ——> (Tryptophanase) indole +Pyruvic acid
Reagent for indole production
Kovac’s
Positive test for indole production?
A red layer forms on top of the media as Kovac’s reagent binds to indole meaning the enzyme tryptophanase is present
How is motility tested for?
Tested in the SIM stab where cloudiness surrounding the stab in the media indicates motile bacteria
Urease hydrolysis reaction
Urea + water ——> (urease) ammonia + CO2
What is the pH indicator in a urea hydrolysis test?
Phenol red
What is a positive and negative urea hydrolysis test and why do they occur?
Positive test = media turns pink, negative media orange/yellow. Occurs as pH increases with the production of ammonia if the bacteria has the enzyme urease
Phenylalanine deaminase reaction
Phenylalanine —→ (Phenylalanine deaminase) Phenylpyruvic acid + NH3 +H2S
Reagent in phenylalanine deaminase reaction
Ferric chloride
What does a positive test look like for phenyalanine deaminase and why does it occur?
If phenylpyruvic acid is present in the media as a result of the phenylalanine deaminase enzyme, ferric chloride will bind to it and form a dark green color. Negative tests remain yellow
What is starch agar composed of?
substrate starch, protein, 1.5% agar
What is starch made of?
Amylose, amylopectin
What is the enzyme that breaks down starch?
Amylase
Why is a protein source required in a starch hydrolysis and gelatin hydrolysis test?
So bacteria can grow regardless of their ability to hydrolyze starch or gelatin
What is the reagent used in a starch hydrolysis test?
Gram’s iodine
What does a negative and positive test look like for a starch hydrolysis test.
Gram’s iodine will bind to remaining starch forming a dark complex indicating that the bacteria can not hydrolyze starch. A clearing around a bacteria indicates that starch is not present and the bacteria possesses the enzyme amylase.
What is gelatin agar made of?
Gelatin, 1.5% agar, protein source
What is the enzyme we are looking for in a gelatin hydrolysis test?
Gelatinase
What is/are the reagent(s) used in a gelatin hydrolysis test?
Mercuric chloride in hydrochloric acid
What does a positive and negative gelatin hydrolysis test look like
Mercuric chloride in hydrochloric acid forms a white precipitate around bacteria with gelatin present. Bacteria with the enzyme gelatinase will have hydrolyzed the gelatin leaving a clearing around the bacteria.
What are the IMVC tests used for?
Differentiate between members of enterobacteriaceae, specifically short rod, gram negative, lactose fermenters
What do MRVP broths contain in their media?
Buffered peptone, glucose
What do MRVP broths test for?
Glucose fermentation
What 2 pathways do bacteria use to ferment glucose in an MRVP test?
Mixed acid fermentation, 2-3, butanediol fermentation
Which pathway does the MR test test for?
Mixed acid fermentation
What is the reagent used in an MR test?
Methyl red
What does a positive and negative test look like in an MR test?
Red = positive for mixed acids, pH <4. yellow = negative for mixed acids, pH >6
What pathway does the VP test test for?
2,3 -butanediol fermentation.
What is the VP test testing for in the media?
Acetoin. Come before 2,3 -butanediol
What are the reagents used in a VP test?
Alpha naphthol and KOH (potassium hydroxide)
What does a positive VP test look like?
Red = positive for acetoin
What does the citrate test test for?
The ability of microbes to use citrate as the sole source of carbon
What is a positive and negative citrate test?
Blue media = positive, green media = negative
What is the pH indicator in a citrate test?
Bromothymol blue
What are the pH’s of a positive and negative citrate test?
Positive = pH >7, negative = pH <7
Parfocal
Object remains in focus as one changes from one objective lens to another, requiring only minor adjustments with the fine adjustment knob to bring the object back into focus
Resolution
The ability of a microscope objective to clearly distinguish two adjacent points lying close together in a magnified specimen
Chromophore
Color bearing ion of a dye
What is the reaction that forms a black precipitate following the cysteine reaction
H2S + FeSO4 —-> FeS + H2SO4, black precipitate is FeS