Proteins, Amino Acids, and Enzymes VIII: Urease Activity

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.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



31. Proteins, Amino Acids,

and Enzymes VIII: Urease

Activity



31



© The McGraw−Hill

Companies, 2002



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes VIII: Urease Activity

1. Complete the following table on urease activity.

Color of

Bacterium



Urea Broth



Disks



Urea Hydrolysis (+ or –)



E. coli



______________



____________



_____________________



K. pneumoniae



______________



____________



_____________________



P. vulgaris



______________



____________



_____________________



S. cholerae-suis



______________



____________



_____________________



187



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



31. Proteins, Amino Acids,

and Enzymes VIII: Urease

Activity



© The McGraw−Hill

Companies, 2002



Review Questions

1. Explain the biochemistry of the urease reaction.



2. What is the purpose of the phenol red in the urea broth medium?



3. When would you use the urease test?



4. Why does the urea disk change color?



5. What is the main advantage of the urea disk over the broth tubes with respect to the detection of urease?



6. What is in urea broth?



7. What color is cerise?



188



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



32. Proteins, Amino Acids,

& Enzymes IX: Lysine &

Ornithine Decarboxylase

Test



E X E RC I S E



32



Proteins,Amino Acids, and Enzymes IX:

Lysine and Ornithine Decarboxylase Test

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame. No mouth

pipetting. Keep all culture tubes upright in a test-tube

rack or in a can.



Materials per Student

24- to 48-hour tryptic soy broth cultures of

Enterobacter aerogenes (ATCC 13048),

Citrobacter freundii (ATCC 8090), Klebsiella

pneumoniae (ATCC e13883), and Proteus

vulgaris (ATCC 13315)

4 Moeller’s lysine decarboxylase broth with

lysine (LDC)

4 lysine iron agar slants (LIA)

4 Moeller’s ornithine decarboxylase broth with

ornithine (ODC)

1 Moeller’s lysine decarboxylase broth without

lysine (DC), which will serve as the control

1 Moeller’s ornithine decarboxylase broth without

ornithine (OD), which will serve as the control

Pasteur pipettes with pipettor

inoculating loop

test-tube rack

sterile distilled water

sterile mineral oil

incubator set at 35°C

8 sterile test tubes

ornithine, lysine, and decarboxylase KEY Rapid

Substrate Tablets and strips (KEY Scientific

Products, 1402 Chisholm Trail, Suite D, Round

Rock, TX 78681; 800–843–1539;

www.keyscientific.com)

Bunsen burner

ninhydrin in chloroform (Dissolve 50 mg

ninhydrin in 0.4 ml of dimethylsulfoxide

[DMSO], then add 25 ml of chloroform to the

DMSO solution.)

10% KOH



Learning Objectives

Each student should be able to

1. Understand the biochemical process of

decarboxylation

2. Tell why decarboxylases are important to some

bacteria

3. Explain how the decarboxylation of lysine can be

detected in culture

4. Perform lysine and ornithine decarboxylase tests



Suggested Reading in Textbook

1. Protein and Amino Acid Catabolism, section 9.9;

see also figure 9.23.



Pronunciation Guide

Citrobacter freundii (SIT-ro-bac-ter FRUN-dee)

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

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

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



Why Are the Above Bacteria Used

in This Exercise?

This exercise gives the student experience using the lysine

and ornithine decarboxylase test to differentiate between

bacteria. Two lysine decarboxylase-positive (Enterobacter

aerogenes and Klebsiella pneumoniae) and two lysine decarboxylase-negative (Proteus vulgaris and Citrobacter

freundii) bacteria, and two ornithine decarboxylase-positive

(E. aerogenes and Citrobacter freundii) and two ornithine

decarboxylase-negative (K. pneumoniae and P. vulgaris)

bacteria were chosen to demonstrate the lysine and ornithine decarboxylase tests.



189



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



32. Proteins, Amino Acids,

& Enzymes IX: Lysine &

Ornithine Decarboxylase

Test



Medical Application

In the clinical laboratory, decarboxylase differential tests

are used to differentiate between organisms in the Enterobacteriacea E.



Principles

Decarboxylation is the removal of a carboxyl group

from an organic molecule. Bacteria growing in liquid

media decarboxylate amino acids most actively when

conditions are anaerobic and slightly acidic. Decarboxylation of amino acids, such as lysine and ornithine, results in the production of an amine and CO2

as illustrated below.



© The McGraw−Hill

Companies, 2002



The lysine decarboxylase test is useful in differentiating Pseudomonas (L.–), Klebsiella (L.+), Enterobacter (L.+), and Citrobacter (L.–) species. The ornithine decarboxylase test is helpful in distinguishing

between Klebsiella (O.–) and Proteus (O.–), and

Enterobacter (O.+) bacteria.

A quick test for ornithine or lysine decarboxylase

is to use the KEY Rapid Substrate Tablets and strips.

These tablets contain the respective amino acids in a

mixture of salts correctly buffered for each test. In addition, a pH indicator is present in the tablet, which

changes color as the decarboxylation reaction progresses. In the lysine decarboxylase test tablet, the indicator is bromcresol purple, which turns purple as the

test becomes positive (figure 32.2). The indicator in

the ornithine decarboxylase test tablet is phenol red,

which turns red in a positive test.



decarboxylase

R



CH



COOH



R



CH2



NH2

An amino acid



An amine



NH2 + CO2

Carbon

dioxide gas



Bacteria that are able to produce the enzymes lysine

decarboxylase and ornithine decarboxylase can decarboxylate lysine and ornithine and use the amines as

precursors for the synthesis of other needed molecules.

In addition, when certain bacteria carry out fermentation, acidic waste products are produced, making the

medium acidic and inhospitable. Many decarboxylases

are activated by a low pH. They remove the acid groups

from amino acids, producing alkaline amines, which

raise the pH of the medium making it more hospitable.

Decarboxylation of lysine or ornithine can be detected by culturing bacteria in a medium containing the

desired amino acid, glucose, and a pH indicator (bromcresol purple, see appendix E). Before incubation, sterile

mineral oil is layered onto the broth to prevent oxygen

from reaching the bacteria and inhibiting the reaction.

The acids produced by the bacteria from the fermentation

of glucose will initially lower the pH of the medium and

cause the pH indicator to change from purple to yellow.

The acid pH activates the enzyme that causes decarboxylation of lysine or ornithine to amines and the subsequent

neutralization of the medium. This results in another

color change from yellow back to purple (figure 32.1).

Lysine iron agar (LIA) is also used for the cultivation and differentiation of members of the Enterobacteriaceae based on their ability to decarboxylate lysine and to form H2S. Bacteria that decarboxylate

lysine turn the medium purple. Bacteria that produce

H2S appear as black colonies.



190



Biochemical Activities of Bacteria



Procedure

First Period (Standard Method)

1. Label four LDC tubes and/or LIA slants with the

names of the respective bacteria (K. pneumoniae,

E. aerogenes, P. vulgaris, and C. freundii) to be

inoculated. Do the same for one control DC tube.

Add your name and date to the tubes.

2. Do the same with the four ODC and one OD tubes.

3. As shown in figure 14.3, aseptically inoculate the

tubes with the proper bacteria.

4. With a sterile Pasteur pipette, layer about 1 ml of

sterile mineral oil on top of the inoculated media.

LIA slants do not need mineral oil.

5. Incubate the cultures for 24 to 48 hours at 35°C.



KEY Test Tablet/Strip Method

1. Label eight sterile test tubes with the respective

bacteria, your name, and date.

2. Pipette 1 ml of sterile distilled water in each tube

for regular tablets and 0.5 ml for ODC test strips.

3. Add a loopful of cell paste or 0.1 ml of thick

bacterial culture to each tube.

4. Add four ornithine test strips to the first four tubes

and four lysine tablets to the other four tubes.

5. Incubate the LDC tubes at 35°C for 24 hours and

the ODC test strips for 4 to 6 hours.

6. A color change to purple (LDC) or red (ODC)

constitutes a positive test; no color change is a

negative test.



Second Period

1. Examine the cultures for color changes in the

medium and record your results in the report for