22.6: Hydrolytic and Miscellaneous Media - Starch, Skim Milk, Gelatin, Indole, Urea, Kligler’s/TSI

Introduction

Many bacteria can produce enzymes that they secrete into the environment. These exoenzymes break down nutrients that are too large to enter the cell. The smaller hydrolyzed products can enter the cell and be metabolized. In this series of tests you will detect the production of the exoenzymes by observing the hydrolysis of the nutrient in question, or the product of hydrolysis itself.

The final test is a combo pack. Kligler’s or Triple Sugar Iron (two very similar tests) can detect sugar utilization and gas production via fermentation, and the ability to produce hydrogen sulfide (\(\ce{H2S}\)). The production of \(\ce{H2S}\) is a unique ability of some bacteria and can be a very identifying factor.

Indole Test--Tryptophan Hydrolysis

Purpose: To detect production of tryptophanase. Part of the IMViC tests.

Media: Tryptone Agar plates

Reagents/indicators: Indole dry slides

Mechanism/reactions: Tryptophanase causes the hydrolysis of Tryptophan → indole + pyruvic acid

Directions: Using a sterile wooden stick (do not use an inoculating wire) pick a small amount of bacteria from a TA plate and touch an area on one section of the dry slide. Look for the color change to pink/red within about 30 sec. DRY SLIDES CAN BE USED FOR MANY TESTS (4 / SQUARE, 4 SQUARES PER SLIDE. USE UP EACH SLIDE BEFORE OPENING A NEW SLIDE PACKET).

Figure \(\PageIndex{1}\): InDole Dryslide, positive(lower squares); J.Gallai CC BY-NC SA

Interpretation:

• (+) = Color change to red within 30 sec.
• (-) = No color change to red

Starch Hydrolysis

Purpose: To detect production of amylase.

Media: Starch Agar plates (1% starch)

Reagents/indicators: Gram’s Iodine

Mechanism/reactions: When iodine comes in contact with starch it turns blue-black.

Directions: Streak starch agar in a straight line. After incubation add Gram’s iodine, drop wise, sparingly, just to cover growth and surrounding area on medium.

Figure \(\PageIndex{2}\): Starch test. Left: negative for amylase after the addition of Gram’s Iodine, the black shows the starch-iodine complex. Right: after the addition of Gram’s iodine, showing the clear zone around the colonies where starch was hydrolyzed. J. Gallai CC BY-NC SA

Interpretation:

• (+) = Colorless zone around colonies where starch has been hydrolyzed by amylases.
• (-) = No zone, medium is blue-black immediately adjacent to growth.

Urease Test

Purpose: To detect production of urease

Media: Urea broth

Reagents/indicators: Phenol Red

Mechanism/reactions: Urease hydrolyzes urea to ammonia and carbon dioxide. Ammonia increases the pH of the culture causing the phenol red to go from yellow to bright pink.

Directions: Inoculate urea broth and incubate for 24 – 48 hours.

Figure \(\PageIndex{3}\): Urease test

Interpretation:

• (+) = Red or bright pink color (pH > 8.4)
• (-) = Yellow color (pH < 6)

Casein Hydrolysis (Skim milk)

Purpose: To detect the production of casease.

Media: Skim Milk Agar

Mechanism/reactions: Casease proteolyses casein into peptides and amino acids.

Directions: Streak agar in a straight line and incubate for 24 – 48 hours.

Figure \(\PageIndex{4}\): Skim milk test, positive(L), negative (R)

Interpretation:

• (+) = Clear zone around growth indicating casein hydrolysis via casease

• (-) = No clear zone.

Gelatin Liquefaction

Purpose: To determine the production of gelatinase.

Media: Nutrient Gelatin Deep (12- 15% gelatin)

Mechanism/reactions: Gelatinase causes the breakdown and liquefaction of gelatin→polypeptides→amino acids.

Directions: Deep stab inoculation, ⅔ of the way down the center of the tube. After incubation, refrigerate for 1 hour before reading.

Figure \(\PageIndex{5}\): Gelatin liquefaction

Interpretation:

• (+) = Liquefaction (after refrigeration)
• (-) = Gels when refrigerated, no liquefaction.

This test does not detect weak positives and concentration of gelatin used may inhibit growth of some organisms.

Triple Sugar Iron Agar (TSI)

(Note that either TSI or KIA will be used and, the difference is in the sugars available in the test)

Purpose: The differentiation of Enterobacteriaceae by their ability to ferment dextrose (glucose), lactose, and sucrose, and produce gas and/or hydrogen sulfide.

Media: TSI contains ferrous sulfate, phenol red, dextrose (glucose), lactose, and sucrose.

Mechanism/reactions:

Fermentation: Phenol red turns yellow in an acid environment.

Dextrose is fermented; the butt of the medium turns yellow and the slant remains red.

Lactose or sucrose is fermented; the butt and slant both turn yellow.

Gas: Gas from fermentation may appear as breaks or cracks in the medium.

Hydrogen Sulfide: A few bacteria are capable of reducing the \(\ce{SO4}\)= to \(\ce{H2S}\) (hydrogen sulfide).  The iron combines with the \(\ce{H2S}\) to form \(\ce{FeS}\) (ferrous sulfide) a black compound.  This will turn the butt black.  Thus, a black butt indicates \(\ce{H2S}\) production.

Directions: Dual inoculation with a needle: streak surface of agar slant, then stab, incubate for 24 – 48 hours.

Interpretation: Slant/Butt, Gas, \(\ce{H2S}\)

• ALK/A: (+) Dextrose = Alkaline slant (red) over acid butt (yellow)

• A/A: (+) Dextrose, Lactose and/or Sucrose= Acid slant over acid butt

• ALK/ALK: No change, alkaline slant over alkaline butt

• G: Gas production

• \(\ce{H2S}\) (+): = Hydrogen Sulfide production = black (produced in acid environment)

Kligler Iron Agar (KIA)

Purpose: The differentiation of Enterobacteriaceae by their ability to ferment dextrose (glucose), lactose, and produce gas and/or hydrogen sulfide.

Media: KIA contains ferrous sulfate, phenol red, dextrose (glucose), lactose.

Mechanism/reactions:

Fermentation: Phenol red turns yellow in an acid environment.

Dextrose is fermented; the butt of the medium turns yellow and the slant remains red.

Lactose is fermented; the butt and slant both turn yellow.

Gas: Gas from fermentation may appear as breaks or cracks in the medium.

Hydrogen Sulfide: A few bacteria are capable of reducing the \(\ce{SO4}\)= to \(\ce{H2S}\) (hydrogen sulfide). The iron combines with the \(\ce{H2S}\) to form (\ce{FeS}\) (ferrous sulfide) a black compound.  This will turn the butt black.  Thus, a black butt indicates \(\ce{H2S}\) production.

Directions: Dual inoculation with a needle: Stab into the center of the deep, then streak surface of agar slant, incubate for 24 – 48 hours.

Figure \(\PageIndex{6}\): Kligler's. Negative(L), Acid/Acid, Alk/Acid/Gas(C), Acid/Acid/\(\ce{H2S}\), Alk/Acid/\(\ce{H2S}\)(R)

Interpretation: Slant/Butt, Gas, \(\ce{H2S}\)

• ALK/A: (+) Dextrose = Alkaline slant (red) over acid butt (yellow)

• A/A: (+) Dextrose and Lactose = Acid slant over acid butt

• ALK/ALK: No change, alkaline slant over alkaline butt

• G: Gas production

• \(\ce{H2S}\) (+): = Hydrogen Sulfide production = black (produced in acid environment)

Materials

• Student stock organisms
• 1 plate Starch Agar/GP
• 1 plate Skim Milk Agar/GP
• 1 tube Gelatin/all organisms
• 1 plate Tryptophan Agar/2 GNRs (Inoculate 2 GNRs on each plate)
• 1 tube Urea broth/All except GNRs
• 1 tube of TSI or Kligler’s/GNR

Procedures

1. Inoculate each GP onto a Starch Agar plate
2. Inoculate each GP onto a Skim Milk Agar plate
3. Perform a stab inoculation with a needle of each of your stocks into a Gelatin tube
4. Inoculate 2 GNRs each onto a Tryptophan Agar plate, inoculate all GNRs total
5. Inoculate all your stocks, except the GNRs, into a Urea broth tube
6. Inoculate each of the GNRs into TSI or Kligler’s (needle inoculate, stab then streak slant)

Results

Give the results for the inoculated bacteria

Bacterium	Starch	Skim milk	Gelatin	Indole	Urea	TSI/Kligler’s S/B G \(\ce{H2S}\)

Conclusion

• Examine your results and state which test or set of tests, if any, would be best to help identify (it would be a good idea to do this for all of your stock cultures!) the following-
  • E. coli
  • Mycobacterium smegmatis
  • B. cereus
  • Lactococcus lactis

Contributors and Attributions

• Kelly C. Burke (College of the Canyons)