Proteins, Amino Acids, and Enzymes IV: Gelatin Hydrolysis

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.



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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.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



© The McGraw−Hill

Companies, 2002



27. Proteins, Amino Acids,

and Enzymes IV: Gelatin

Hydrolysis



27



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes IV: Gelatin Hydrolysis

1. Complete the following table on gelatin hydrolysis.

Gelatin Hydrolysis (+ or –)

Bacterium



Tube Results



KEY Strips



E. aerogenes



__________________



__________________



E. coli



__________________



__________________



P. vulgaris



__________________



__________________



2. Sketch and describe what is happening in each tube with respect to gelatin hydrolysis.



E. aerogenes



E. coli



P. vulgaris



167



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



27. Proteins, Amino Acids,

and Enzymes IV: Gelatin

Hydrolysis



© The McGraw−Hill

Companies, 2002



Review Questions

1. How can gelatin hydrolysis be beneficial to certain bacteria?



2. What is gelatin?



3. What is unique about gelatin at 35°C versus 5°C?



4. Why did you refrigerate the gelatin cultures before observing them for liquefaction?



5. Can gelatin hydrolysis be correlated with the pathogenicity of a bacterium? Explain your answer.



6. Why is gelatin liquefied in the presence of gelatinase?



7. How does a KEY Rapid Gelatin Test Strip work?



168



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



28. Proteins, Amino Acids,

and Enzymes V: Catalase

Activity



E X E RC I S E



28



Proteins,Amino Acids, and Enzymes V:

Catalase Activity

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. No mouth

pipetting. Three percent hydrogen peroxide is caustic to

the skin and mucous membranes. Keep all culture tubes

upright in a test-tube rack.



Pronunciation Guide

Enterococcus faecalis (en-te-ro-KOK-kus fee-KAL-iss)

Micrococcus luteus (my-kro-KOK-us LOO-tee-us)

Staphylococcus aureus (staf-il-oh-KOK-kus ORE-ee-us)



Why Are the Above Bacteria Used

in This Exercise?



Materials per Student

18- to 24-hour tryptic soy broth cultures of

Staphylococcus aureus (ATCC 25923),

Enterococcus faecalis (ATCC 19433), and

Micrococcus luteus (ATCC 9341)

tryptic soy agar slants

3% hydrogen peroxide (H2O2)(caustic) or Difco’s

SpotTest Catalase Reagent

Bunsen burner

inoculating loop

Pasteur pipette with pipettor

incubator set at 35°C

test-tube rack

wax pencil

clean glass slides

wooden applicator stick (or Nichrome wire loop)



In this exercise, the student will learn to perform the catalase

test. The catalase test is very useful in differentiating between groups of bacteria. The authors have chosen the following three bacteria to accomplish the above objective.

Staphylococcus aureus (L. aureus, golden) is a grampositive coccus that is catalase positive when grown in an

aerobic environment. S. aureus is mainly associated with the

human skin and mucous membranes of warm-blooded vertebrates, but is often isolated from food products, dust, and

water. Enterococcus faecalis (L. faecium, of the dregs, of

feces) is a catalase negative, gram-positive coccus. E. faecalis occurs widely in the environment, particularly in feces

of vertebrates. Micrococcus luteus (L. luteus, golden yellow)

is another gram-positive coccus that also is catalase positive.

M. luteus occurs primarily on mammalian skin and in soil,

but commonly can be isolated from food products and air.



Learning Objectives



Principles



Each student should be able to



Some bacteria contain flavoproteins that reduce O2,

resulting in the production of hydrogen peroxide

(H 2 O 2 ) or superoxide (O 2 – ). These are extremely

toxic because they are powerful oxidizing agents and

destroy cellular constituents very rapidly. A bacterium

must be able to protect itself against such O2 products

or it will be killed.

Many bacteria possess enzymes that afford protection against toxic O2 products. Obligate aerobes and

facultative anaerobes usually contain the enzymes superoxide dismutase, which catalyzes the destruction



1. Understand the biochemical process of hydrogen

peroxide detoxification by aerobic bacteria

through the production of the enzyme catalase

2. Describe how catalase production can be

determined

3. Perform a catalase test



Suggested Reading in Textbook

1. Oxygen Concentration, section 6.4.



169



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



of superoxide, and either catalase or peroxidase,

which catalyze the destruction of hydrogen peroxide as

follows:

2O– + 2H+ superoxide

2

dismutase

2H2O2 catalase or

peroxidase



O2

Oxygen

2H2O

Water



+



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Companies, 2002



28. Proteins, Amino Acids,

and Enzymes V: Catalase

Activity



H2O2



Figure 28.1 Catalase Test on Slants. (a) Staphylococcus aureus,

catalase positive. Notice the bubbles of oxygen (tube on the left).

(b) Enterococcus faecalis, catalase negative; note the absence of

bubbles (tube on the right).



Hydrogen peroxide

+



O2

Free oxygen



Most strict anaerobes lack both enzymes and therefore

cannot tolerate O2.

Catalase production and activity can be detected

by adding the substrate H2O2 to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. If

catalase was produced by the bacteria, the above

chemical reaction will liberate free O2 gas. Bubbles of

O2 represent a positive catalase test; the absence of

bubble formation is a negative catalase test.

Catalase activity is very useful in differentiating

between groups of bacteria. For example, the morphologically similar Enterococcus (catalase negative) and

Staphylococcus (catalase positive) can be differentiated using the catalase test (figure 28.1).



(a)



(b)



Figure 28.2 Catalase Test on Slides. A positive catalase

reaction (left slide) shows gas bubbles; a negative catalase reaction

reveals an absence of gas bubbles (right slide).



Procedure

First Period

1. Label each of the tryptic soy agar slants with the

name of the bacterium to be inoculated, your

name, and date.

2. Using aseptic technique (figure 14.3), heavily

inoculate each experimental bacterium into its

appropriately labeled tube by means of a streak

inoculation.

3. Incubate the slants at 35°C for 18 to 24 hours.



Second Period

1. To test for catalase, set the slant in an inclined

position and pipette several drops of a 3% solution

of H2O2 over the growth on the slant or use 3 to 5

drops of Difco’s SpotTest catalase reagent.

2. The appearance of gas bubbles (figure 28.1a)

indicates a positive test; the absence of gas

bubbles is a negative test (figure 28.1b).

3. Based on your observations, determine and record

in the report for exercise 28 whether or not each

bacterium was capable of catalase activity.

4. Note: An alternative procedure for doing the

catalase test is to remove growth from a slant

using a wooden applicator stick or Nichrome wire

loop and place the growth on a glass slide. The

cells are then mixed in a drop of 3% H2O2 or a



170



Biochemical Activities of Bacteria



drop of Difco’s SpotTest catalase reagent.

Immediate bubbling indicates a positive catalase

test (figure 28.2).



HINTS AND PRECAUTIONS

(1) Dispose of the hydrogen peroxide slides in the appropriate container filled with disinfectant. (2) When using

a slant for other purposes in the same laboratory period,

it is possible to save material by adding H2O2 to the slant

after finishing with it. (3) Extreme care must be exercised if a colony is taken from a blood agar plate. Erythrocytes contain catalase, and a transfer of only a few

blood cells can give a false-positive reaction. (4) Always

use fresh hydrogen peroxide, since it is unstable.