Urease Test and Microbial Identification
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Questions and Answers

What is the purpose of phenol red in urea medium?

  • To act as a pH indicator, signaling urea hydrolysis. (correct)
  • To solidify the medium for stab inoculation.
  • To inhibit the growth of non-urease producing organisms.
  • To provide a nitrogen source for bacterial growth.

A positive urease test results in a decrease in the pH of the medium.

False (B)

Why is it important to use a young (18-24 hour) culture for the urease test?

To ensure the bacteria are actively growing and producing ample urease enzyme.

In a motility test using urea medium, if the organisms remain at the site of inoculation, they are considered ______.

<p>non-motile</p> Signup and view all the answers

What is the recommended incubation temperature for the urease test?

<p>35-37°C (A)</p> Signup and view all the answers

In a urease test, why is zinc dust added if the broth doesn't turn red and no gas is produced?

<p>To determine if nitrite has been oxidized to nitrate, invalidating the test. (C)</p> Signup and view all the answers

A positive urease test should always show a rapid color change to red within 2 minutes of adding reagents, along with gas production.

<p>False (B)</p> Signup and view all the answers

What storage conditions are crucial for urea-containing medium to prevent autohydrolysis?

<p>The medium must be stored at 2 to 8°C in the dark.</p> Signup and view all the answers

When performing overnight urease tests using a medium that contains ______, the alkaline reaction may be due not to urease but to hydrolysis of this.

<p>peptone</p> Signup and view all the answers

Match the following test results with the appropriate organism or description:

<p>Positive urease test = Proteus mirabilis Negative urease test = Escherichia coli Red color after zinc addition = Nitrite oxidized to nitrate Alkaline reaction in peptone medium = Hydrolysis of peptone if performed overnight</p> Signup and view all the answers

In a Triple Sugar Iron Agar (TSIA) test, what color indicates acid production due to the fermentation of sugars?

<p>Yellow (A)</p> Signup and view all the answers

The Oxidase test identifies bacteria based on their ability to ferment lactose.

<p>False (B)</p> Signup and view all the answers

What specific metabolic process in TSIA is indicated by the medium turning black?

<p>metabolism of sodium thiosulfate</p> Signup and view all the answers

Cytochrome c oxidase catalyzes the final electron transfer from cytochrome c to a(n) ______ molecule.

<p>oxygen</p> Signup and view all the answers

Match the component of TSIA with its function:

<p>Phenol Red = pH indicator Sodium Thiosulfate = Indicates $H_2S$ production Ferric Citrate = Reacts with $H_2S$ to form a black precipitate Glucose = Fermented first, but is quickly depleted</p> Signup and view all the answers

In a motility test using soft agar, how does one differentiate motile from non-motile bacteria?

<p>Motile bacteria create a diffuse growth pattern extending from the inoculation point, while non-motile bacteria remain confined. (B)</p> Signup and view all the answers

The urease test, when performed directly on gastric biopsy samples, is used as a rapid test to detect the presence of E. coli.

<p>False (B)</p> Signup and view all the answers

What indicates a positive result in the nitrite reduction test after adding sulfanilic acid and alpha-naphthylamine reagents?

<p>absence of red coloration</p> Signup and view all the answers

In the motility test, __________ bacteria will only grow in the soft agar tube in the area where they are inoculated.

<p>non-motile</p> Signup and view all the answers

Match the following components often found in microbiological media with their primary role:

<p>Pancreatic digest of casein = Provides amino acids and peptides Peptic digest of animal tissue = Source of nitrogen, carbon, and vitamins Yeast extract = Supplies B vitamins and growth factors NaCl = Maintains osmotic balance</p> Signup and view all the answers

In the urease test, what does the absence of red color and gas production after the addition of zinc dust indicate?

<p>The nitrite has not been oxidized to nitrate, allowing the test to proceed. (D)</p> Signup and view all the answers

A positive urease test result must show a color change from colorless to red within 2 minutes after reagent addition.

<p>False (B)</p> Signup and view all the answers

Why is it important to store urea at 2 to 8°C in the dark?

<p>Urea is light sensitive and might undergo autohydrolysis.</p> Signup and view all the answers

In the urease test, if a medium containing peptone is used for overnight tests, the alkaline reaction may be due to hydrolysis of _____ rather than urease activity.

<p>peptone</p> Signup and view all the answers

Match the following organisms with their typical urease test result:

<p>Proteus mirabilis = Positive Escherichia coli = Negative</p> Signup and view all the answers

A bacterium that ferments glucose but not lactose or sucrose in a TSIA test will result in which of the following outcomes?

<p>Red slant, yellow butt (K/A) (A)</p> Signup and view all the answers

In a TSIA test, if a bacterium ferments lactose and/or sucrose, the resulting slant and butt will both turn yellow due to acid production.

<p>True (A)</p> Signup and view all the answers

What is the purpose of stabbing the butt of the test tube in the TSIA procedure?

<p>To create an anaerobic environment for detecting fermentation at the bottom of the tube.</p> Signup and view all the answers

In the TSIA test, bacteria that produce H2S break down sodium __________, leading to its decrease in the culture media.

<p>thiosulfate</p> Signup and view all the answers

Match the media components with their main role in the TSIA test:

<p>Peptone = Provides nitrogen and carbon sources Lactose and Sucrose = Differential carbohydrates Sodium Thiosulfate = Substrate for H2S production Phenol Red = pH indicator</p> Signup and view all the answers

The Indole test detects the activity of which enzyme?

<p>Tryptophanase (A)</p> Signup and view all the answers

The Methyl Red test identifies bacterial ability to produce alkaline end products from glucose fermentation.

<p>False (B)</p> Signup and view all the answers

What is the purpose of using Ehrlich’s reagent (ethyl alcohol instead of isoamyl alcohol) in the Indole test?

<p>To perform the test on non-fermenters and anaerobes.</p> Signup and view all the answers

An excessive amount of _______ in a test can give a weakly positive reaction.

<p>KOH</p> Signup and view all the answers

Which of the following organisms is NOT explicitly mentioned?

<p>Escherichia coli (D)</p> Signup and view all the answers

The Voges-Proskauer (VP) test identifies which specific metabolic byproduct?

<p>Acetylmethylcarbinol (Acetoin) (B)</p> Signup and view all the answers

The Citrate utilization test is specifically used to differentiate Gram-positive bacteria.

<p>False (B)</p> Signup and view all the answers

In the VP test, Barritt's reagents consist of alpha-naphthol and ________.

<p>KOH</p> Signup and view all the answers

Which of the following bacterial genera typically gives a negative result on the Citrate utilization test?

<p>Escherichia (C)</p> Signup and view all the answers

What is the purpose of performing the Citrate test in conjunction with other IMViC tests?

<p>To differentiate Gram-negative bacilli within the Enterobacteriaceae family.</p> Signup and view all the answers

Which specific culture media is recommended for performing the VP test?

<p>MR-VP Broth (D)</p> Signup and view all the answers

Match each bacterial genus with its typical Citrate utilization test result:

<p>Klebsiella = Positive Escherichia = Negative Proteus = Variable</p> Signup and view all the answers

A positive VP test indicates that the bacteria can ferment glucose.

<p>True (A)</p> Signup and view all the answers

Edwardsiella typically gives a ______ reaction in Citrate Utilization Test.

<p>positive</p> Signup and view all the answers

Which of the following reagents is NOT used in performing Citrate Utilization Test?

<p>Alpha-naphthol (C)</p> Signup and view all the answers

What indicates a negative result in the Citrate utilization test?

<p>No growth and the slant remains green (D)</p> Signup and view all the answers

What two products are generated from oxaloacetic acid?

<p>Pyruvic acid and $CO_2$ (D)</p> Signup and view all the answers

In the Citrate utilization test, how is a positive result visually indicated?

<p>Growth on the medium with a color change (D)</p> Signup and view all the answers

What color change in the Citrate Utilization test indicates a positive reaction?

<p>Green to blue (A)</p> Signup and view all the answers

What does an excess of sodium from sodium citrate, carbon dioxide, and water produce?

<p>Sodium Carbonate ($Na_2CO_3$) (D)</p> Signup and view all the answers

What is the initial substrate that is metabolized in the Citrate utilization test?

<p>Citrate (B)</p> Signup and view all the answers

What is the final pH of the medium in a positive Citrate Utilization test?

<p>6.9 ± 0.2 at 25°C (C)</p> Signup and view all the answers

What does the growth of an organism in the Citrate Utilization test indicate?

<p>Utilization of citrate (A)</p> Signup and view all the answers

What must occur for a Citrate test to be considered positive?

<p>Citrate metabolism (D)</p> Signup and view all the answers

What enzyme breaks down citrate into oxaloacetate and acetate?

<p>Citrase (A)</p> Signup and view all the answers

What is the original color of the slant that shows a negative Citrate test reaction?

<p>Green (C)</p> Signup and view all the answers

Which end product causes a color change in a positive Citrate test?

<p>Sodium carbonate (D)</p> Signup and view all the answers

What is the role of Dipotassium Phosphate in Citrate Utilization test?

<p>Buffer (B)</p> Signup and view all the answers

What is the first step of Citrate Utilization if it results in the production of pyruvate?

<p>Breaking down citrate into oxaloacetate and acetate (C)</p> Signup and view all the answers

Which of the following reagents is used in the VP test?

<p>Barritt’s reagents (D)</p> Signup and view all the answers

What causes an increase in the alkalinity of the medium when the organism is positive for citrate utilization test?

<p>Formation of ammonia (D)</p> Signup and view all the answers

What indicates a positive result in the Voges-Proskauer test?

<p>Formation of a red-pink color at the surface of the medium (A)</p> Signup and view all the answers

What is the first step in performing the Voges-Proskauer test after bacterial growth?

<p>Transferring 2 mL of broth to a clean test tube (C)</p> Signup and view all the answers

What culture age is recommended for the sample bacteria in Voges-Proskauer?

<p>18 to 24 hours old (D)</p> Signup and view all the answers

What incubation temperature is typically used for the Voges-Proskauer test?

<p>35 ± 2°C (B)</p> Signup and view all the answers

How should the tubes be incubated for the Voges-Proskauer test?

<p>Aerobically (A)</p> Signup and view all the answers

What is Reagent A in the Voges-Proskauer test?

<p>5% α-naphthol solution (C)</p> Signup and view all the answers

What type of culture is recommended for inoculation?

<p>18-24 hour culture (D)</p> Signup and view all the answers

How are citrate agar tubes typically inoculated?

<p>By streaking the surface of the slant (C)</p> Signup and view all the answers

In the Citrate Utilization Test, what does luxuriant growth on the slant without an accompanying color change potentially indicate?

<p>A false negative (C)</p> Signup and view all the answers

Why is it important to use a sterile inoculating needle?

<p>To prevent contamination. (C)</p> Signup and view all the answers

Why should the slant not be stabbed?

<p>To maintain an aerobic environment. (D)</p> Signup and view all the answers

What should be done if no color develops after the initial incubation period?

<p>Re-incubate for an additional 24 hours and retest. (B)</p> Signup and view all the answers

Why should inoculation not be done from broth cultures?

<p>Due to the risk of carryover of media. (D)</p> Signup and view all the answers

Why should the caps of the test tubes be left loosened?

<p>To ensure adequate aerobic environment. (A)</p> Signup and view all the answers

What is the length of time that the tube should be vigourously shaken for?

<p>30 mintues (B)</p> Signup and view all the answers

What reagent is used for the tube method when testing aerobic organisms for indole production?

<p>Kovac’s reagent (C)</p> Signup and view all the answers

What indicates a positive result in the rapid spot test for indole?

<p>Development of blue color within 20 seconds (B)</p> Signup and view all the answers

What is the purpose of moistening the filter paper in the rapid spot test?

<p>To facilitate the reaction between the reagent and the bacterial colony (C)</p> Signup and view all the answers

What is the recommended culture age for a well-isolated colony used in the rapid spot test from an 18-24-hour culture?

<p>18-24 hours (A)</p> Signup and view all the answers

What is the final pH of buffered peptone used in Methyl Red Test?

<p>6.9±0.2 (B)</p> Signup and view all the answers

When is Ehrlich’s reagent used for the tube method in indole testing?

<p>For anaerobes and weak indole producers (A)</p> Signup and view all the answers

What is the role of hydrochloric acid in Kovac’s reagent?

<p>To dissolve and stabilize p-dimethylaminobenzaldehyde (A)</p> Signup and view all the answers

According to the Methyl Red test, what is the percentage (v/v) of concentrated HCl?

<p>10% (D)</p> Signup and view all the answers

The Methyl Red (MR) test requires a growth medium with an adequate amount of which component?

<p>Tryptophan (A)</p> Signup and view all the answers

Which medium should not be used for the Methyl Red test because it destroys tryptophan?

<p>Mueller-Hinton Agar (C)</p> Signup and view all the answers

What can interfere with the spot indole test by inducing false-negative results?

<p>Nitrate (B)</p> Signup and view all the answers

The indole test is used to differentiate Proteus mirabilis from other species of what genus?

<p>Proteus (D)</p> Signup and view all the answers

The indole test differentiates indole-positive E. coli from indole-negative species of which genera?

<p>Enterobacter and Klebsiella (A)</p> Signup and view all the answers

Which extraction method provides the most sensitive results in the Indole test?

<p>Xylene (D)</p> Signup and view all the answers

The indole test differentiates Klebsiella pneumoniae (indole negative) from Klebsiella what?

<p>oxytoca (A)</p> Signup and view all the answers

The quality control for a positive Methyl Red (MR) test requires what specific organism?

<p>Escherichia coli (ATCC25922) (C)</p> Signup and view all the answers

Flashcards

Urease Enzyme

An enzyme produced by some organisms, especially those causing urinary tract infections.

Urea Medium

A medium containing urea and a pH indicator to detect urease activity.

Phenol Red

A pH indicator used in urea medium to detect changes in pH due to urease activity. Starts at pH 6.9.

Urease Test Inoculation

The method of applying a bacterial sample to the urea medium using a sterile needle.

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Non-motile organisms in medium

If an organism is non-motile, it will remain at the site of inoculation, exhibiting a lack of spread.

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Why add zinc dust?

Zinc dust is added to determine if nitrite has not been oxidized to nitrate, which would invalidate the test.

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Proteus mirabilis

A positive urease test control organism.

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Escherichia coli

A negative urease test control organism.

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False positives in urease test

Alkaline reactions can be due to the hydrolysis of peptone rather than urease activity.

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Urea storage conditions

Urea is light-sensitive and can undergo autohydrolysis if not stored properly.

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Yeast Extract in Media

Used to differentiate Pseudomonas aeruginosa from Enterobacteriaceae.

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TSIA Test

A test based on the ability of different bacterial genera to metabolize specific carbohydrates and a sulfur compound.

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Acid Production in TSIA

Indicates the fermentation of sugars, producing acid and decreasing the pH of the medium.

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Cytochrome c oxidase

A large transmembrane protein that catalyzes the final electron transfer to oxygen in aerobic respiration.

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Oxidase Test

Detects bacteria that can transfer a terminal electron to molecular oxygen using cytochrome c oxidase.

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Color Change in Medium

Indicates a pH change occurred in the medium, often due to bacterial metabolic activity.

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Pancreatic/Peptic Digest

Nutrient sources often included in microbiological media to support bacterial growth.

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Yeast Extract

Used to encourage or facilitate the growth of microorganisms.

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Nitrite Reduction Test

Used to determine if an organism can reduce nitrate or nitrite.

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Sulfanilic Acid and alpha-Naphthylamine

Reagents added to detect nitrite (NO2-) after bacterial growth in a nitrate reduction test.

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Negative Nitrite Reduction

Absence of red color and gas in broth; zinc dust confirms no oxidation to nitrate.

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Positive Nitrite Reduction with Zinc

Turns mixture red, indicating nitrite has been completely reduced to nitrogen gas.

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Positive Control result for Urease Test

No color change to red within 2 minutes and gas production in Durham tube.

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Urea Medium Storage

Medium must be stored at 2 to 8°C in the dark to prevent autohydrolysis.

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Peptone Hydrolysis Interference

Alkaline reactions caused by peptone breakdown instead of urease activity.

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TSIA Medium

A medium used to differentiate bacteria based on their ability to ferment carbohydrates (glucose, lactose, sucrose) and produce hydrogen sulfide (H2S).

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TSIA: Red slant/Yellow butt

A red slant/yellow butt (K/A) indicates that the bacteria ferments glucose but not lactose or sucrose.

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TSIA: Yellow slant/Yellow butt

A yellow slant/yellow butt (A/A) indicates that the bacteria ferments lactose and/or sucrose.

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TSIA: Red slant/Red butt

A red slant/red butt (K/K) indicates that the bacteria is not able to ferment any of the tested sugars.

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Sodium Thiosulfate in TSIA

A sulfur source in TSIA medium that can be broken down by bacteria to produce H2S, indicated by a black precipitate.

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Indole Test

Detects if bacteria can produce indole from tryptophan using tryptophanase enzymes.

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Ehrlich's Reagent

Ehrlich's reagent (ethyl alcohol instead of isoamyl alcohol) is used to test non-fermenters and anaerobes for indole production.

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Methyl Red (MR) Test

Identifies if bacteria produce stable acid end products via mixed-acid fermentation of glucose.

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Reagent Amounts

An excessive amount can give a weakly positive reaction, so use the reagents in the appropriate amount.

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Bromothymol blue

Bromothymol blue is a pH indicator.

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VP Test

A biochemical test that detects an organism's ability to metabolize pyruvate into acetoin.

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VP Test Reagents

Alpha-naphthol and KOH are used to enhance and intensify results in the VP test.

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MR-VP Broth

A broth used for performing the VP test, containing glucose.

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Citrate Test

Differentiates organisms based their ability to use citrate as a sole carbon source.

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Citrate Test Use

Used to differentiate Gram-negative bacilli of the Enterobacteriaceae family.

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Citrate-Positive Bacteria

Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, and Providencia.

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Citrate-Negative Bacteria

Escherichia, Shigella, Morganella, and Yersinia.

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Proteus Citrate Reaction

Can give variable results in citrate tests.

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VP Test Reagent A

5% Alpha-naphthol Solution (Barritt’s Reagent A)

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VP Test Reagent B

40% KOH or NaOH solution

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Salmonella Typhimurium

A Gram-negative bacterium; positive reaction in citrate test, changing medium from green to blue.

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Luxuriant Growth

The ability of an organism to grow well in a medium, showing active metabolism.

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Positive Citrate Reaction

Indicates an organism can utilize citrate as its sole carbon source.

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Citrase

The enzyme that breaks down citrate into oxaloacetate and acetate.

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Oxaloacetate Breakdown

Oxaloacetate broken down into pyruvate and carbon dioxide.

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Ammonium Salt Metabolism

Metabolism of ammonium salts yielding ammonia or sodium carbonate, increasing alkalinity.

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Negative Citrate Reaction

A negative citrate test result, indicated by the medium remaining green.

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Barritt’s Reagent B

40% KOH or NaOH solution for enhancing and intensifying results in the VP test.

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Inoculating Loop

A sterile tool used to transfer microorganisms from a culture.

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Well-isolated Colonies

Visible clusters of bacteria grown on a solid medium, originating from a single cell or a group of cells.

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Incubation

Growing microorganisms in a controlled environment to allow them to multiply.

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Citrate Utilization Test

A broth used to determine if an organism can utilize citrate as its sole carbon source.

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Positive Citrate Test

Indicated by the development of a Prussian blue color on the slant of the medium.

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Negative Citrate Test

No color change or the medium remains green. Growth may or may not be visible.

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Equivocal results

In citrate test. Test should be repeated with a smaller inoculum, if the agar remains unchanged on further incubation.

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Citrate Metabolism

Metabolic pathway where citrate is converted to oxaloacetic acid, which then becomes pyruvic acid and carbon dioxide.

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Na2CO3 Formation

The medium becomes alkaline due to the excess of sodium being used.

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Citrate Metabolism Result

Indicates the organism can metabolize citrate, resulting in alkaline products and a color change in the medium.

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VP Test Function

A test to determine if an organism produces acetoin from pyruvate.

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Citrate Test Method

Inoculate the organism on a slant and watch for the medium to turn blue.

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Growth in Citrate Test

The growth of the organism indicates a positive citrate test result.

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Lack of Isolated Colonies

Indicates insufficient growth or improper inoculation technique.

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Inoculating a Colony

Using a sterile needle to pick a single, well-isolated colony from a fresh culture.

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Vigorous Shaking of Tube

This action ensures even mixing during incubation.

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Stabbing the slant

When stabbing, bacteria is being deprived of necessary oxygen.

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Re-incubating Broth

A negative test can become positive with extended incubation, if the bacteria is slow-growing.

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Citrate Agar Inoculation

The citrate agar slant is streaked on the surface to allow for a positive result, and not stabbed

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Loosened Caps

Leaving caps loose ensures oxygen exchange for aerobic bacteria.

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Avoid Broth Inoculation

To avoid carryover of nutrients that could skew results.

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Rapid Spot Test

A rapid test to detect indole production by bacteria.

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Kovac’s Reagent

Reagent used to test for indole production, especially for aerobic organisms.

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Ehrlich’s Reagent

Reagent used to test for indole production, especially for anaerobes and weak indole producers.

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Blue Color Development

Indicates the presence of indole, signaling a positive result in the spot test.

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No Color or Pink Color

Indicates a negative result for indole production.

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Red color in MR test

Indicates a positive result for mixed-acid fermentation.

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Hydrochloric Acid in Kovac's Reagent

Used in the rapid spot test to dissolve p-dimethylaminobenzaldehyde.

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MR-VP Use

Used to differentiate organisms among the Enterobacteriaceae.

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Tryptophan Importance (MR)

An adequate amount of tryptophan is needed for accurate results.

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Mueller-Hinton Agar (MR)

Not appropriate for the MR test because tryptophan is destroyed during acid hydrolysis of casein.

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Nitrate Disk Interference

It can interfere with the spot indole test by inducing false-negative results.

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Indole Test Use

Used for differentiating Proteus mirabilis from other Proteus species.

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E. coli Differentiation

Differentiates indole-positive E. coli from indole-negative Enterobacter and Klebsiella.

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Klebsiella Differentiation

The test further differentiates K.pneumoniae(indole negative) from K.oxytoca (indole positive)

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Citrobacter Differentiation

Further separates C. freundii (indole negative) from C. koseri (indole positive)

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Study Notes

Urease Test

  • Indicates the identification of Proteus species, distinguishing them from non-lactose-fermenting Enterobacteriaceae

Principle of Urease Test

  • During the test, organisms utilize the urea as the sole source of nitrogen, producing sufficient ammonia to overcome the buffering capacity of the medium

Motility Test

  • Flagellum possesses a rigid, helical structure and actual motility results from its rotation

Nitrite Reduction Test

  • Nitrate reduction by bacteria relies on nitrate reductase
  • Presence of gas is visible in the Durham tube

Principle of Lysine Iron Agar Test

  • Lysine decarboxylation is an anaerobic process in the media butt
  • Lysine deamination is an aerobic process on the slant of the media

Limitations of Lysine Iron Agar Test

  • Certain species or strains may show delayed reactions or completely fail to ferment the carbohydrate

Catalase Test

  • Oxygen is released as byproduct of hydrogen peroxide decomposition

Principle of Catalase Test

  • Different mechanisms are found breaking down products to non-toxic substances in pathogenic organisms

Coagulase Test

  • Bound coagulase is called the clumping factor and is detected rapidly using a slide test
  • Requires several colonies and lacks sensitivity
  • Some strains of S. aureus may appear Catalase-negative by drop method so the test should be repeated with the tube method

Oxidase Test

  • Use fresh culture of bacteria for testing
  • Avoid testing bacteria grown on media with dyes (like EMB, MAC medium)
  • Don't test strict anaerobes
  • Use freshly made oxidase reagent
  • Store the oxidase reagent in a dark place at 20°C
  • Avoid overflooding the plate with an oxidase reagent
  • Avoid the use of nichrome wire loops

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Description

Explore the urease test principles and its applications in identifying bacteria based on urease production. Understand factors influencing test results, including medium composition and incubation conditions. Learn to interpret results and correlate them with specific organisms.

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