Micro - Biochemical Testing II Flashcards
Are biochemical tests to determine the genotype or phenotype of an organism?
Phenotype
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
Single enzyme tests (6)
Catalase
Coagulase
Oxidase
Indole
Urease
PYR
Catalase converts ___ into…
Hydrogen peroxide
Water and oxygen
This group of organisms do not have catalase
Obligate anaerobes
-lethal for them
Catalase differentiates these 3 major genera of organisms: Staph, Strep, Listeria. What are the test results for each?
Staphylococcus and Listeria = positive
Streptococcus = negative
Weak bubbling or bubbling that takes place a few minutes later is catalase ___
Negative
-positive must be IMMEDIATE bubbling
When should we perform a coagulase test?
For gram-positive cocci that are catalase positive
-need to determine if organism is Staph aureus
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
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
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
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
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
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
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
Positive and negative QC cells for catalase test are
Positive control = Staph aureus
Negative control = Strep pyogenes
Positive and negative QC cells for oxidase test are
Positive control = Pseudomonas aeruginosa
Negative control = E coli
The indole test detects the presence of the enzyme ___, which hydrolyzes the amino acid ___ into ___
Tryptophanase
Tryptophan
Indole
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
Bromcresol purple has this color at acidic pH and this color at alkaline pH
Yellow
Purple
Bromcresol blue has this color at acidic pH and this color at alkaline pH
Yellow
Blue
Phenol red has this color at acidic pH and this color at alkaline pH
Yellow
Red/pink
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
