Biochemical Testing Flashcards
TSIA - purpose
- Used to differentiate Enterobacteriaceae
- Differentiates them from other Gram-negative rods such as Pseudomonas
All other sugar must be converted to glucose before they enter the EMP. (T/F)
True. All other sugar must be converted to glucose before they enter the EMP.
Glucose utilization occurs anerobically on the slant where O2 is not available and on the butt where there is aerobic conditions. (T/F)
Glucose utilization occurs aerobically on the slant where O2 is available and on the butt where there is anaerobic condition.
Incubation period for TSIA
6 hours
Describe different processes occurring in TSIA medium for:
- Lactose/sucrose fermenters
- Non lactose/sucrose fermenters
- Gas and/or H2S producers
Lactose/sucrose fermenters
1. Once the glucose fermenting bacterium has reduced all of the available glucose to pyruvate, it will further metabolize pyruvate via TCA cycle to produce acid end products.
2. The acid in the medium cause the ph indicator phenol red, to change its colour to yellow. Both the slant and butt with a glucose fermenter will appear yellow.
3. After the depletion of glucose, the organism (that has the correct enzymes) starts to degrade lactose/sucrose
and continues to make acid end products.
Non lactose/sucrose fermenters
4. If the organism cannot use lactose/sucrose in the medium, it must shift to protein utilization as energy source.
5. As the protein breakdown occurs only in aerobic condition, the byproduct of protein and amino acid metabolism i.e NH3 changes the pH environment of the medium from the neutral to alkaline in the slant region where the condition is aerobic.
Gas and H2S producers
6. Gas production can be detected when holes are formed or the medium is broken into several fragments.
When do we call a TSIA rxn acid/acid?
– Butt and slant that still remain yellow for lactose and sucrose fermentors (degrades) after 18 hours of incubation and the reaction is called acid/acid.
When do we call a TSIA rxn alkali/acid
– The colour of slant appears red & is called alkali/acid while the colour of the butt is still yellow due to anaerobic glucose breakdown.
Production of what causes blackening of medium in TSIA?
– H2S production by organism results in the blackening of the medium.
H2S is produced from an ingredient in TSIA medium which is?
Sodium thiosulphate
An ingredient called sodium thiosulphate is contained in TSIA medium, what does it combine with to form ferrous sulphide?
This colour is due to the production of H2S from an ingredient of the medium, sodium thiosulphate,
- which then combines with another component of the medium, ferrous ammonium sulphate resulting in the formation of black, insoluble compound, ferrous sulphide.
What black, insoluble compound is formed as a result of H2S production in TSIA?
Ferrous sulphide
Red/yellow colour in TSIA rxn indicates?
Alkali/Acid
Utilizes Glucose only and peptone
Yellow/Yellow colour in TSIA rxn indicates?
Acid/Acid
Utilizes Glucose, Lactose and sucrose
Red/Red colour in TSIA rxn indicates?
Alkali/Alkali
No sugars are utilized, utilize peptone
H2S gas production is indicated by ?
Blackening of media
Gas production is indicated by?
Bubbles in butt
E. coli gives acid/Acid with gas production without H2S production. What does this mean?
This means E. coli utilizes all sugar anaerobically with production of gas during fermentation.
Pseudomonas spp gives alkaline/alkaline (Red/Red), what does this mean?
Pseudomonas spp does not utilize any sugar
Escherichia, Klebsiella, Enterobacter
Acid slant/ Acid butt (A/A)
Gas +, H2S -
Shigella, Serratia
Alkaline slant/Acid butt (K/A)
Gas -, H2S -
Salmonella, Proteus
Alkaline slant/ Acid butt (K/A)
Gas +, H2S +
Pseudomonas
Alkaline slant/ alkaline butt (K/K)
Gas -, H2S -
Urease test - purpose
Urease test is used to ID which organisms?
- Allows for Proteus to be distinguished from non-lactose-fermenting members of the Enterobacteriaceae.
- Rapid test on gastric biopsy samples to detect the presence of H. pylori.
The urea test is part of the battery of tests to identify the following:
Gram-negative enteric pathogens, including Yersinia spp.
Fastidious
Gram-negative rods—Brucella, Helicobacter pylori, and Pasteurella.
Gram-positive rods—Corynebacterium and Rhodococcus spp.
Yeasts—Cryptococcus spp.
Urease test - principle and controls
- Differentiates organisms based on their ability to hydrolyze urea with enzyme urease, utilizing urea as sole source of nitrogen
Product = ammonia
Positive test: Proteus mirabilis.
Negative test: Escherichia coli.
Urease test - results
Positive (+) = all pink within 24 hrs; rapid urea hydrolysis;strong urease production
Weak Positive (w+) = orange or yellow (24 hrs); partially pink (24 hrs - 6 days); slow urea hydrolysis; weak urease
Negative (–) = orange or yellow at 24 hrs and 24 hrs - 6 days;No urea hydrolysis;urease absent
Urease positive organisms
PUNCHKiSS MTB Y Proteus mirabilis Ureaplasma Nocardia Cryptococcus Helicobacter pylori (urease ++) Klebsiella Staph. saprophyticus Staph. epidermidis
Mycobacteria
Trichophyton
Brucella
Yersinia pestis is urease-negative, and Yersinia enterocolitica and Yersinia pseudotuberculosis are urease +
Motility test - purpose
- Used to detect bacterial motility
Motility is a differential characteristic of which family of bacteria?
Enterobacteriaceae
How can we observe motility?
Motile = diffuse growth radiating from central stab line
The non-motile bacteria will only grow in the soft agar tube and only the area where they are inoculated.
What is motility?
Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility.
Motility by bacterium is demonstrated in ____ _____ agar medium.
Why this medium?
Motility by bacterium is demonstrated in semi solid agar medium.
This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness
Some organisms will not display sufficient growth in the semi solid medium to make an accurate determination, and additional follow-up testing is required. (T/F)
True
Motility test controls
Positive: Escherichia coli
Negative: Staphylococcus aureus
List motile bacteria
Motile bacteria ---Motile with petrichous flagella Cute Baby SLEePing Clostridia (all except C. perfringes and C. tetani) Bacillus (Except B. anthracis) Salmonella Listeria monocytogen E. coli Proteus
---Motile with polar flagella Very Protective Solution HCL is polar- Vibrio Pseudomonas Spirochetes H. pylori Campylobacter Legionella
—Other motile bacteria
Citrobacter
Serratia
Enterobacter
Purpose
- Used to differentiate Enterics
- performed as a part of the IMViC test that is used to differentiate the members of the Enterobacteriaceae family.
- used as a traditional method to distinguish indole-positive E. coli from indole-negative Enterobacter and Klebsiella.
- The test also differentiates Proteus mirabilis from other Proteus species.
- differentiates K. pneumoniae(indole negative) from K. oxytoca (indole positive) and Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive).
Principle
Identifies bacteria capable of producing indole from tryptophan using the enzyme tryptophanase
Indole test results interpretation
Indole rapid test = pink = positive
Kovac’s reagent - added to tube, forming a liquid layer
Positive = formation of red color
Indole - Negative = no color
What causes red colour in indole test?
indole combines with kovac’s reagent to form this colour
Criteria for indole testing
Fresh growth of a Gram-negative rod on a medium that does not contain dyes and contains tryptophan, e.g., BAP or CHOC.
Anaerobic Gram-positive rods.
Anaerobic Gram-negative rods.
Media that contain dyes (e.g., EMB, MAC) shouldn’t be used in indole testing (T/F)
True
The growth medium must contain an adequate amount of tryptophan. What is an example of a growth medium not recommended due to low amounts of tryptophan? And why?
Do not use Mueller-Hinton agar for the test, because tryptophan is destroyed during the acid hydrolysis of casein.
List indole +, indole - organisms
Indole +ve bacteria
Citrobacter diversus
E. coli
Klebsiella oxytoca
Indole -ve bacteria Enterobacter Serratia Citrobacter freundii Klebsiella pneumoniae Pseudomonas Shigella Salmonella
Indole variable
Proteus
Citrobacter diversus is indole _ while Citrobacter freundii is indole _
Citrobacter diversus is indole + while Citrobacter freundii is indole -
What test can we use to differentiate E. coli from Enterobacter and Klebsiella pneumoniae?
Indole test
E. coli is indole _ while Enterobacter and K. pneumoniae are indole _
E. coli is indole + while Enterobacter and K. pneumoniae are indole -
Spot indole test controls
Positive: Escherichia coli
Negative: Klebsiella pneumoniae
How do we determine lactose fermenters? By use of what Agar?
MacConkey Agar contains lactose, it also inhibits gram positive growth
If gram -ve organism ferments lactose the medium will turn pink
List lactose fermenters and non lactose fermenters
Lactose fermenters: E. coli, Klebsiella, Enterobacter, Citrobacter, Serratia
Non lactose fermenters: Pseudomonas, Shigella, Salmonella, Proteus
Principle and Purpose of Citrate test
- Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy.
- This test is among a suite of IMViC Tests that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.
What is the enzyme used by bacteria to break down citrate and what product is formed?
Citrate-permease,
- converts citrate to pyruvate.
Pyruvate can then enter the organism’s metabolic cycle for the production of energy
What happens when the bacteria metabolizes citrate?
The resulting shift in pH does what to the medium?
When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity.
The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.
Describe positive and negative citrate utilization rxns
Positive Reaction: Growth with color change from green to intense blue along the slant.
Examples: Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia etc.
Negative Reaction: No growth and No color change; Slant remains green.
Examples: Escherichia, Shigella, Yersinia etc.
Control organisms for Citrate test
Citrate Positive: Klebsiella pneumoniae
(growth; blue color)
Citrate Negative: Escherichia coli
(no growth or trace of growth)
List Citrate positive and negative organisms
Citrate positive
Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia
Citrate negative
Escherichia, Shigella, Yersinia
Salmonella is citrate _ while Shigella is citrate _
Salmonella is citrate + while Shigella is citrate -
Oxidase test - Principle and Purpose
- The oxidase test detects the presence of a cytochrome oxidase system that will catalyse the transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine.
The dye is reduced to deep purple color.
- This test is used to ASSIST in the identification of Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of which produce the enzyme cytochrome oxidase.
Steps in Oxidase test
- Cytochrome containing organisms produce an intracellular oxidase enzyme.
- – This oxidase enzyme catalyzes the oxidation of cytochrome c. - Oxidase positive bacteria possess cytochrome oxidase
- Cytochrome oxidase catalyses the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen).
- The test reagent, acts as an artificial electron acceptor for the enzyme oxidase.
- The oxidised reagent forms the coloured compound indophenol blue.
Organisms which contain cytochrome c as part of their respiratory chain are oxidase-_______ and turn the reagent ____/______
Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it __________, and are oxidase-________.
Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple.
Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless, and are oxidase-negative.
Cytochrome system present in aerobic organisms can utilize __ as final _______ receptor
End product = H2O or H2O2 (broken down by catalase)
In oxidase test, the ____ _______ acts as the final electron acceptor for _______ enzyme.
End product = coloured compound __________ ____
Cytochrome system present in aerobic organisms (can utilize O2 as final Hydrogen receptor)
End product = H2O or H2O2 (broken down by catalase)
In oxidase test, the test reagent acts as the final electron acceptor for oxidase enzyme.
End product = coloured compound indophenol blue
Positive and negative controls for Oxidase test
Positive Control: Pseudomonas aeruginosa
Negative Control: Escherichia coli
Oxidase Positive Organisms
PANCH Big VacuuM PeopLe
Pseudomonas, Aeromonas, Neisseria, Campylobacter, Helicobacter pylori,
Brucella, Vibrio, Moraxella, Pasteurella, Legionella pneumophila
Coagulase test - purpose
- differentiate Staphylococcus aureus (+) which produce the enzyme coagulase, from S. epidermis and S. saprophyticus (-) which do not produce coagulase.
i. e Coagulase Negative Staphylococcus (CONS).
