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).
What is coagulase?
Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin.
Staphylococcus aureus produces two forms of coagulase which are?
Bound and free
Bound coagulase (clumping factor) is bound to the ________ ____ ____ and reacts directly with fibrinogen.
This results in an alternation of fibrinogen so that it ________ on the staphylococcal cell, causing the cells to ______ when a bacterial suspension is mixed with the ______.
Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts directly with fibrinogen.
This results in an alternation of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with the plasma.
Bound coagulase doesn’t require coagulase-reacting factor (T/F)
True
Free coagulase involves the activation of ______ ________-reacting factor (CRP), to from a ________-CRP complex.
This complex in turn reacts with _________ to produce the fibrin clot.
Free coagulase involves the activation of plasma coagulase-reacting factor (CRP), to from a coagulase-CRP complex.
This complex in turn reacts with fibrinogen to produce the fibrin clot.
Positive and negative results for coagulase test
Fibrin Clot of any size- Positive
No Clot- Negative
Coagulase + and - organisms
Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria like S. pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae, S. hyicus
Coagulase Negative Organisms: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc.
Catalase test - principle and purpose
- Demonstrate presence of catalase
- DIFFERENTIATE morphologically similar Enterococcus or Streptococcus (catalase negative) and Staphylococcus (catalase positive/catalase producing)
- DIFFERENTIATE aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive
- ASSIST in ID of Enterobacteriaceae
What is catalase?
an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2).
The enzyme catalase mediates the breakdown of ________ _________ into oxygen and water.
Bacteria protect themselves from the lethal effect of ________ _________ which is accumulated as an end product of aerobic _________ metabolism with the use of catalase
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water.
Bacteria protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism with the use of catalase
Positive and negative catalase rxn results
Positive: Copious bubbles produced, active bubbling
Negative: No or very few bubbles produced.
Catalase + and - organisms
Catalase +ve Staphylococci, Listeria, Corynebacterium diphtheriae, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus
Catalase -ve
Streptococcus
Enterococcus
+ and - controls Catalase test
Positive: Staphylococcus aureus
Negative: Enterococcus faecalis
What is hemolysis?
Hemolysis is the breakdown of red blood cells (RBC). A substance that causes hemolysis is a hemolysin.
Beta-hemolysis (β-hemolysis) is associated with complete ____ of red cells surrounding the colony.
Beta hemolysis is caused by two hemolysins _ and _; the former is inactive in the presence of _______.
Thus, stabbing of the plate increases the intensity of the ________ reaction.
S is an ______-stable cytotoxin.
Beta-hemolysis (β-hemolysis) is associated with complete lysis of red cells surrounding the colony.
Beta hemolysis is caused by two hemolysins O and S; the former is inactive in the presence of oxygen.
Thus, stabbing of the plate increases the intensity of the hemolysis reaction.
S is an oxygen-stable cytotoxin.
Beta-hemolytic organisms are generally commensals of throat and causes opportunistic infections.
Examples: Streptococcus pyogenes, or Group A beta-hemolytic Strep (GAS).
Weakly beta-hemolytic species: Streptococcus agalactiae, Clostridium perfringens, Listeria monocytogenes
Alpha-hemolysis (α-hemolysis) is a ______ or “green” hemolysis.
Alpha hemolysis is caused by _______ ______ produced by the bacterium, oxidizing ________ to green methemoglobin.
It exhibit incomplete haemolysis with 1-2 mm wide.
Examples: Streptococcus _________ and a group of oral streptococci (Streptococcus _______)
Alpha-hemolysis (α-hemolysis) is a partial or “green” hemolysis.
Alpha hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin.
It exhibit incomplete haemolysis with 1-2 mm wide.
Examples: Streptococcus pneumoniae and a group of oral streptococci (Streptococcus viridans)
Gamma hemolytic colonies show neither typical alpha nor beta haemolysis.
There may be, however, slight _________ in the medium. The streptococci included in this group are usually not ________.
Examples: Enterococcus ______ (formerly called “Group D Strep”)
Gamma hemolytic colonies show neither typical alpha nor beta haemolysis.
There may be, however, slight discolouration in the medium. The streptococci included in this group are usually not pathogenic.
Examples: Enterococcus faecalis (formerly called “Group D Strep”)
Mannitol Salt Agar (MSA) is used as a ________ and differential medium for the ________ and identification of Staphylococcus aureus.
Mannitol Salt Agar (MSA) is used as a selective and differential medium for the isolation and identification of Staphylococcus aureus.
Selective media - define
It encourages the growth of a group of certain bacteria while inhibiting the growth of others
Mannitol is the fermentable ________, fermentation of which leads to ____ production, detected by phenol ___ indicator.
Mannitol is the fermentable carbohydrate, fermentation of which leads to acid production, detected by phenol red indicator.
________ positive staphylococci (e.g., Staphylococcus aureus) produce ______ colonies and a surrounding yellow medium while _______ negative staphylococci produce ___ colonies and no color change of the phenol ___ indicator.
Coagulase positive staphylococci (e.g., Staphylococcus aureus) produce yellow colonies and a surrounding yellow medium while coagulase negative staphylococci produce red colonies and no color change of the phenol red indicator.
Match the following results of MSA: Staphylococcus aureus Staphylococci other than S. aureus (e.g. Staphylococcus epidermidis ) Streptococci Micrococci Gram-negative bacteria
No growth to trace growth. Colorless or Red colonies with red zones. Large white to orange. No growth to trace growth. Yellow colonies with yellow zones.
Staphylococcus aureus : Yellow colonies with yellow zones.
Staphylococci other than S. aureus (e.g. Staphylococcus epidermidis ) : Colorless or Red colonies with red zones.
Streptococci : No growth to trace growth.
Micrococci : Large white to orange.
Gram-negative bacteria : No growth to trace growth.
Positive and negative controls for MSA
Positive Control: Staphylococcus aureus
Negative Control: Escherichia coli
Further biochemical tests are necessary for the identification of S. aureus or other spp, why?
Several Staphylococcus species other than aureus are mannitol positive
Presumptive Staphylococcus aureus must be confirmed with a coagulase test before MSA (T/F)
True
SSAgar purpose
- Selective and differential medium for the isolation of Salmonella and some Shigella species
- Aids in the differentiation of lactose and non-lactose-fermenters
This medium is not recommended for the primary isolation of Shigella (T/F)
True
The inclusion of ____ Salts, Sodium ______ and Brilliant _____ serve to inhibit gram-positive, coliform organisms and inhibit swarming ______ spp., while allowing Salmonella spp. to grow.
The inclusion of Bile Salts, Sodium Citrate and Brilliant Green serve to inhibit gram-positive, coliform organisms and inhibit swarming Proteus spp., while allowing Salmonella spp. to grow.
Bacterial colonies will appear colorless with black centers for this bacteria, identify and state why this happens.
, resulting in .
Salmonella will not ferment lactose, but produce hydrogen sulfide (H2S) gas.
Bacterial colonies will appear colorless, identify and state why this happens.
Shigella do not ferment lactose or produce hydrogen sulfide gas
Bacterial growth with a pink color, identify and state why this happens.
Coliform bacteria such as E. coli will ferment the lactose in the media
They do not produce any hydrogen sulfide.
Large, mucoid, pale, opaque cream to pink colonies, identify and state why this happens.
Enterobacter and Klebsiella ferment lactose and don’t produce hydrogen sulfide
Identifying diff between E. coli and Enterobacter and Klebsiella via SSA
Enterobacter and Klebsiella appears larger than E. coli
+ and - SSA test
Positive
Salmonella enteriditis = Colorless colonies with black center
Salmonella typhi = Colorless colonies with black center
Salmonella typhimurium = Colorless colonies with black center
Shigella flexneri = Colorless colonies
Negative
Enterococcus faecalis = Inhibited
Escherichia coli = Inhibited
Enterobacter aerogenes = partially inhibited. Cream-pink
CAMP test is used to distinguish the species Streptococcus agalactiae from other species of beta-hemolytic Streptococcus. S. agalactiae, a member of the Lancefield Group B streptococci,
used to distinguish the species Streptococcus agalactiae from other species of beta-hemolytic Streptococcus.
It is used to identify Listeria monocytogenes which also produces a positive CAMP reaction.
CAMP factor -
diffusible extracellular hemolytic heat-stable protein
Principle of CAMP test
Certain organisms (including group B streptococci) produce a CAMP factor that cooperates with the beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells.
The group B streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar.
Positive CAMP test result
Positive: Arrowhead zone of beta- hemolysis adjacent to the place where the two streak lines (of two organisms) come into proximity.
Negative: No enhancement of hemolysis.
+ and - controls - CAMP test
Positive: Streptococcus agalactiae —enhanced arrowhead hemolysis.
Negative: Streptococcus pyogenes —beta-hemolysis without enhanced arrowhead formation.
Colonies of Listeria monocytogenes have a narrow zone of beta-hemolysis on sheep blood agar, what does this mean for CAMP test?
L. monocytogenes may be confused with group B beta-hemolytic streptococci, if catalase and gram stain are not performed.
Hippurate hydrolysis test
Hippurate hydrolysis test is used to detect the ability of bacteria to do the foll:
hippuricase
substrate hippurate (hydrolysis) ---> glycine and benzoic acidHippurate hydrolysis - purposes
- Most frequently used in the identification of Gardnerella vaginalis, Streptococcus agalactiae, Campylobacter jejuni, and Listeria monocytogenes.
- Differentiation of β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci.
- Aids in the separation of Campylobacter jejuni and Campylobacter coli strains.
When hippurate is hydrolysed by an organism _______ and _______ ____ are formed.
Glycine is ________ (removal of amino group) by the oxidizing agent ninhydrin.
The end products of the ninhydrin oxidation react to form a ______-colored product.
When hippurate is hydrolysed by an organism glycine and benzoic acid are formed.
Glycine is deaminated (removal of amino group) by the oxidizing agent ninhydrin.
The end products of the ninhydrin oxidation react to form a purple-colored product.
+ and - results - Hippurate test
Positive: A positive test is indicated by the appearance of a deep blue/violet color in 30 minutes.
Negative: Colorless or slightly yellow pink color
String test procedure summarized
Suspend 18 - 24 hr growth from Heart Infusion Agar (HIA) or other non-inhibitory medium in a drop of 0.5% aqueous solution of sodium deoxycholate.
Positive String test - interpretation
If the result is positive, the bacterial cells will be lysed by the sodium deoxycholate or Sodium taurocholate, the suspension will lose turbidity, and DNA will be released from the lysed cells causing the mixture to become viscous.
A mucoid “string” is formed when an inoculating loop is drawn slowly away from the suspension.
String test - uses
- Separate Vibrio spp. from Aeromonas spp. and P. shigelloides.
- Differentiate Vibrio cholerae (string test positive) from other Vibrio spp. (string test negative).
+ and - string test result appearances
Positive Test: Appearance of String (thread like mucoid) when the loop is pulled upwards.
Negative Test: No appearance of String when the loop is pulled upwards.
+ and - string test organisms as controls
+
Vibrio cholerae
-
Aeromonas spp. and P. shigelloides
What are the important components of litmus test?
The lactose (milk sugar), litmus (pH indicator), and casein (milk protein) contained within the medium can all be metabolized by different types of bacteria.
Principle of litmus test
Litmus test - uses
The test differentiates microorganisms based on reduction, fermentation, clot formation, digestion, and the formation of gas.
- Differentiates members of the Enterobacteriacaeae from other gram-negative bacilli based on the enterics’ ability to reduce litmus.
- Commonly used to differentiate members within the genus Clostridium. (C perfringes is litmus +)
By producing acid from the fermentation of ______ present inmilka bacterium may also cause themilkto curdle or ____ in the bottom of the tube.
Fermentation of lactose is demonstrated when the litmus turns ___ as a result of ____ production.
If sufficient acid is produced, _____ in the milk is _______, solidifying the milk
By producing acid from the fermentation of lactose present inmilka bacterium may also cause themilkto curdle or clot in the bottom of the tube.
Fermentation of lactose is demonstrated when the litmus turns pink as a result of acid production.
If sufficient acid is produced, casein in the milk is coagulated, solidifying the milk
Litmus media reactions are not specific (T/F), if true what is the next step?
You should do additional tests for definitive identification of microorganisms.
Positive litmus milk results can look like?
Acid pH: pink to red color
Alkaline pH: purplish- blue color
Reduction: white
Acid curd: hard curd with clear supernatant (whey)
Digestion: Dissolution of clot with clear, grayish, watery fluid and a shrunken, insoluble pink clot
Rennet curd: soft curd followed by peptonization (alkaline pH, supernatant brown)
Gas production: bubbles in coagulated milk
White (R) - litmus was reduced + became clear
Pink (A) - lactose fermented + acidic
Bluish (K) - Basic pH, no lactse fermentation
Curd forms (C)
Gas in curd (G)
Brown ring (P) - casein protein broken down
Negative litmus milk test result
Negative test:
Color and consistency remain same.
Another name for Nagler’s reaction
Lecithinase test
Nagler’s rxn uses
- Used to identify organisms that liberate phospholipases (lecithinases) e.g. Clostridium perfringens.
- helps in the differentiation of C. perfringens from other Clostridium spp that also produce lecithinase (C.baratti, C.absonum, C.bifermantans, C.sordelli, and C.novyi) by neutralization of lecithin c activity by an antitoxin.
Bacillius cereus also exhibits strong lecithinase activity but can be differentiated by ?
- Strong hemolytic property on sheep blood agar and motility.
List Bacillus spp that are Leicithinase + and -
Lecithinase positive or weakly positive: B. thuringiensis and B.anthracis
Lecithinase negative: Bacillus sphaericus
Nagler’s rxn explain
Bacterial lecithinase break down this lecithin (a normal component of egg yolk) to insoluble diglycerides resulting in
an opaque halo, surrounding the colony when grown on the egg yolk agar medium.
Modified egg yolk agar is a differential and enriched medium, how?
used in the isolation and presumptive differentiation of different species based on their lecithinase and lipase production and proteolytic activity.
The degradation of lecithin present in the egg yolk results in the formation of ______ ________ around the colonies.
The lipase enzyme hydrolyzes the fats within the egg yolk, which results in an ________ ______ on the colony surface.
The degradation of lecithin present in the egg yolk results in the formation of opaque precipitate around the colonies.
The lipase enzyme hydrolyzes the fats within the egg yolk, which results in an iridescent sheen on the colony surface.
How is C. perfringes different from other Clostridium spp in the Lecithinase test?
The alpha (α) toxin of C. perfringens has phospholipase activity
Can DNA enter cells?
Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is way too large to enter the cell membrane.
How do bacterial cells utilize extracellular DNA?
In order to utilize external DNA, bacteria cells secrete exoenzymes (DNases) outside of the cell that hydrolyze DNA into nucleotides.
The nucleotides can then move across the cell membrane via transport proteins.
The cell can use nucleotides to make nucleic acids and to use as a source of nitrogen, phosphate and carbon.
DNase test purpose
To differentiate organisms based on the production of deoxyribonuclease.
What kind of agar is DNase agar?
DNase agar is a differential medium
What indicator is used in DNase test and how is it bound to DNA?
Methyl green as an indicator.
Methyl green is a cation which binds to the negatively-charged DNA.
DNase test results with colour indicator
Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments.
When the DNA is broken down, it no longer binds to the ______ green, and a clear ____ will appear around the areas where the DNase-producing organism has grown.
Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments.
When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around the areas where the DNase-producing organism has grown.
DNase agar without indicator
In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing of the agar after addition of ___ (oligonucleotides dissolves in acid but ___ _____ are insoluble).
The acid precipitates unhydrolyzed DNA making the medium opaque.
Therefore, DNase producing colonies ________ DNA and produce a clear zone around the growth.
In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing of the agar after addition of HCL (oligonucleotides dissolves in acid but DNA salts are insoluble).
The acid precipitates unhydrolyzed DNA making the medium opaque.
Therefore, DNase producing colonies hydrolyze DNA and produce a clear zone around the growth.
Positive and negative DNase test results
Positive: Clear zone around inoculation line
Negative: If no degradation of DNA occurs, the medium remains green.
Uses of DNase test
- Differentiate Staphylococcus aureus which produces the enzyme deoxyribonuclease from other Staphylococci which do not produce DNase.
- Useful if plasma is not available to perform coagulase test or when the result of coagulase tests are difficult to interpret.
- Used to distinguish Serratia (positive) from Enterobacter sp.
- Moraxella catarrhalis (positive) from Neisseria
+ and - DNase producing organisms
Positive: Staph aureus, Serratia, Moraxella catarrhalis
Negative: Staph epidermidis, Enterobacter, Neisseria
The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. What are their functions?
Permease allows lactose to enter the bacterial cell wall,
Beta-galactosidase - breaks down lactose into glucose and galactose
The glucose and galactose (products) can then be metabolized by the bacteria. However, some organisms lack permease and appear as late or non-lactose-fermenters.
How can we overcome this in testing more accurately for lactose fermenting organisms?
The ONPG test is considered to be a very sensitive test for lactose-fermentation.
O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, acts as the substrate for the beta-galactosidase to ascertain the particular enzyme activity
Purpose of ONPG test
To determine the ability of an organism to produce β-galactosidase enzyme.
- differentiating members of the Enterobacteriaceae and other microorganisms based on beta-D-galactosidase activity.
- distinguishes late lactose fermenters from non–lactose fermenters of Enterobacteriaceae
O-nitrophenyl-beta-D-galactopyranoside (ONPG) is an artificial substrate structurally similar to lactose
What is the main difference between ONPG is lactose?
Unlike lactose, the substrate O-nitrophenyl-beta-D-galactopyranoside (ONPG) is capable of penetrating the bacterial cell without the presence of permease.
+ and - ONPG test results
Positive: Development of a yellow colouration (presence of β-galactosidase)
Note: The fluid and disc will turn any shade of yellow if positive for galactosidase enzyme.
Negative: No colour development (absence of enzyme)
All organisms tested must be inoculated from what kind of medium?
a lactose-containing medium (e.g., TSI or MacConkey).
Lactose fermenters are ONPG (positive/negative)
Late lactose fermenters are ONPG (positive/negative)
Non lactose fermenters are ONPG (positive/negative)
Lactose fermenter (ONPG Positive): E. coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease.
Late lactose fermenter (ONPG Positive): Citrobacter spp
Non lactose fermenter (ONPG Negative): Salmonella spp; Shigella spp; Proteus spp; Providencia spp and Morganella spp do not produce β-galactosidase so can not ferment lactose.
Match the foll:
Lactose fermenter (ONPG Positive):
Late lactose fermenter (ONPG Positive):
Non lactose fermenter (ONPG Negative):
Salmonella spp; Shigella spp; Proteus spp; Providencia spp and Morganella spp do not produce β-galactosidase so can not ferment lactose.
Citrobacter spp
E. coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease.
Lactose fermenter (ONPG Positive): E. coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease.
Late lactose fermenter (ONPG Positive): Citrobacter spp
Non lactose fermenter (ONPG Negative): Salmonella spp; Shigella spp; Proteus spp; Providencia spp and Morganella spp do not produce β-galactosidase so can not ferment lactose.
+ and - controls for ONPG test
Positive control: Escherichia coli
Negative control: Proteus vulgaris
Hanging drop method - process
This preparation gives good views of microbial motility.
- A drop of culture is placed on a coverslip that is encircled with petroleum jelly
- The coverslip and drop (fluid containing bacteria) are then inverted over the well of a depression slide.
- The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation.
Hanging drop method can be used for ?
Presumptive diagnosis of Vibrio Cholerae from rice watery stools. = Darting motility
Presumptive diagnosis of L monocytogenes = tumbling motility in hanging drop preparations
Some bacteria have the ability to utilize ______ and convert it to a stable ____ like lactic acid, acetic acid or formic acid as the end product.
These bacteria initially metabolise glucose to ______ _____, which is further metabolized through the ‘mixed acid pathway to produce the stable (acid/alkali).
Methyl red test - effect of acid
The acid so produced (increases/decreases) the pH to 4.5 or below, which is indicated by a change in the colour of methyl red from yellow to ___.
Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic acid, acetic acid or formic acid as the end product.
These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized through the ‘mixed acid pathway to produce the stable acid.
Methyl red test - effect of acid
The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the colour of methyl red from yellow to red.
Positive and negative results for methyl red test
Positive Reaction: A distinct red color (A) Examples: E. coli Citrobacter Proteus Salmonella Yersinia
Negative Reaction: A yellow color (B) Examples: Klebsiella Serratia Enterobacter Vibrio cholerae
Controls
Klebsiella pneumoniae —MR negative (yellow)
Escherichia coli —MR positive (red)
The methyl red test must not be performed unless the medium has been incubated for how long?
It is important that a light inoculum be used. If an inoculum is too heavy, what can occur?
Minimum of 48 hours
Tests that are run too early may result in false-positive interpretation.
Bacterial growth may be inhibited and result in invalid test results.
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl carbinol from (glucose/sucrose) fermentation.
If present, acetylmethyl carbinol is converted to
_____ in the presence of alpha- naphthol,
strong alkali (40% KOH), and O2.
This brings out a (red/yellow) complex.
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl carbinol from glucose fermentation.
If present, acetylmethyl carbinol is converted to
diacetyl in the presence of alpha- naphthol,
strong alkali (40% KOH), and O2.
This brings out a red complex.
+ and - results VP test
Positive Reaction: A pink-red color at the surface
Negative Reaction: A lack of a pink-red color
+ and - controls VP test
Positive Control: Klebsiella (formerly Enterobacter) aerogenes
Negative Control: Escherichia coli
+ and - VP test organisms
Positive VP organisms
Enterobactericieae family:
Klebsiella, Enterobacter, Serratia
Viridans group streptococci (except Streptococcus vestibularis), Listeria, Vibrio eltor, Vibrio alginolyticus
Negative VP organisms
Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc.
