Experiments Investigating Exoenzymes, Carbohydrate Fermentation, Nitrate Reduction and Urea Hydrolysis

EXPERIMENTS INVESTIGATING EXOENZYMES AND THE MAJOR FOOD GROUPS:

STARCH HYDROLYSIS AND GELATIN HYDROLYSIS
MATERIALS

1. Starch Agar Plates -- 1 per group

2. Gelatin Tubes -- 2 per group

CULTURES

1. Bacillus subtilis slants

2. Escherichia coli slants

3. Pseudomonas aeruginosa slants

4. Streptococcus bovis slants

ACTIVITIES

Experiment 1 - The Utilization of Starch

Work in groups of 2

One starch plate per group

Divide the plate into 4 equal sections labeled A , B, C, D

Spot the microbes in the center of the appropriate section:

A = B. subtilis

B = E. coli

C = P. aeruginosa

D = S. bovis

After 48 hours incubation, flood the plate with gram's iodine. Take a wooden applicator stick and scrape away some growth from each of the organisms so that the medium shows. Check for the presence of starch which is indicated by the formation of a blue-black color.

Experiment 2 - The Utilization of Gelatin

Work in groups of 2

Two gelatin tubes per group

Inoculate (stab) with:

E. coli

P aeruginosa

Since gelatin is liquid at 37^o C, gelatinase activity can be evaluated after 48 hours incubation by placing the tubes in the refrigerator for about 10 minutes. Be careful not to agitate the tubes!

CARBOHYDRATE FERMENTATION

MATERIALS

1. Glucose Tubes (Phenol red broth) -- 2 per group

2. Lactose Tubes -- 2 per group

3. Sucrose Tubes -- 2 per group

CULTURES

1. Proteus vulgaris slants

2. Staphylococcus aureus slants

3. Also the organisms from the starch and gelatin experiments

Experiment 3

Work in groups of 2

Use two tubes of each carbohydrate per group

Choose two organisms per group

Inoculate (stab) a set of carbohydrates with each organism

After 48 hours incubation, observe for acid production. Also check to see if any gas has been produced. Instructor can bring out some uninoculated tubes for comparison.

NITRATE REDUCTION

MATERIALS

1. Nitrate Broths -- 3 per group

2. Zinc powder

3. Sulfanilic Acid Reagent in dropper bottles

4. Alpha-napthylamine Reagent in dropper bottles

CULTURES

1. Escherichia coli slants

2. Pseudomonas aeruginosa slants

Experiment 4

Work in groups of 2

Use three nitrate tubes of per group

Inoculate one with E. coli and another with P. aeruginosa. Leave the third tube uninoculated.

After 48 hours incubation, perform the test for nitrite as follows:

Add two drops of Sulfanilic Acid

Add two drops of Alpha-Napthylamine

Check for color change

If no color change develops, add a pinch of Zinc and note if the color changes.

Be sure to read about this test in Leboffe and Pierce!

UREA HYDROLYSIS

MATERIALS

1. Urea Agar Slants -- 2 per group

CULTURES

1. Escherichia coli slants

2. Proteus vulgaris slants

Experiment 5

Use prepared urea agar slants.

Work in groups of 2

Use two urea tubes of per group

Inoculate one with E. coli and another with P. vulgaris.

After 48 hours incubation, observe for ammonia production (basic conditions).

Instructor can provide an uninoculated urea for comparison.