Proteins, Amino Acids, and Enzymes V: Catalase Activity

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



+



© The McGraw−Hill

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.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



28. Proteins, Amino Acids,

and Enzymes V: Catalase

Activity



28



© The McGraw−Hill

Companies, 2002



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes V: Catalase Activity

1. Complete the following table on catalase activity.

Bacterium



Presence of Bubbling (catalase positive)



Absence of Bubbling (catalase negative)



E. faecalis



____________________________________



____________________________________



S. aureus



____________________________________



____________________________________



M. luteus



____________________________________



____________________________________



171



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



28. Proteins, Amino Acids,

and Enzymes V: Catalase

Activity



Review Questions

1. What is the importance of catalase to certain bacteria?



2. Do anaerobic bacteria require catalase? Explain your answer.



3. Write a balanced equation for the degradation of H2O2 in the presence of catalase.



4. What two groups of bacteria can be differentiated with the catalase test?



5. What are three products that result when flavoproteins reduce O2?

a.

b.

c.

6. What are several bacteria that produce catalase?



7. What is the substrate of the catalase reaction?



172



Biochemical Activities of Bacteria



© The McGraw−Hill

Companies, 2002



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



29. Proteins, Amino Acids,

and Enzymes VI: Coagulase

and DNase Activity



E X E RC I S E



29



Proteins,Amino Acids, and Enzymes VI:

Coagulase and DNase Activity

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame and water

bath. One normal HCl can cause severe skin burns and

irritate and burn the respiratory and digestive systems.

No mouth pipetting. Keep all culture tubes upright in a

test-tube rack or in a can.



Pronunciation Guide

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

S. epidermidis (e-pee-DER-meh-diss)



Why Are the Above Bacteria Used

in This Exercise?



Materials per Student

24- to 48-hour tryptic slant cultures of

Staphylococcus aureus (ATCC 25923) and

Staphylococcus epidermidis (ATCC 14990)

citrated rabbit plasma

DNase test agar plates

water bath at 35°C

inoculating loop

Bunsen burner

1 N hydrochloric acid (caustic)

small test tubes

1-ml Pasteur pipettes with pipettor

wax pencil

incubator set at 35°C

test-tube rack



This exercise shows the student how to differentiate pathogenic species of staphylococci from nonpathogenic species

by performing catalase and DNase tests. The two most

commonly encountered staphylococci will be used: Staphylococcus aureus and S. epidermidis. S. aureus is coagulase

and DNase positive, whereas S. epidermidis is coagulase

and DNase negative.



Medical Application

In the clinical laboratory, coagulase activity is used to distinguish between pathogenic (coagulase ϩ) and nonpathogenic (coagulase Ϫ) staphylococci.



Learning Objectives

Each student should be able to

1. Understand the biochemistry of the enzymes

coagulase and DNase

2. Explain how coagulase and DNase confer a

survival advantage to bacteria that produce these

enzymes

3. Describe how pathogenic species of staphylococci

can be differentiated from nonpathogenic species

4. Perform coagulase and DNase tests



Suggested Reading in Textbook

1. The Staphylococcaceae, section 23.4.



Principles

Coagulases are enzymes that clot blood plasma by a

mechanism that is similar to normal clotting. Although

coagulase activity is not required for pathogenicity,

this enzyme is a good indicator of the pathogenic potential of S. aureus. Coagulase-producing staphylococci (termed coagulase positive) form a fibrin clot

around themselves and avoid attack by the host’s defenses. In the coagulase test (figure 29.1), coagulasepositive staphylococci will cause the plasma to clot by

using coagulase to initiate the clotting cascade. Citrate

and EDTA are usually added to act as anticoagulants



173



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



29. Proteins, Amino Acids,

and Enzymes VI: Coagulase

and DNase Activity



Figure 29.1 Steps in the Coagulase Test. (a) Positive reaction is indicated by clouding and solidification of plasma due to Staphylococcus

aureus. (b) Negative reaction with coagulase-negative Staphylococcus epidermidis.



Pipette 0.5 ml

of citrated

rabbit plasma

into each

tube



Inoculate with

S. aureus



Inoculate with

S. epidermidis



Incubate at

37°C for 1 hour



(a)



and prevent false-positive results. Cultures should be

considered coagulase negative if they are unclotted

after 4 hours.

In addition to coagulase production, most pathogenic strains of staphylococci produce a nuclease enzyme called DNase. DNase degrades host DNA and

increases the pathogenicity of staphylococci that possess it. To demonstrate the presence of DNase, agar

containing dissolved DNA is spot-inoculated with

staphylococci. A zone of clearing around the colony

indicates a positive DNase test. This clearing occurs

because the large DNA molecule has been degraded by

the enzyme, and the end products dissolve in the added

acid. Intact DNA does not dissolve in weak acid but

rather is precipitated by it; thus, the medium around

colonies that do not produce DNase becomes opaque.



174



Biochemical Activities of Bacteria



(b)



Procedure

First Period

Coagulase Test



1. Add 0.5 ml of citrated rabbit plasma to two

small test tubes. With the wax pencil, label the

tubes with the respective bacteria, your name,

and date.

2. Inoculate one tube with enough S. aureus paste to

make a cloudy suspension. Inoculate the other

tube with S. epidermidis. Alternatively, one can

add about 5 drops of thick 18- to 24-hour broth

culture to each tube.

3. Incubate both tubes at 35°C for 1 to 4 hours in a

water bath. Afterward, examine both tubes for the

presence or absence of clouding and clots.