Micro - Biochemical Testing II Flashcards by Kevin Tran

Are biochemical tests to determine the genotype or phenotype of an organism?

Phenotype

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Two types of general biochemical tests as they relate to speed of the test

Rapid test

spot tests = immediate reaction
same-day tests = 4 hours or less

Conventional tests
-single tube tests usually within 18-24 hrs

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Single enzyme tests (6)

Catalase

Coagulase

Oxidase

Indole

Urease

PYR

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Catalase converts ___ into…

Hydrogen peroxide

Water and oxygen

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This group of organisms do not have catalase

Obligate anaerobes

-lethal for them

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Catalase differentiates these 3 major genera of organisms: Staph, Strep, Listeria. What are the test results for each?

Staphylococcus and Listeria = positive

Streptococcus = negative

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Weak bubbling or bubbling that takes place a few minutes later is catalase ___

Negative

-positive must be IMMEDIATE bubbling

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When should we perform a coagulase test?

For gram-positive cocci that are catalase positive

-need to determine if organism is Staph aureus

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What organism (genus/species) does the slide coagulase test SCREEN for? What test do we use to CONFIRM if it’s this organism?

Staph aureus
-most virulent Staph

Tube coagulase test

read every 30 mins for 4 hrs
incubate and read after 18 hrs if negative

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Which coagulase test is used to detect bound coagulase and which is used to detect free coagulase?

Slide coagulase test = bound coagulase

Tube coagulase test = free coagulase

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Both slide and tube coagulase tests are based on this mechanism that causes coagulation

Bound coagulase on cell wall or free coagulase converts fibrinogen to insoluble fibrin clot

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When doing the tube coagulase test, why should we check on the tube every half hour for up to 4 hours to determine if there’s coagulation?

Staphycoccus produces staphylokinase, which dissolves the fibrin clot
-causes false negative results

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Oxidase is an enzyme that is involved in what types of reactions?

Redox reactions

catalyzes transport of electrons from donor compounds (NADH) to electron acceptors
Kovac’s reagent acts as artificial electron acceptor and changes color

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The oxidase test is used to differentiate gram-positive or gram-negative bacteria? Differentiates what groups of organisms? Enterobacteriaceae are catalase positive or negative?

Gram-negative

Non-enterics from enterics

Negative

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An organism that is oxidase positive will convert the reagent, ___, into this color… A negative result looks like this…

Kovac’s

Blue-purple

No color change

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Positive and negative QC cells for catalase test are

Positive control = Staph aureus

Negative control = Strep pyogenes

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Positive and negative QC cells for oxidase test are

Positive control = Pseudomonas aeruginosa

Negative control = E coli

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The indole test detects the presence of the enzyme ___, which hydrolyzes the amino acid ___ into ___

Tryptophanase

Tryptophan

Indole

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The indole test is used in the differentiation of this group of organisms. The test is useful for the identification of these two organisms in the group that test positive…

Anaerobes/Enterobacteriaceiae

E. coli
Proteus vulgaris

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Bromcresol purple has this color at acidic pH and this color at alkaline pH

Yellow

Purple

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Bromcresol blue has this color at acidic pH and this color at alkaline pH

Yellow

Blue

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Phenol red has this color at acidic pH and this color at alkaline pH

Yellow

Red/pink

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Neutral red has this color at acidic pH and this color at alkaline pH. What common agar plate is it normally used in?

Red

Yellow

MacConkey agar

There are 3 types of indole tests. A positive result looks like this… A negative result looks like this…

Color change
-blue-green or bright pinkish red

Colorless

The urease enzyme is used to break down ___ into ___

Urea Ammonia and CO2 - CO2 released - ammonia reacts with water to form ammonium hydroxide, which raises pH

Is a positive urease test acidic or alkaline pH? What is the pH indicator for the urease test?

Alkaline Phenol red

Urease test - what color is the positive tube? What color is the negative tube? What is a weak positive reaction considered?

Pink Yellow Weak positive = positive -any pink is positive for test

The PYR test is to ID this organism (genus). The test is also used to differentiate ___ (genus) from Group D Streptococcus

Group A Streptococcus (S. pyogenes) Enterococcus -both are positive for PYR test

PYR test - a positive result looks like this

Red/pink color

The carbohydrate fermentation tests are useful to differentiate this family of organisms...

Enterobacteriaceae

Carbohydrate fermentation - pH indicator... Gas in trapped in inverted...

Phenol red Durham tube

Carbohydrate fermentation - positive result looks like... Negative result looks like...

Yellow (with/without gas production) Red

Amino acid degradation results in pH that is... Why?

Alkaline Due to actions of dehydrolyase, decarboxylase or deaminase

Amino acid degradation is used to differentiate these species (3) from the rest of Enterobacteriaceae. Are they all positive or negative for the test?

Proteus Morganella Providencia Positive -rest of enterics are negative

Amino acid degradation - decarboxylase positive looks like... Decarboxylase negative looks like...

Purple Yellow (from glucose fermentation)

How do we create an anaerobic condition for amino acid degradation test tube (ie lysine decarboxylase)

Add mineral oil

Phenylalanine deaminase test - what color is produced once ferric chloride is added?

Green

MR test checks for production of... VP test checks for production of...

Mixed acids Neutral end products (acetoin, diacetyl)

Can an organism be positive for both MR and VP?

No, either MR or VP is positive, never both

A positive MR or VP test is the appearance of a ___ color upon addition of their respective reagents

Red - neg MR = yellow/orange - neg VP = brown/copper

Citrate, malonate, and acetate are tests that utilize...

Their respective substrates as the sole carbon source | -citrate most common

Citrate test - pH indicator? What does the positive result look like (2)? What does the negative result look like?

Bromothymol blue Growth or blue color Green

Salt tolerance test - pH indicator... How much salt does the media contain?

Bromocresol purple 6.5% sodium chloride - high salt inhibitory to many organisms - contains glucose

What is the term for a salt loving organism?

Halophile | -ie Enterococcus

Salt tolerance test - what does a positive result look like (2)? What does a negative result look like?

Turbidity in medium OR Yellow Purple

Bile esculin hydrolysis - positive result look like... Negative result looks like...

Black slant - indicates esculin hydrolysis - bile is inhibitory to most pathogens No color change -brown

Optochin sensitivity test - what is optochin? What organism is the test confirmatory for (genus/species)? What similar group of organisms does the test rule out (what type of hemolysis)?

Antibacterial agent Streptococcus pneumoniae = alpha hemolytic Rules out other alpha hemolytic streptococci -not done for beta or gamma-hemolytic

Is Streptococcus pneumoniae optochin sensitive or insensitive?

Sensitive - look for zone of inhibition around disk

Which group of organisms are vancomycin resistant? Which are not?

Gram-negative = resistant Gram-positive = susceptible

Novobiocin resistance is used to screen for what organism (genus/species)? Is it resistant or susceptible to the antibiotic?

Staphylococcus saprophyticus -UTI pathogen Resistant

Does Haemophilus grow on blood agar or chocolate agar? Why or why not?

Grows only on chocolate agar (hemolysed RBCs). Cannot grow on blood agar since it can't break down RBCs (unless satelliting around another organism)

Haemophilus requires these two factors for growth... Which factor(s) does H. influenziae require for growth? Which factor(s) does H. parainfluenzae require for growth?

X or V or both, depending on species Both X and V V -has X

Haemophilus exhibits satelliting around this organism when co-cultured on a blood plate... Why?

S aureus S aureus breaks down RBCs, allowing Haemophilus to grow around it -make small streaks of S aureus on blood agar plate with Haemophilus lawn of growth

What is CAMP? What organism is it made by?

Extracellular protein Group B streptococci (S. agalactiae) - weakly beta hemolytic - Group A streptococci (S. pyogenes) do not make CAMP

What does CAMP interact with? What species makes this other compound? What does the interaction do?

Beta lysin toxin Staph aureus Synergistic effect - enhanced hemolysis on blood agar

Carbohydrate fermentation uses how many tubes per sugar tested? OF oxidative/fermentation uses how many tubes per sugar tested?

1 2 -cover one tube with mineral oil for fermentation conditions