Proteins,Amino Acids, and Enzymes III: Casein Hydrolysis

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



26. Proteins, Amino Acids,

and Enzymes III: Casein

Hydrolysis



Figure 26.1 Proteolytic Hydrolysis.

Protease

H



O



H



O



H



H



O



C



C



O

N



C



H



R1



N



C



H



C



C



R2



+ H2O



N



C



H



R1



C



+

O–



H3N+



R2



Peptide

bond

Peptide



Procedure

First Period

1. Melt the tubes of plate count agar by placing them

in the boiling water bath. After melting, place the

tubes in the 48° to 50°C water bath for 10

minutes.

2. With a wax pencil, mark the bottom of a petri

plate into three sections: label one E. coli, the

second, B. subtilis, and the third, P. aeruginosa.

Add your name and date to the plate.

3. Pipette 2 ml of warm (48° to 50°C) sterile skim

milk into the petri plate. Add the melted agar and

mix thoroughly by moving the plate in a circular

motion. Allow this medium to gel on a cool, level

surface.

4. As shown in figure 14.3, aseptically spotinoculate (figure 26.2a) each third of the petri

plate with the appropriate bacterium as per the

label. Place a loopful of culture on the center of

each section and spread it in a circular fashion to

cover an area about the size of a dime or less (5 to

18 mm in diameter).

5. Incubate the plate in an inverted position at 35°C

for 24 to 48 hours.



Carboxyl

component



Amino

component



Figure 26.2 Procedure for Determining Casein

Hydrolysis. (a) Spot inoculation of a plate count agar with milk

plate. (b) Plate exhibiting two zones of proteolysis.



Plate count agar



0.5 cm



(a)



Zones of

proteolysis



Bacterial growth

(no proteolysis)



(b)



Second Period

1. Examine the plate count agar for the presence or

absence of a clear zone (zone of proteolysis)

surrounding the growth of each of the bacterial

test organisms. You can see the clear zones best

against a black background.

2. Based on your observations, determine and record

in the report for exercise 26 which of the bacteria

were capable of hydrolyzing the casein. Also,

measure the zone of hydrolysis for each colony.



162



Biochemical Activities of Bacteria



HINTS AND PRECAUTIONS

Aseptic technique must be followed because possible

contaminating microorganisms from the air or body

might be capable of hydrolyzing casein and thus lead to

erroneous results.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



26. Proteins, Amino Acids,

and Enzymes III: Casein

Hydrolysis



26



© The McGraw−Hill

Companies, 2002



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes III: Casein Hydrolysis

1. Complete the following table on casein hydrolysis.

Bacterium



Casein Hydrolysis (+ or –)



Diameter of Hydrolysis Zone



E. coli



____________________________________



____________________________________



B. subtilis



____________________________________



____________________________________



P. aeruginosa



____________________________________



____________________________________



2. Draw your skim milk agar plates showing the growth patterns of the above three bacteria. Label accordingly.



163



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



26. Proteins, Amino Acids,

and Enzymes III: Casein

Hydrolysis



© The McGraw−Hill

Companies, 2002



Review Questions

1. Define the following terms:

a. protein

b. hydrolysis

c. casein

d. protease

e. amino acid

f. peptide bond

g. proteolysis



2. How can plate count agar that contains milk be used to demonstrate proteolysis?



3. Why are some bacteria able to grow on plate count agar that contains milk even though they do not produce

any proteases?



4. Draw the chemical reaction for proteolytic hydrolysis.



5. Why was sterile skim milk used in this experiment?



6. Why is milk white?



164



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



27. Proteins, Amino Acids,

and Enzymes IV: Gelatin

Hydrolysis



E X E RC I S E



27



Proteins,Amino Acids, and Enzymes IV:

Gelatin Hydrolysis

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. No mouth pipetting. Keep all culture tubes upright in a test-tube rack.



Pronunciation Guide

Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAHjen-eez)

Escherichia coli (esh-er-I-ke-a KOH-lee)

Proteus vulgaris (PRO-te-us vul-GA-ris)



Materials per Student

24- to 48-hour tryptic soy broth cultures of

Enterobacter aerogenes (ATCC 13048, gel. +),

Escherichia coli (ATCC 11229, gel. – ), and

Proteus vulgaris (ATCC 13315, gel. +)

4 nutrient gelatin deep tubes

Bunsen burner

inoculating loop

1-ml pipettes with pipettor

refrigerator or ice-water bath

test-tube rack

incubator set at 35°C

3 KEY Rapid Gelatin Test Strips (KEY Scientific

Products, 1402 Chisholm Trail, Suite D, Round

Rock, TX 78681; 800–843–1539)

3 sterile test tubes

wax pencil



Learning Objectives

Each student should be able to

1. Understand how the proteolytic enzyme

gelatinase liquefies gelatin

2. Explain why some bacteria hydrolyze gelatin

3. Describe how gelatin liquefaction is tested for in

the laboratory, and perform a gelatin hydrolysis

test



Suggested Reading in Textbook

1. Complex Media, section 5.7; see also box 5.1 and

table 5.5.



Why Are the Above Bacteria Used

in This Exercise?

This exercise shows the student how to differentiate between bacteria based on their ability to produce the enzyme

gelatinase. The authors have chosen the following three

bacteria to accomplish this objective. Enterobacter aerogenes is gelatinase positive, but gelatin is very slowly liquefied by most strains. In contrast, Proteus vulgaris also is

gelatinase positive and liquefies gelatin very rapidly. Escherichia coli is gelatinase negative.



Medical Application

In the clinical laboratory gelatin hydrolysis is used to distinguish between the pathogenic Staphylococcus aureus

(ϩ) and the nonpathogenic S. epidermidis (slow–). It can

also be used to distinguish Listeria monocytogenes (– for

gelatin hydrolysis and one cause of bacterial meningitis)

from some species of Corynebacterium.



Principles

When boiled in water, the connective tissue collagen

(which is stringy, insoluble, and indigestible) changes

into gelatin, a soluble mixture of polypeptides. Certain

bacteria are able to hydrolyze gelatin by secreting a proteolytic enzyme called gelatinase. The resulting amino

acids can then be used as nutrients by the bacteria. Since



165



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



27. Proteins, Amino Acids,

and Enzymes IV: Gelatin

Hydrolysis



hydrolyzed gelatin is no longer able to gel, it is a liquid.

The ability of some bacteria to digest gelatin is an important characteristic in their differentiation. For example, when grown on a gelatin medium (Thiogel),

Clostridium perfringens causes liquefaction, whereas

Bacteroides fragilis does not. Gelatin hydrolysis can

also be used to assess the pathogenicity of certain bacteria. The production of gelatinase can often be correlated

with the ability of a bacterium to break down tissue collagen and spread throughout the body of a host.

Gelatin liquefaction (the formation of a liquid) can

be tested for by stabbing nutrient gelatin deep tubes. Following incubation, the cultures are placed in a refrigerator or ice bath at 4°C until the bottom resolidifies. If

gelatin has been hydrolyzed, the medium will remain liquid after refrigeration. If gelatin has not been hydrolyzed,

the medium will resolidify during the time it is in the refrigerator (figure 27.1). Nutrient gelatin may require up

to a 14-day incubation period for positive results.

Another way to test for gelatinase is by the use of

KEY Rapid Test Strips. These strips are used in the

rapid test (within 24 to 48 hours) for gelatin liquefaction. Liquefaction is demonstrated by the bacterium’s

ability to remove, with gelatinase, the outer layer of

the strip when the gelatin test strip is immersed in a

suspension of bacterial cells. If gelatin is removed, the

strip changes to a blue color, and the test is positive; if

there is no color change, the test is negative.



Procedure

First Period

1. Label three nutrient gelatin deeps with your name,

date, and the bacterium to be inoculated. Label

the fourth tube “control.”

2. Using aseptic technique (see figure 14.3),

inoculate three of the deeps with the appropriate

bacterium by stabbing the medium f of the way

to the bottom of the tube.

3. Incubate the four tubes for 24 to 48 hours or

longer at 35°C. The incubation time depends on

the species of bacteria; some may require

incubation for up to 2 weeks. If the latter is the

case, observe on days 7 and 14.



© The McGraw−Hill

Companies, 2002



Figure 27.1 Hydrolysis of Gelatin. If gelatin is hydrolyzed by

the enzyme gelatinase, it does not gel when cooled but remains a

liquid. Thus it flows when the culture is tilted backward (right

tube). A negative control tube is on the left. Notice that the solid

gelatin does not flow when the tube is tilted.



2. Drop one gelatin test strip into each tube.

3. Incubate for 24 to 48 hours at 35°C.



Second Period

1. Remove the nutrient gelatin deep tubes from the

incubator and place them in the refrigerator at

4°C for 30 minutes or in an ice bath for 3 to 5

minutes.

2. When the bottom resolidifies, remove the tubes

and gently slant them. Notice whether or not the

surface of the medium is fluid or liquid. If the

nutrient gelatin is liquid, this indicates that gelatin

has been hydrolyzed by the bacterium. If no

hydrolysis occurred, the medium will remain a

gel. The uninoculated control should also be

negative.



KEY Rapid Test

1. Observe the color of the three gelatin test strips.

Liquefaction will appear first along the surface of

the suspension. A blue color is a positive test; no

color change is a negative test.

2. Based on your observations, determine and record

in the report for exercise 27 which of the three

bacteria were capable of hydrolyzing gelatin.



KEY Rapid Test

1. Into three small test tubes, pipette d ml (or about 2025 drops with a Pasteur pipette) of a heavy bacterial

suspension or suspend paste in d to 1 ml of water.

With a wax pencil, label each of the tubes with the

appropriate bacterium, your name, and date.



166



Biochemical Activities of Bacteria



HINTS AND PRECAUTIONS

Do not shake the tubes when moving them to a refrigerator; gelatin digestion may have occurred only at the

surface.