Proteins, Amino Acids, and Enzymes VI: Coagulase and DNase Activity

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.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



29. Proteins, Amino Acids,

and Enzymes VI: Coagulase

and DNase Activity



A positive coagulase test is represented by any

degree of clotting, from a loose clot suspended in

plasma to a solid clot (figure 29.1a).



© The McGraw−Hill

Companies, 2002



Figure 29.2 DNase Test. (a) Spot inoculate half of a DNase

test agar plate with S. aureus and the other half with S. epidermidis.

(b) DNase lysis (S. aureus, right side of plate) and no lysis

(S. epidermidis, left side of plate).



DNase Test



1. Divide a DNase test agar plate in half by marking

the bottom with a wax pencil. Add your name and

date.

2. On d of the plate, heavily spot-inoculate S. aureus

over a 0.5-cm area (figure 29.2a). Do the same

with S. epidermidis on the other half of the plate.

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



DNase test agar



Staphylococcus

epidermidis



0.5 cm



Staphylococcus

aureus



Second Period

1. Using a Pasteur pipette with bulb, flood the

DNase test agar plate with 1 N HCl. A zone of

clearing around the colony indicates a positive

DNase test (i.e., the DNA in the medium has been

degraded) (figure 29.2b).

2. Based on your observations, determine and record

in the report for exercise 29 whether or not each

bacterium was coagulase and DNase positive or

negative.



HINTS AND PRECAUTIONS

(1) Do not shake or vortex the citrated rabbit plasma when

inoculating it with the bacteria. (2) In a positive DNA test,

the zone of clearing should appear immediately.



( ) (a)



(b)



Proteins, Amino Acids, and Enzymes VI: Coagulase and DNase Activity



175



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



© The McGraw−Hill

Companies, 2002



29. Proteins, Amino Acids,

and Enzymes VI: Coagulase

and DNase Activity



29



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes VI: Coagulase and DNase Activity

1. Complete the following table on coagulase and DNase activity.

Biochemical Results



S. aureus



S. epidermidis



Coagulase test (+ or –)



________________________



________________________



DNase test (+ or –)



________________________



________________________



2. Make a drawing of your DNase test agar plate observations.



177



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



29. Proteins, Amino Acids,

and Enzymes VI: Coagulase

and DNase Activity



© The McGraw−Hill

Companies, 2002



Review Questions

1. How does the enzyme coagulase function?



2. How does the enzyme DNase function?



3. False-positive coagulase tests have been reported for some bacteria that can metabolize citrate. Explain.



4. Does a coagulase-positive staphylococcus also have to be DNase positive? Explain why or why not.



5. What is the function of the 1 N HCl added to the DNase plates after incubation?



6. Describe a positive DNase test.



7. Describe a negative DNase test.



178



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



30. Proteins, Amino Acids,

and Enzymes VII: Oxidase

Test



E X E RC I S E



30



Proteins,Amino Acids, and Enzymes VII: Oxidase Test

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. No mouth

pipetting. The oxidase reagent is caustic. Avoid contact

with eyes and skin. In case of contact, immediately flush

eyes or skin with plenty of water for at least 15 minutes.



Materials per Student

young 24-hour tryptic soy broth cultures of

Alcaligenes faecalis (ATCC 8750),

Escherichia coli (ATCC 25922), and

Pseudomonas aeruginosa (ATCC 27853)

tryptic soy agar plates

tetramethyl-p-phenylenediamine dihydrochloride

(oxidase reagent)

Bunsen burner

platinum or plastic loops

wax pencil

Pasteur pipette with pipettor

Oxidase Disks or Dry Slides (Difco); Oxidase

Test Strips (KEY Scientific Products); SpotTest

Oxidase Reagent (Difco)

wooden applicator sticks

Whatman No. 2 filter paper



Learning Objectives

Each student should be able to

1. Understand the biochemistry underlying oxidase

enzymes

2. Describe the experimental procedure that enables

one to distinguish between groups of bacteria

based on cytochrome oxidase activity

3. Give examples of oxidase-positive and oxidasenegative bacteria

4. Perform an oxidase test



Suggested Reading in Textbook

1. The Electron Transport Chain, section 9.5; see

also figures 9.13–9.15.

2. Rapid Methods of Identification, section 36.2; see

also table 36.3.



Pronunciation Guide

Alcaligenes faecalis (al-kah-LIJ-e-neez fee-KAL-iss)

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

Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jinOH-sah)



Why Are the Above Bacteria Used

in This Exercise?

This exercise gives the student experience in performing

the oxidase test. The oxidase test distinguishes between

groups of bacteria based on cytochrome oxidase activity.

Three bacteria will be used. Alcaligenes faecalis (L. faecium, of the dregs, of feces) is a gram-negative, aerobic

rod (coccal rod or coccus) that possesses a strictly respiratory type of metabolism with oxygen as the terminal

electron acceptor. It is thus oxidase positive. Escherichia

coli is a facultatively anaerobic gram-negative rod that

has both respiratory and fermentative types of metabolism and isoxidase negative. Pseudomonas aeruginosa is

a gram-negative, aerobic rod having a strictly respiratory

type of metabolism with oxygen as the terminal electron

acceptor and thus is oxidase positive.



179



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



30. Proteins, Amino Acids,

and Enzymes VII: Oxidase

Test



Medical Application

The oxidase test is a useful procedure in the clinical laboratory because some gram-negative pathogenic species of bacteria (such as Neisseria gonorrhoeae, P. aeruginosa, and

Vibrio species) are oxidase positive, in contrast to species in

the family Enterobacteriaceae, which are oxidase negative.



Principles

Oxidase enzymes play an important role in the operation of the electron transport system during aerobic respiration. Cytochrome oxidase (aa3 type) uses O2 as an

electron acceptor during the oxidation of reduced cytochrome c to form water and oxidized cytochrome c.

The ability of bacteria to produce cytochrome oxidase can be determined by the addition of the oxidase

test reagent or test strip (tetramethyl-p-phenylenediamine dihydrochloride or an Oxidase Disk, p-aminodimethylaniline) to colonies that have grown on a

plate medium. Or, using a wooden applicator stick, a

bacterial sample can either be rubbed on a Dry Slide

Oxidase reaction area, on a KEY test strip, or filter

paper moistened with the oxidase reagent. The light

pink oxidase test reagent (Disk, strip, or Slide) serves

as an artificial substrate, donating electrons to cytochrome oxidase and in the process becoming oxidized to a purple and then dark purple (figure 30.1)

compound in the presence of free O2 and the oxidase.

The presence of this dark purple coloration represents

a positive test. No color change or a light pink coloration on the colonies indicates the absence of oxidase and is a negative test.



Procedure

First Period

1. With a wax pencil, divide the bottom of a tryptic

soy agar plate into three sections and label each



180



Biochemical Activities of Bacteria



© The McGraw−Hill

Companies, 2002



with the name of the bacterium to be inoculated,

your name, and date.

2. Using aseptic technique (see figure 14.3), make a

single streak-line inoculation on the agar surface

with the appropriate bacterium.

3. Incubate the plate in an inverted position for 24 to

47 hours at 35°C.



Second Period

1. Add 2 to 3 drops of the oxidase reagent to the

surface of the growth of several isolated colonies

of each test bacterium or to some paste that has

been transferred to a piece of filter paper. Using

another colony, place an Oxidase Disk on it. Add a

drop of sterile water. If Dry Slides or test strips are

available, use a wooden applicator stick to transfer

a sample to the slide, test strip, or filter paper

moistened with oxidase reagent. Alternatively, drop

a KEY oxidase test strip onto the surface of a slant

culture and moisten it with water if necessary.

2. Observe the colony or sample for the presence or

absence of a color change from pink to purple,

and finally to dark purple. This color change will

occur within 20 to 30 seconds. Color changes

after 20 to 30 seconds are usually disregarded

since the reagent begins to change color with time

due to auto-oxidation. Oxidase-negative bacteria

will not produce a color change or will produce a

light pink color.

3. Based on your observations, determine and record

in the report for exercise 30 whether or not each

bacterium was capable of producing oxidase.

HINTS AND PRECAUTIONS

(1) Students should note the color change immediately

following the addition of oxidase reagent. Color changes

after 20 seconds are not valid. (2) Using Nichrome or

other iron-containing inoculating devices may cause

false-positive reactions. (3) If bacterial paste is transferred with an applicator stick, put the stick in a jar of

disinfectant or a Biohazard bag immediately after use.