Proteus Panel Overview

• Key Tests Analyzed:

  • SIM Stab

  • Urea Hydrolysis

  • Phenylalanine Deamination

SIM Stab Test

• Components of SIM Stab:

  • Sulfur reduction

  • Indole production

  • Motility

• Characteristics of Medium:

  • Semisolid medium containing 0.75% agar (half the percentage of regular agar)

  • Used primarily for assessing motility of microorganisms

• Purpose:

  • Assess the capability of microbes to reduce sulfur or to use sulfur as a final electron acceptor, indicating the type of respiration (aerobic or anaerobic).

Sulfur Reduction

• Reaction Pathway:

  • Cysteine → (enzyme cysteine desulfurase) → pyruvic acid + NH₃ + H₂S (hydrogen sulfide)

• Subsequent Reaction:

  • H₂S + FeSO₄ → FeS (black precipitate) + H₂SO₄

• Significance of Black Precipitate:

  • Indicates the presence of cysteine desulfurase, typically associated with the genus Proteus

  • This reaction is also known as the putrefaction of cysteine.

Indole Production (Tryptophan Hydrolysis)

• Reaction Mechanism:

  • Tryptophan + H₂O → (enzyme tryptophanase) → pyruvic acid + Indole

• Testing Method:

  • Add Kovac’s reagent to the stab test.

• Beneficial Observations:

  • Formation of a red layer on top of the SIM stab indicates indole presence.

  • Positive reactions are observed in E. coli and Proteus.

• Alternate Test for Indole:

  • Tryptone broth method: shake broth, add 8-10 drops of Kovac’s reagent, do not shake afterward.

  • E. coli and Proteus are confirmed positive for indole production.

Motility Testing

• Assessment Method:

  • Cloudiness surrounding the stab indicates motility.

  • Presence of peritrichous flagella is often credited for motility in microorganisms.

• General Finding:

  • All Gram-negative rod microbes tested are noted to be motile.

Urea Hydrolysis

• Chemical Reaction:

  • Urea + H₂O → (enzyme urease) → Ammonia + CO₂

• pH Indicator in Media:

  • Phenol red serves as a pH indicator

  • Media starts light orange; presence of urease turns the medium hot pink.

• Noteworthy Characteristic of Proteus:

  • Specifically identified as a urease producer, capable of hydrolyzing urea to raise pH in its environment.

Phenylalanine Deamination

• Chemical Reaction:

  • Phenylalanine → (enzyme phenylalanine deaminase) → phenylpyruvic acid + NH₃

• Testing Procedure:

  • Add 10% ferric chloride to the phenylalanine slant surface.

• Interpretation of Reaction:

  • Presence of phenylpyruvic acid will bind with ferric chloride, resulting in a dark hunter green color on the agar slant.

Diffusible Enzymes Assay

• Methods:

  • Conducted via plate hydrolysis tests

  • Enzymes assessed include:

  • Starch hydrolysis

  • Gelatin hydrolysis

• Purpose:

  • Illustrate the bacteria’s ability to produce exoenzymes that diffuse into media.

• Hydrolysis Reactions Include:

  • Urea hydrolysis and tryptophan hydrolysis are additional examined reactions.

Starch Hydrolysis

• Composition of Starch Agar:

  • Contains starch (substrate), a protein source, and 1.5% agar.

  • Starch consists of amylose and amylopectin components.

• Key Enzyme Investigated:

  • Amylase, which breaks down amylose into simpler sugar units.

• Role of Protein Source:

  • Ensures growth of all bacteria, regardless of starch hydrolyzing capability.

• Testing Reagent:

  • Iodine (e.g., Gram’s iodine) is used for testing.

• Result Interpretation:

  • Flood the plate with iodine; clearing around bacteria indicates a positive starch hydrolysis test.

Gelatin Hydrolysis

• Components of Gelatin Agar:

  • Gelatin (protein source), and 1.5% agar.

• Focus on Enzyme:

  • Gelatinase acts on gelatin polymer.

• Importance of Protein Source:

  • Ensures all bacteria can grow, with a concentration of 0.4% gelatin agar.

• Testing Reagent:

  • Mercuric chloride in hydrochloric acid (HgCl₂/HCl); forms a white precipitate in reaction with gelatin.

• Sign of Successful Hydrolysis:

  • Clearing around bacteria signifies a positive gelatin hydrolysis test.

IMViC Battery of Tests

• Components of the IMViC Tests:

  • Tryptone broth (indole test)

  • MRVP broths (two tests: Methyl Red and Voges Proskauer)

  • Citrate slant (citrate utilization test)

• Purpose:

  • Differentiate among Enterobacteriaceae members, particularly Gram-negative short rod enteric lactose fermenters.

• General Characteristics of Enterobacteriaceae:

  • Gram-negative, short rods

  • Examples include: E. coli, Enterobacter, Serratia, and Proteus

  • Ferments glucose, but lactose fermentation is inconsistent

  • Acts as facultative anaerobes, capable of reducing nitrate and producing catalase.

Tryptophan Hydrolysis and Indole Production

• Chemical Reaction Recap:

  • Tryptophan + H₂O → (tryptophanase) → pyruvic acid + Indole

• Kovac’s Reagent Test:

  • Indicates presence of indole with a red layer on stub when present. E. coli, Proteus, Serratia included in tests.

• Conclusion on Indole Production:

  • E. coli and Proteus are positive; Serratia is negative despite red color possibly being due to pigment.

MRVP Tests: Methyl Red vs. Voges Proskauer

• Rationale for Two Broths:

  • Allows inoculating a single broth and dividing for tests on different metabolic pathways.

• Composition of Broths:

  • Contains buffered peptone and glucose, lacking phenol red but present with a phosphate buffer; serves as a protein source.

  • Primarily checks glucose fermentation through two pathways: mixed acid fermentation pathway and 2,3-butanediol fermentation pathway.

• Observation:

  • Microbe will select one pathway for glucose fermentation, not both simultaneously.

Methyl Red Test

• Objective:

  • Tests organism’s ability to produce mixed acids via mixed acid fermentation pathway.

• Process:

  • Grow in MRVP broth for at least 72 hours; afterwards, shake and add 15 drops of methyl red.

• Indicator Functionality of Methyl Red:

  • pH < 4 yields red color

  • pH > 6 yields yellow

  • pH between 4 and 6 yields orange color

• Results Interpretation:

  • Red coloration indicates positive result for mixed acids; E. coli and Proteus are noted to be positive.

Voges Proskauer Test

• Contrast to Methyl Red:

  • Focuses on neutral end products formation.

• Target Substance:

  • Acetoin is monitored, which precedes 2,3-butanediol in production pathways.

• Testing Method:

  • Add α-naphthol and KOH in a 3:1 ratio; red indicates positive reaction for acetoin.

• Expected Outcomes:

  • Enterobacter and Serratia are expected to be positive for Voges Proskauer test due to negative results from Methyl Red.

Citrate Utilization Test

• Purpose:

  • Assesses the microbe's ability to utilize citrate as its sole carbon source.

• Interpretation of Color Change:

  • Blue coloration indicates a positive result (capable of citrate utilization), whereas green indicates negative result.

• Mechanism:

  • If a microbe can utilize citrate, it will also use nitrogen from ammonium salts, cleaving it and resulting in ammonia output.

• Indicator Used:

  • Bromthymol blue, with green (pH < 7) and blue (pH > 7) color changes indicating metabolic activity.