Proteins,Amino Acids, and Enzymes VII: Oxidase Test

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.



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



Figure 30.1 Oxidase Test. Note the purple to dark purple color after the colonies have been added to filter paper moistened with

oxidase reagent.



Biochemistry within bacteria



2 reduced cytochrome c + 2H+ +



1/2 O

2



cytochrome

oxidase



2 oxidized cytochrome c + H2O



Biochemistry on filter paper (disk/slide)



H 3C



CH3



H 3C



CH3

N+



N



2 oxidized cytochrome c +



+ 2 reduced cytochrome c



N+



N

H 3C



CH3



H 3C



Tetramethyl-p-phenylenediamine

(reagent)



CH3



Wurster's blue

(dark purple)



Proteins, Amino Acids, and Enzymes VII: Oxidase Test



181



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



© The McGraw−Hill

Companies, 2002



30. Proteins, Amino Acids,

and Enzymes VII: Oxidase

Test



30



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes VII: Oxidase Test

1. Complete the following table on the oxidase test.

Color of Colonies after Adding



Oxidase Production (+ or –)



Bacterium



Reagent



Disk or Slide



Reagent



Disk or Slide



A. faecalis



____________



____________



____________



____________



E. coli



____________



____________



____________



____________



P. aeruginosa



____________



____________



____________



____________



183



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



30. Proteins, Amino Acids,

and Enzymes VII: Oxidase

Test



© The McGraw−Hill

Companies, 2002



Review Questions

1. What metabolic property characterizes bacteria that possess oxidase activity?



2. What is the importance of cytochrome oxidase to bacteria that possess it?



3. Do anaerobic bacteria require oxidase? Explain your answer.



4. What is the function of the test reagent in the oxidase test?



5. The oxidase test is used to differentiate among which groups of bacteria?



6. Why should nichrome or other iron-containing inoculating devices not be used in the oxidase test?



7. Are there limitations to the oxidase test?



184



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



31. Proteins, Amino Acids,

and Enzymes VIII: Urease

Activity



E X E RC I S E



31



Proteins,Amino Acids, and Enzymes VIII:

Urease Activity

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. Keep all culture tubes upright in a test-tube rack or in a can.



Materials per Student

24- to 48-hour tryptic soy agar slants of

Escherichia coli (ATCC 11229), Klebsiella

pneumoniae (ATCC e13883), Proteus vulgaris

(ATCC 13315), and Salmonella cholerae-suis

(ATCC 29631)

5 urea broth tubes

Bunsen burner

test-tube rack

inoculating loop

incubator set at 35°C

urea disks (Difco) or urease test tablets (KEY

Scientific Products)

4 sterile test tubes

wax pencil

sterile forceps



Learning Objectives

Each student should be able to

1. Understand the biochemical process of urea

hydrolysis

2. Determine the ability of bacteria to degrade urea

by means of the enzyme urease

3. Tell when the urease test is used

4. Perform a urease test



Suggested Reading in Textbook

1. Pseudomonas and the Enterobacteriaceae, section

22.3; see also figure 22.8 and tables 22.6, 22.7.



Why Are the Above Bacteria Used

in This Exercise?

In this exercise, the student will perform a urease test to determine the ability of bacteria to degrade urea by means of

the enzyme urease. The authors have chosen two ureasepositive bacteria (Klebsiella pneumoniae and Proteus vulgaris) and two urease-negative bacteria (Escherichia coli

and Salmonella cholerae-suis).



Medical Application

In the clinical laboratory, members of the genus Proteus can

be distinguished from other enteric nonlactose-fermenting

bacteria (Salmonella, Shigella) by their fast urease activity. P.

mirabilis is a major cause of human urinary tract infections.



Principles

Some bacteria are able to produce an enzyme called

urease that attacks the nitrogen and carbon bond in

amide compounds such as urea, forming the end products ammonia, CO2, and water (figure 31.1).

Urease activity (the urease test) is detected by

growing bacteria in a medium containing urea and using

a pH indicator such as phenol red (see appendix E).

When urea is hydrolyzed, ammonia accumulates in the

medium and makes it alkaline. This increase in pH

causes the indicator to change from orange-red to deep

pink or purplish red (cerise) and is a positive test for

urea hydrolysis. Failure of a deep pink color to develop

is a negative test.



Pronunciation Guide

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

Klebsiella pneumoniae (kleb-se-EL-lah nu-mo-ne-ah)

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

Salmonella cholerae-suis (sal-mon-EL-ah coler-ah

SU-is)



Procedure

First Period

1. Label each of the urea broth tubes with the name of

the bacterium to be inoculated, your name, and date.



185



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



31. Proteins, Amino Acids,

and Enzymes VIII: Urease

Activity



Figure 31.1 Urea Hydrolysis. (a) Uninoculated control. (b) Weakly positive reaction (delayed positive). (c) Very rapid positive reaction.

(d) Negative reaction.

Biochemistry within bacteria

H 2N



C



O + 2H2O



urease



CO2



+



H 2O



+



2NH3



H 2N



Urea



Carbon

dioxide



Water



Water



Ammonia



Biochemistry within tubes



Ammonia + phenol red



(a)



(b)



2. Using aseptic technique (see figure 14.3),

inoculate each tube with the appropriate

bacterium by means of a loop inoculation.

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



deep pink



(c)



(d)



Figure 31.2 KEY Test for Urea. After incubation, a pink to red

color constitutes a positive test (tube on the left). If the original straw

color persists, the test is negative (tube on the right).



Urea Disks or Tablets

1. Add 0.5 ml (about 20 drops) of sterile distilled

water to four sterile test tubes for the Difco disk

or 1 ml distilled water for the KEY tablet.

2. Transfer one or two loopfuls of bacterial paste to

each tube. Label with your name and date.

3. Using sterile forceps, add one urea or urease disk

tablet to each tube.

4. Incubate up to 4 hours at 35°C. Check for a color

change each hour. (The KEY test may be

incubated up to 24 hours if necessary.)



2. Based on your observations, determine and record

in the report for exercise 31 whether each

bacterium was capable of hydrolyzing urea.



Second Period

1. Examine all of the urea broth cultures and urea

disk or urease tablet tubes to determine their color

(figures 31.1 and 31.2).



186



Biochemical Activities of Bacteria



HINTS AND PRECAUTIONS

Some bacteria have a delayed urease reaction that may

require an incubation period longer than 48 hours.